Complement factor H dysfunction in atypical hemolytic ... - Helda

Complement factor H dysfunction in atypical hemolytic ... - Helda

Academic Dissertation

Complement factor H dysfunction in atypical

hemolytic uremic syndrome

Markus Lehtinen

Department of Bacteriology and Immunology

Haartman Institute

University of Helsinki


To be publicly discussed with permission of the Medical Faculty of the University of Helsinki, in the

Small Auditorium of the Haartman Institute on March 18th, 2011, at 12 o’clock noon.


T. Sakari Jokiranta

MD, PhD, Adjunct Professor

Department of Bacteriology and Immunology

Haartman Institute

University of Helsinki


Olli Lassila

MD, PhD, Professor

Department of Medical Microbiology and Immunology

University of Turku

Sarah Butcher

PhD, Professor

Institute of Biotechnology

University of Helsinki


Anna Blom

PhD, Professor

Department of Laboratory Medicine

University of Lund

©2011 by Markus Lehtinen

Printed at Unigrafia OY

ISBN 978-952-92-8586-0 (paperback)

ISBN 978-952-10-6817-1 (pdf)

To my family

Minna, Iina, and Enna


Original publications ............................................................................................................................... 7

Abbreviations .......................................................................................................................................... 8

Abstract ................................................................................................................................................. 10

1 Review of the literature ................................................................................................................ 12

1.1 Main functions of the complement system .......................................................................... 12

1.2 Activation of the complement system .................................................................................. 12

1.3 Amplification and terminal pathway of complement ........................................................... 15

1.4 Regulation of the complement system ................................................................................. 16

1.5 Complement in innate immune response ............................................................................ 22

1.6 Complement in adaptive immunity ...................................................................................... 24

1.7 Complement in cell death ..................................................................................................... 25

1.8 Crosstalk of complement with coagulation system .............................................................. 27

1.9 Atypical hemolytic uremic syndrome ................................................................................... 29

2 Aims of the study .......................................................................................................................... 32

3 Materials and Methods ................................................................................................................. 33

3.1 Materials ............................................................................................................................... 33

3.2 Methods ................................................................................................................................ 33

4 Results ........................................................................................................................................... 39

4.1 Rational mutagenesis of FH19-20 ......................................................................................... 39

4.2 Self - non-self discrimination by FH19-20 ............................................................................. 48

4.3 aHUS mutations disturb target recognition function of FH19-20 by various mechanisms .. 52

5 Discussion...................................................................................................................................... 56

5.1 Sequence and structure analysis in mutagenesis of FH19-20 .............................................. 56

5.2 Self – non-self discrimination by FH19-20 ............................................................................ 56

5.3 Molecular pathogenesis of aHUS .......................................................................................... 58

5.4 Dysfunction of FH19-20 in aHUS ........................................................................................... 58

6 Acknowledgments ......................................................................................................................... 61

7 References .................................................................................................................................... 64

Original publications


Lehtinen MJ, Meri S, and Jokiranta TS. Interdomain contact regions and angles

between adjacent short consensus repeat domains. Journal of Molecular Biology.

344(5):1385-96, 2004.


Jokiranta TS, Jaakola VP, Lehtinen MJ, Pärepalo M, Meri S, and Goldman A. Structure

of complement factor H carboxyl-terminus reveals molecular basis of atypical

haemolytic uremic syndrome. EMBO Journal. 25(8):1784-94, 2006.


Lehtinen MJ, Rops AL, Isenman DE, van der Vlag J, and Jokiranta TS. Mutations of

factor H impair regulation of surface-bound C3b by three mechanisms in atypical

hemolytic uremic syndrome. Journal of Biological Chemistry. 284(23):15650-8, 2009.

IV Kajander T, Lehtinen MJ, Hyvärinen S, Bhattacharjee A, Leung E, Isenman DE, Meri S,

Goldman A, and JokirantaTS. Dual interaction of factor H with C3d and

glycosaminoglycans in host-nonhost discrimination by complement. Proceedings of

the National Academy of Sciences USA. Early Edition Feb 1, 2011.





aHUS Atypical hemolytic uremic syndrome


Alternative pathway

BCR B cell receptor



C1-Inh C1 inhibitor

C1qR C1q receptor

C3aR C3a receptor

C4bp C4 binding protein

C5aR C5a receptor


Cofactor activity


Cluster of differentiation


Classical pathway


Complement receptor

CRP C-reactive protein

CS-A Chondroitin sulfate A

DAA Decay accelerating activity

DAF Decay accelerating factor (CD55)


Dendritic cell

DDD Dense deposit disease


Endothelial cell


Sheep erythrocyte




Factor B

FcγR Fc gamma receptor


Factor D


Factor H

FHL-1 Factor H like protein 1

FHR Factor H related protein


Factor I

GAG Glycosaminoglycan

GBM Glomerular basement membrane

GEnC Glomerular endothelial cell

GlcNAc N-acetyl-glucosamine


Heparan sulfate


Immune complex

iC3b Inactive C3b

iC4b Inactive C4b


Interdomain contact region






Lectin pathway



MAC Membrane attack complex

MASP MBL-associated serine protease

MBL Mannan binding lectin

MCP Membrane cofactor protein (CD46)

mGEnC mouse glomerular endothelial cell


NF-kappa B Nuclear factor kappa B

NMR Nuclear magnetic resonance



PBS Phosphate buffered saline



PTX3 Long pentraxin 3

RBC Red blood cell

RCA Regulators of complement activation

RLA Radioligand assay


Sialic acid

SAP Serum amyloid P component

SCR Short consensus repeat domain

SDS-PAGE Sodium dodecyl sulfate – polyacrylamide gel electrophoresis

SPR Surface Plasmon resonance


T-cell dependent

TGF-β Transforming growth factor β


Tissue factor

THBD Thrombomodulin


T-cell independent

TLR Toll-like receptor

TMB Tetramethylbenzidine

TNF-α Tumor necrosis factor α

vWF von Willebrand factor


Ångström (0.1 nm)



Complement is a group of plasma proteins that recognizes and attacks invading microbes leading to

opsonization, lysis, and induction of inflammation. It also facilitates clearance of apoptotic and

necrotic cells by opsonization and enhances B-cell responses to antigens. Complement is initiated via

different activation pathways that converge at the C3 step, leading to C3b deposition on the target.

C3b deposition initiates the amplification of the complement that leads to release of anaphylatoxins

and to the formation of lytic membrane attack complexes (MAC). One of the complement activation

pathways - the alternative pathway (AP) - can in principle activate on any surface, self or non-self,

and is therefore tightly regulated by the endogenous regulatory proteins. In atypical hemolytic

uremic syndrome (aHUS) the AP regulation on self surfaces is insufficient and leads to complement

attack against erythrocytes, platelets, and endothelial cells resulting usually in end-stage renal

disease. In this disease, impaired regulation of the AP activation and complement amplification

result from either point mutations in AP proteins or autoantibodies that interfere with their


The AP protein that is most often dysfunctional in aHUS is factor H (FH). FH is the main regulator of

AP activation in plasma, and on the self surfaces it is one of the key regulators inhibiting AP

activation and complement amplification. FH is composed of 20 homologous short consensus repeat

domains (SCRs) that are also found in many other complement regulators. The N-terminal domains

1-4 (FH1-4) mediate together with factor I the inactivation of C3b on the self surfaces. The C-

terminal domains 19 and 20 (FH19-20) are critical for the ability of FH to discriminate between C3b

opsonized self and non-self surfaces. Self surfaces are rich in anionic sialic acids (SA) and

glycosaminoglycans (GAGs) such as heparan sulfate that are not usually present in non-pathogenic

microbes. FH19-20 contains binding sites for both the C3d part of C3b and self surface polyanions

that enhances avidity of FH to C3b on self surfaces and thus C3b inactivation. Factor H mutations

that have been described from aHUS patients segregate to FH19-20 and impair self surface

recognition by FH.

The aims were to study rational mutagenesis of SCR domains, molecular mechanism of FH19-20

mediated self – non-self discrimination and to investigate why point mutations in FH19-20 lead to

aHUS. To address these questions crystallographic structures of FH19-20 and FH19-20 in complex

with C3d (FH19-20:C3d) were solved and rational mutagenesis to FH19-20 was planned based on

sequence and structure analysis of SCRs and FH19-20. Point mutations that had aHUS association or

were located in the FH19-20-C3d or heparin binding interfaces were introduced into recombinant

FH19-20. Functional defects caused by these mutations were studied by analyzing the binding of the

FH19-20 mutant proteins to C3d, C3b, heparin, and mouse glomerular endothelial cells (mGEnCs)

using radioligand assays, affinity chromatography, enzyme immunoassays, and surface plasmon


The results revealed two independent binding interfaces between FH19-20 and C3d - the FH19 site

and the FH20 site. Superimposition of the FH19-20:C3d complex on the previously published C3b

and FH1-4:C3b structures showed that the FH20 site on C3d was partially occluded, but the FH19 site

was fully available. Furthermore, the binding of FH19-20 via the FH19 site to C3b did not block the

binding of the functionally important FH1-4 domains and kept the FH20 site free. The results of


structural analysis also showed that the simultaneous binding of the FH19-20 via the FH19 site to

C3b and via the FH20 site to C3d was possible.

It was shown by binding assays that when FH19-20 was bound to C3b via the FH19 site, FH20 domain

could bind to heparin. In addition the heparin binding and mGEnC binding sites on FH20 were found

to be nearly identical indicating that the FH20 mediates binding to cells and heparin similarly. It was

further shown that FH19 site and FH20 were both necessary to recognize non-activator cell model of

self-surfaces. These findings indicate that simultaneous binding of FH19 site to C3b and FH20 to

anionic self structures are the key interactions in self surface recognition by FH and explains how FH

discriminates between self and non-self.

The aHUS associated mutations on FH19-20 were found to disrupt binding of the FH19 or FH20 site

to C3d/C3b, or to disrupt binding of FH20 to heparin or mGEnC. Any of these dysfunctions leads to

loss of FH avidity to C3b bearing self surfaces explaining the molecular pathogenesis of aHUS cases

where mutations are found within FH19-20.

In conclusion, the results have revealed the molecular mechanism of FH19-20 mediated self – nonself

discrimination by FH. It is based on simultaneous binding of FH19 site to C3b and binding of

FH20 to anionic self structures that increases FH avidity to C3b and enhances downregulation of

complement activation. The aHUS-associated mutations in FH19-20 disrupt the self – non-self

discrimination mechanism of FH that can lead to thrombocytopenia, microangiopathic hemolytic

anemia, and acute renal failure - the triad of typical characteristics in aHUS. Understanding of the

molecular pathogenesis of aHUS will help in designing correct treatment for the patients and in

developing new therapeutic approaches for aHUS.


Review of the literature

1 Review of the literature

1.1 Main functions of the complement system

The complement system is composed of about 35 plasma or membrane proteins that form a

cascade-like activation, amplification, effector, and control system (Müller-Eberhard 1988). During

an infection, microbes trigger complement system via lectin pathway (LP), classical pathway (CP),

and alternative pathway (AP) that all converge to the amplification of the cascade and opsonization

of the microbe with C3b. Opsonization facilitates phagocytosis and stimulates B-cell responses, and

the amplification leads to release of anaphylatoxins that attract and activate the cells of the innate

immune system (Hugli et al. 1978; Fearon et al. 2000; Köhl 2006). The end point of the complement

cascade is the formation of membrane attack complex (MAC) that can insert into target membranes

and lead to lysis of the microbe (Cole et al. 2003).

Another major function of complement is to facilitate clearance of apoptotic and necrotic cells (Poon

et al. 2010). Complement activation leads to opsonization of the non-viable cells, but the activation

is restricted and does not lead to lysis of self cells and generation of heavy inflammatory response. In

addition complement facilitates clearance of immune complexes (IC) from circulation by solubilizing

and transporting them to the reticuloendothelial system (Schifferli et al. 1986).

1.2 Activation of the complement system

Complement is continuously monitoring blood and lymph for the presence of its targets e.g.

microbes and necrotic cells. The targets are recognized mainly by three activation pathways CP, LP,

and AP. Recent results have suggested that properdin (P) may also act as a target-recognition

molecule leading to complement activation (Hourcade 2006). These pathways recognize different

structures on the targets but all lead to C3b deposition on the surface (Figure 1). Complement can

also be activated extrinsically by coagulation system proteases such as thrombin, which has been

shown to cleave C3 (Spath et al. 1976; Huber-Lang et al. 2006).

Figure 1. Schematic presentation of the activation of complement cascade. After recognizing the

target all the three pathways converge to covalent attachment of C3b on the surface.


Review of the literature

1.2.1 Alternative pathway

AP activation requires only the plasma proteins factor B (FB), factor D (FD), and C3 to initiate the

attack on any surface (Figure 2) (Schreiber et al. 1978). The activation is not really triggered but is

rather continuous by spontaneous conversion of C3 to metastable C3* that reacts with water and

forms C3b like C3(H 2 O)(Lachmann et al. 1975; Pangburn et al. 1983). C3(H 2 O) binds FB and forms a

C3(H 2 O)B complex that is a target for protease FD (Pangburn et al. 1981). FD cleaves FB in a complex

that leads to release of Ba fragment and to formation of the enzyme C3(H 2 O)Bb. The Bb fragment is

attached transiently in C3(H 2 O)Bb that has a half-life of 90 seconds restricting the operation time of

the enzyme and complement activation (Medicus et al. 1976; Pangburn et al. 1986). Properdin (P)

can act as positive regulator of complement by binding to C3(H 2 O)Bb that stabilizes the enzyme

(Fearon et al. 1975). C3H 2 OBb is a C3 convertase enzyme that cleaves C3 to C3a and to metastable

C3b* that has a short-lived and highly reactive thioester moiety (Law et al. 1977; Sim et al. 1981).

Importantly, C3b* can react with –OH or –NH groups that are universally present in almost every

biological macromolecule, giving explanation to the versatile recognition capability of the AP

(Pangburn et al. 1980a; Pangburn et al. 1980b). A covalent bond is formed between C3b and the

target that directs the downstream complement cascade events to that site.

Figure 2. Alternative pathway activation. Spontaneous hydrolysis of C3 to C3* (box 1) initiates the

formation of C3(H 2 O)Bb that converts C3 to metastable C3b* (box 2). C3b* can attach to surface or

react with water and form fluid phase C3b(H 2 O)Bb that can cleave more C3 (box 3). Properdin has

been omitted from the figure for clarity. The dotted arrows indicate enzymatic activity.

1.2.2 Classical pathway

The action of the AP is complemented by CP and LP that utilize pattern recognition molecules to

identify their targets. The CP is initiated by the collectin C1q that recognizes multiple ligands like lipid

A that is part of lipopolysaccharide (LPS) on microbial surfaces (Loos et al. 1974). CP is activated

indirectly by binding of C1q to clusters of IgG1 or IgG3, or IgM on microbial surfaces or in immune

complexes. CP is also activated on dying cells that are opsonized with C-reactive protein (CRP) or


Review of the literature

display phosphatidylserine, chromatin, and/or DNA on their surface that are all C1q ligands (Agnello

et al. 1970; Claus et al. 1977; Robey et al. 1985; Paidassi et al. 2008).

C1q monomer is composed of collagen-like tail and fibrinogen like head domain that form a

hexameric C1q that resembles a bunch of tulips (will be referred as C1q) (Strang et al. 1982). C1q is

bound to two C1r and to two C1s proteases that together form C1 complex (C1qr 2 s 2 ) in plasma or on

the target (Gigli et al. 1976). Binding of C1q to the target auto-activates C1r that in turn activates C1s

(Sim et al. 1977). Active C1s enzyme cleaves plasma C4 to C4a and C4b (Sim et al. 1977). C4a that is

released to plasma in proteolysis is not anaphylatoxic and is not known to have any other function

either (Gorski et al. 1979; Meuer et al. 1981). C4b on the other hand has highly reactive and shortlived

thioester (like C3b) that mediates covalent binding of C4b to the target (Dodds et al. 1996). C4b

acts as an acceptor for C2 that is cleaved by C1r to fragments C2a and C2b (Sim et al. 1977). The

complex C4b2b (C4b2a in old nomenclature), i.e. the classical pathway C3 convertase, cleaves

plasma C3 to C3b and C3a (Cooper 1975).

1.2.3 Lectin pathway

The LP is initiated by mannan binding lectin (MBL), or one of the L-, H-, or M-ficolins that are

homologous to C1q (Thiel 2007). LP recognizes microbial sugar structures, like lipoteichoic acid (LTA)

and peptidoglycan of gram positive bacteria, GlcNAc and GalNAc of gram negative bacteria, and

mannose/mannan in yeast cell walls, leading to complement attack (Ikeda et al. 1987; Lynch et al.

2004; Matsushita 2009; Zhang et al. 2009). It also recognizes DNA and C-reactive protein (CRP) on

the surfaces of dying cells and facilitates their clearance (Claus et al. 1977; Ng et al. 2007). The

functions of the CP and LP are thus similar, but the ligands on the targets are different, and

importantly only the CP recognizes antibodies (Wallis et al. 2010).

MBL or one of the ficolins forms a complex with one of the MBL-associated serine proteases 1, 2, or

3 (MASPs) of which MASP-2 has been shown to cleave C4 and C2 and to deposit C3b on the target

(Takayama et al. 1994; Thiel et al. 1997; Matsushita et al. 2000a). MASP-1 can apparently cleave C2

and C3 but not C4 and also to activate of MASP-2 (Chen et al. 2004; Takahashi et al. 2008). This

indicates a distinct function for MASP-1 from MASP-2, but its role is still elusive. The function of

MASP-3 is unknown, but it does not cleave C2 or C4 (Dahl et al. 2001; Zundel et al. 2004). MASPs are

homologous to C1r and C1s but do not bind C1q. Also C1r and C1s do not bind MBL or ficolins that

makes the activation of the pathways clearly distinct but functionally complementary (Harmat et al.

2004; Wallis et al. 2010).

1.2.4 Properdin pathway

The recently rediscovered properdin pathway utilizes properdin as a pattern recognition molecule

(Pillemer et al. 1954; Hourcade 2006). Properdin has been shown to bind to dying cells, certain

microbes and malignant cells where it acts as a receptor for C3b, C3bB and C3bBb, initiating the

amplification of the complement system (Kemper et al. 2008). The significance of the properdin

pathway is still elusive, but it has raised a lot of attention as a novel activation pathway.


Review of the literature

1.3 Amplification and terminal pathway of complement

Activation via AP, CP, and LP leads to the main amplification step of complement that is necessary

for proceeding to the terminal pathway and in the generation of many antimicrobial effector

functions (Suankratay et al. 1998; Brouwer et al. 2006; Harboe et al. 2008).

1.3.1 Amplification

C3b deposition on the surface starts the main amplification step of the complement that uses the

components of the alternative pathway C3, FB and FD (Figure 3). Target bound C3b acquires FB from

plasma that is cleaved by FD and the complex C3bBb is formed (Lesavre et al. 1979). C3bBb, i.e. the

alternative pathway C3 convertase, is an enzyme that cleaves C3 to C3a and C3b and thus amplifies

C3b deposition on the surface. C3bBb, like fluid phase C3(H 2 O)Bb, is transient with half life of 90 s

(Medicus et al. 1976; Pangburn et al. 1986). C3a is released to the environment where it informs

inflammatory cells (e.g. macrophages and neutrophils) that complement activation is taking place

(Cochrane et al. 1968). The high density of C3b molecules and C3bBb complexes enhances binding of

the multimeric properdin to C3b and C3bBb, thus increasing the half-life of the C3bBbP

approximately 10-fold and further enhancing complement activation (Fearon et al. 1975). Although

the classical pathway C3-convertase C4b2b can also bind properdin, the role of CP is considered to

be less significant in amplification (Harboe et al. 2004).

Figure 3. The main amplification step of the complement system and the terminal pathway.

Complement activation leads to deposition of C3b on the surfaces. The generation of C3bBb(P)

amplifies C3 conversion to C3b (box 1) and leads to propagation of the cascade by binding back to

C3bBb(P) on the surface (box 2). C3b 2 Bb(P) acts as C5 convertase that initiates the terminal pathway

(box 3). The dotted arrows indicate enzymatic activity.

1.3.2 Terminal pathway

The terminal pathway of complement is initiated when ample amounts of C3b is created and some

of it binds back to C3bBb(P) or C4b2b(P) complexes that change their substrate specificity to C5 and

cleave it to C5a and C5b (Takata et al. 1987; Kinoshita et al. 1988; Rawal et al. 2001). Importantly,

C5a that is released to plasma is a powerful anaphylatoxin and chemotaxin, and more potent than

C3a released earlier in the cascade (Meuer et al. 1981). Unlike C3b or C4b, C5b is not capable of

covalent binding to the surface but acts as a platform for sequential C6 and C7 binding (Gewurz et al.

1968; Podack et al. 1978a; Law et al. 1980). C5b-7 inserts into the membrane of the target cell and

acquires C8 from plasma (Hammer et al. 1975; Podack et al. 1980). C8 can protrude into the lipid

bilayer and act as an initiator of attachment of 16 to 18 C9 molecules that create a cylindrical pore in


Review of the literature

the membrane (Podack et al. 1982; Tschopp et al. 1982). This C5b-9 or membrane attack complex

(MAC) allows diffusion of small molecules through the membrane that leads to rapid lysis of the

target cell (Podack et al. 1984b). Formation of MAC together with opsonization of the target with

C3b and the release of C3a and C5a are the main consequences of the amplification and terminal


1.4 Regulation of the complement system

The complement system is capable of initiating and augmenting the inflammatory response and

lysing cells as described above. In order to prevent unwanted damage to self cells the complement

system needs to be tightly regulated. Regulatory proteins may act during activation, amplification,

and effector steps of the cascade that enables fine tuning of the activation in different physiological


1.4.1 Regulator proteins

Many complement regulatory proteins are encoded by the regulators of complement activation

(RCA) gene cluster in chromosome 1 (Carroll et al. 1988; Rodríguez de Córdoba et al. 1999). All the

RCA proteins are composed of 4 to 30 homologous short consensus repeat (SCR) domains that have

probably arisen by gene duplication events (Pardo-Manuel et al. 1990; Krushkal et al. 2000). SCR

domains are small, approximately 60 amino acid protein domains that typically have short betastrands

forming a bead-like structure stabilized by two characteristic disulphide bridges (Figure 4)

(Norman et al. 1991). The SCR domains are usually arranged in proteins in a head-to-tail fashion

making RCA proteins resemble beads-in-string (Perkins et al. 1991). The binding sites in SCR- bearing

proteins may span over several domains and therefore the orientation between the SCRs is critical

for the function of the protein.

The RCA proteins include fluid phase proteins C4b binding protein (C4bp), factor H (FH), factor H-like

protein 1 (FHL-1), and six factor H related (FHR) proteins 1, 2, 3, 4A, 4B, and 5, and membrane bound

regulators complement receptor 1 (CR1, CD35), membrane cofactor protein (MCP, CD46), and decay

accelerating factor (DAF, CD55). Complement receptor 2 (CR2, CD21) is also encoded by the same

locus, but has not been shown to regulate complement activation. FHL-1 is an alternatively spliced

and truncated gene product of FH that has the first seven SCRs and four extra amino acids in the C-

terminus (Scwaeble et al. 1987). Genes for other complement regulators are scattered in the

genome and are composed mainly of other types of domains. These include membrane proteins

CD59 (protectin) and thrombomodulin (THBD), and plasma proteins carboxypeptidase B, sMAP,

MAP-1, vitronectin, clusterin, and C1 inhibitor (C1-Inh) (Reid et al. 1989).

All the membrane bound regulators (RCA and non-RCA) are quite ubiquitously expressed on self cells

to protect them, but for example MCP is not expressed on erythrocytes (Cole et al. 1985). Fluid

phase regulators protect also self-cells, but importantly also act on non-cellular self-structures.

Furthermore, fluid phase regulators restrict spreading of complement activation from the site of

attack and prevent the activation of complement in plasma.


Review of the literature

Figure 4. Schematic presentation of the exon structure of DAF gene and the crystal structure of

four SCRs in DAF. Typically one exon codes a single SCR domain but some domains are coded by two

exons. In proteins, SCRs are aligned like beads on a string. For example, FH is composed of 20 SCRs,

CR1 of 30 SCRs, and MCP and DAF of 4 SCRs. DAF PDB ID: 1OJV (Lukacik et al. 2004).

1.4.2 Means of regulation

The regulator proteins function by competing with the complement components for the binding

sites. In addition, they may have cofactor activity (CA) or decay accelerating activity (DAA) (Figure 5).

The decay of the transient C3 and C5 convertases can be accelerated by for example FH and DAF

(DAA). Factor I (FI) is a soluble protease that cleaves C3b and C4b to their inactive forms iC3b and

iC4b by releasing C3f or C4f fragments to the fluid phase (Harrison et al. 1980a; Harrison et al.

1980b). FI needs C4bp, CR1, MCP, FH, or FHL-1 as a cofactor to perform the proteolysis. These

proteins are said to have cofactor activity (CA). CR1 is not particularly efficient in generating iC3b,

but is the only regulator that acts as a cofactor for the third cleavage that generates C3dg and C3c,

which is released to fluid phase (Ross et al. 1982; Seya et al. 1985). C3dg can be further cleaved by

plasma proteases to C3d that is thought to have similar biological activity than C3dg.

Figure 5. Principles of decay accelerating and cofactor activities. A. Proteins that have decay

accelerating activity can accelerate the decay of C3 and C5 convertases and thus inhibit complement

activation. An example of DAA is illustrated with DAF and C3bBb. B. Proteins that have cofactor

activity act as cofactors for protease FI that inactivates C3b. An example of CA is illustrated with

MCP. The DAA and CA functions are similar in fluid phase and on surfaces.

1.4.3 Factor H and self – non-self discrimination

The properties of surfaces that are different in humans and microbes determine whether

complement activation is terminated or not. It has been shown that C3b prefers to attach to certain


Review of the literature

carbohydrates and amino acid hydroxyl groups, but this is not sufficient for target discrimination

(Tack et al. 1980; Meri et al. 1990b; Sahu et al. 1994; Sahu et al. 1995). Rather, the surface

properties determine the rate of C3b deposition. Thus target discrimination is mediated by FH that

forms a ternary complex with C3b and the surface (Carreno et al. 1989; Meri et al. 1990b).

Accordingly, it has been shown that the presence of negatively charged heparin or sialic acids on the

self cell surfaces leads to approximately 10-fold increase in the avidity of FH to C3b-surfaces and to

higher cofactor activity of FH (Weiler et al. 1976; Fearon 1978; Pangburn et al. 1978; Kazatchkine et

al. 1979b; Meri et al. 1990b).

1.4.4 Anionic self surface structures

Heparin, a highly sulfated form of heparan sulfate (HS), has been used widely in complement

research as a model of the cell surface glycosaminoglycans (GAGs). HS is a major contribution to the

negative charge of the endothelial cell surface glycocalyx and extracellular matrices, like glomerular

basement membrane (GBM) (Rops et al. 2004b; Weinbaum et al. 2007). Soluble heparin is released

and synthesized only in mast cells, but HS in the glycocalyx of the cells has heparin-like domains

where FH is thought to bind.

Chondroitin sulfate A (CS-A) is the main GAG on platelets (Okayama et al. 1986). Upon platelet

activation, the fully sulfated form of CS-A is exposed on the surface and is also released from α-

granules (MacPherson 1972; Ward et al. 1979; Okayama et al. 1986). Factor H also protects platelets

from complement attack (Devine et al. 1987).

Sialic acids or neuraminic acids also contribute to the biology and the high negative charge of the self

surfaces in addition to GAGs. Sialic acids are a diverse group of nine-carbon sugars with a negative

charge that are present as terminal sugars in glycans of lipids and proteins (Angata et al. 2002). Sialic

acid biology and specificity of the interactions with proteins are still very much elusive (Varki 2008).

However, target surfaces bearing sialic acids, especially erythrocytes, are recognized by FH, which

subsequently restricts complement activation on these surfaces (Fearon 1978; Pangburn et al. 1978).

1.4.5 C3b, GAG and sialic acid binding sites on FH

The major functions of FH have been located on certain domains by several structural and functional

methods (Figure 6). The domains 19 and 20 of factor H (FH19-20) have been shown to be crucial for

self – non-self discrimination (Pangburn 2002; Ferreira et al. 2006), to bind C3d, iC3b and C3b

(Sharma et al. 1996; Jokiranta et al. 2000; Pangburn et al. 2004), and to bind heparin (Gordon et al.

1995; Blackmore et al. 1998; Herbert et al. 2006). The sialic acid and CS-A binding sites that are

important in self surface recognition have been localized to FH16-20 (Ram et al. 1998) and FH15-20,

respectively (Jokiranta et al. 2005). These binding sites are probably located in FH19-20, since these

domains are critical for protecting sialic acid-rich sheep erythrocytes (Pangburn 2002) and CS-A rich

platelets (Jokiranta et al. 2005; Vaziri-Sani et al. 2005). FH19-20 is also a hot-spot for aHUS

associated mutations in FH (Pérez-Caballero et al. 2001).

FH1-4, or minimally FH1-3, binds to C3b, possesses CA and DAA functions, and also competes with

FB on binding to C3b (Alsenz et al. 1984; Gordon et al. 1995; Kühn et al. 1995a; Kühn et al. 1996a;


Review of the literature

Wu et al. 2009). Third binding site for C3b on FH mediates binding to the C3c part of C3b, but the

results on the location have been controversial. FH8-20 has been shown to bind C3c, whereas in the

same assay, FH1-5 and FH19-20 did not bind C3c, and FH2-6, FH8-11, and FH15-18 did not bind C3b

(Jokiranta 2000). Others have shown that FH6-8, FH6-10, FH7-8 and FH8-15 bind C3b, whereas FH15-

18, FH10-15, FH10-12, FH11-14, FH12-13, and FH13-15 do not interact with C3b ((Sharma et al.

1996; Schmidt et al. 2008)). Thus it seems that the binding site would be located at FH8 and might

expand to FH7 and FH9 as well, but the role of other domains or existence of even a fourth binding

site cannot be ruled out.

FH has a well defined heparin binding site on FH7 in addition to FH19-20 (Gordon et al. 1995;

Blackmore et al. 1998; Herbert et al. 2006; Prosser et al. 2007). A third heparin binding site has been

mapped to domain 9 and also suggested to be in domain 13 (Pangburn et al. 1991; Ormsby et al.

2006), but a recent report has contradicted the existence of these sites (Schmidt et al. 2008). The

mid-part of the FH has been shown to be compact and partly folded back and thus functional sites

may be hidden in the full protein (Aslam et al. 2001; Oppermann et al. 2006).

Figure 6. Schematic presentation of binding sites in FH. The SCR domains of FH are presented as

circles.The solid line presents the binding site and dashed line presents possible role of additional

domains in binding.

1.4.6 Regulation of complement activation

Factor H is the main regulator of AP activation that is initiated in the fluid phase. Deficiency of FH

leads to consumption of C3 from plasma and to Dense Deposit Disease (DDD) (Smith et al. 2007).

DDD and aHUS both affect kidney function and are caused by dysfunctional factor H. However, in

DDD complement is activated in plasma and glomerular basement membrane, while in aHUS

complement is activated mainly on cell surfaces. Factor H functions in the fluid phase by competing

with factor B for C3b binding, by acting as a cofactor in cleavage of C3b to iC3b and accelerates

spontaneous decay of the fluid phase C3 convertases, C3b(H 2 O)Bb and C3(H 2 O)Bb (Weiler et al.

1976; Whaley et al. 1976; Pangburn et al. 1977; Kazatchkine et al. 1979a; Farries et al. 1990). FHL-1

performs CA and DAA like FH but the physiological significance of FHL-1 has not been shown - it

could be modulation of activation, since plasma concentration of FHL-1 is about 10 % of FH

concentration (Misasi et al. 1989; Kühn et al. 1995b; Kühn et al. 1996b). C4bp has also been shown

to cleave fluid phase C3b and thus may fine tune AP activation (Nagasawa et al. 1977; Seya et al.

1985; Blom et al. 2001).


Review of the literature

The main regulator of the CP and LP is C1-Inh, which binds to C1r, C1s (Ratnoff et al. 1969), and

MASPs (Matsushita et al. 2000b). C1-Inh is a suicide protease inhibitor that irreversibly binds to its

targets (Laurell et al. 1978) and disassembles the C1 and probably also MBL/ficolin/MASP complexes

(Ziccardi et al. 1979; Presanis et al. 2004). The activation of the LP is also regulated by MAP-1 and

sMAP proteins that are alternatively spliced isoforms of the MASP-1 and MASP-2 genes, respectively

(Matsushita et al. 2000a; Skjøedt et al. 2010). MAP-1 and sMAP lack the serine protease domains of

the MASPs and act as competitive inhibitors for MASPs on MBL and ficolin binding. On surfaces C1-

Inh, sMAP and MAP-1 function less efficiently than in the fluid phase, allowing complement

activation (Wallis et al. 2010).

CP and LP activation lead to deposition of C4b and C4b2b on the cell surfaces. C4b is cleaved to

inactive C4b (iC4b) by C4bp, MCP, or CR1 that act as cofactors for FI (Scharfstein et al. 1978; Iida et

al. 1983; Seya et al. 1986), and the decay of the C4b2b complexes is accelerated by C4bp, DAF, and

CR1 (Gigli et al. 1979; Nicholson-Weller et al. 1982; Iida et al. 1983). C4bp also binds to C4b and

inhibits convertase formation (Blom et al. 1999).

1.4.7 Regulation of amplification and inhibition of MAC formation on surfaces

The amplification step of the complement is redundantly regulated by many proteins (Figure 7). The

decay of C3bBb is accelerated by FH, FHL-1, CR1, and DAF (Weiler et al. 1976; Whaley et al. 1976;

Fearon 1979; Nicholson-Weller et al. 1982; Kühn et al. 1995a), and the deposited C3b is inactivated

by FH, FHL-1, CR1 and MCP that act as cofactors for factor I mediated cleavage (Pangburn et al.

1977; Medicus et al. 1983; Seya et al. 1986; Kühn et al. 1995c). Factor H also competes with factor B

on binding to C3b (Kazatchkine et al. 1979a; Farries et al. 1990).

Binding of properdin to C3bBb or C4b2b does not probably block the DAA or CA activity of the

regulatory proteins, but rather inhibits their actions by stabilizing the convertase. Properdin has

been shown to inhibit FH and FI mediated cleavage of C3b in C3bBbP (Medicus et al. 1976) and FH

and DAF have been shown to accelerate decay of C3bBbP (Weiler et al. 1976; Whaley et al. 1976;

Nicholson-Weller et al. 1982; Hourcade 2006).

The decay of C5-convertases (C3b 2 Bb(P) and C3bC4b2b(P)) is accelerated by FH (Kuhn 1996), CR1

(Krych-Goldberg 1999), C4bp (Rawal 2007) and DAF (Kuttner-Kondo 2001). MCP and CR1, but not FH

and C4bp, can act as cofactors for FI in cleaving C3b dimers on C5 convertases (Seya 1991) and C4bp,

but not FH, has CA for C3bC4b2b (Meri et al. 1990c; Rawal et al. 2007).

Recently, the action of the C5 convertase was suggested to be inhibited also by FHR-1 (Heinen et al.

2009). The functions of the other five FHR proteins are not well established but they may also act on

C5 convertases (Zipfel et al. 2009). The actions of C5 convertases that lead MAC formation are

inhibited on the self cell membranes by CD59 and vitronectin (S-protein), which interfere with C9

polymerization (Bhakdi et al. 1988; Meri et al. 1990a).


Review of the literature

Figure 7. Regulation of the main amplification loop. FH, DAF, and CR1 accelerate the decay of C3

and C5 convertases. FH, MCP, and CR1 act as cofactors for factor I in cleaving C3b to iC3b. The

amplification loop and the terminal pathway are shown in grey.

1.4.8 Restriction of complement activation in the fluid phase

The function and lifetime of the inflammatory mediators C3a and C5a is regulated by the soluble

protease carboxypeptidase-N. Carboxypeptidase-N cleaves C3a and C5a to the C3adesArg and

C5adesArg forms, which lack terminal arginines (Erdos et al. 1962; Bokisch et al. 1969). The desArg

forms exhibit reduced inflammatory and chemotactic functions. Consequently, the action of

carboxypeptidase-N is to create a chemical gradient that guides the inflammatory cells to the site of

complement activation, and to limit the inflammatory response at more remote sites where the

peptides have been taken (for example, by diffusion).

Some C3b and C4b that are not surface-bound but remain in the fluid phase are cleared mainly by

the fluid phase regulators FH and C4bp (Seya et al. 1995). In addition, the CR1 that is expressed on

red blood cells (RBCs) captures C4b, C3b, iC3b, C3b 2 -IgG, and C3b-ICs (Fearon 1979; Kinoshita et al.

1986; Schifferli et al. 1986). CR1 can also process iC3b to C3dg, an ability unique to this regulator

(Ross et al. 1982).

C5b, C5b6, and C5b-7 generated by amplification may drift from the original site of the activation

and insert into membranes of bystander cells or form MAC in the fluid phase. These actions are

regulated by vitronectin and clusterin, which bind to soluble C5b-7 (sC5b-7), sC5b-8, and sC5b-9

leading to inhibition of MAC formation (Podack et al. 1978b; Podack et al. 1979; Podack et al. 1984a;

Jenne et al. 1989; Tschopp et al. 1993).

Taken together, the main functions of complement regulators are (1) to limit the activation in the

fluid phase and on viable self-cells and noncellular self-surfaces, (2) to regulate the extent of

activation by controlling the amplification, (3) to limit complement attack to the target or its

immediate vicinity by inactivating the components released into fluid phase, and (4) to limit the

effects of the anaphylatoxins, and inflammation in general, to the region where activation occurs.


Review of the literature

1.5 Complement in innate immune response

In an infection, the microbe is opsonized with C1q, MBL, ficolins, C3b, iC3b, and C3dg, and

anaphylatoxins C3a and C5a are generated in response to activation (Figure 8, box 1). Neutrophils,

macrophages, and dendritic cells that are important in the innate immune response bear C3a and

C5a receptors (C3aR and C5aR) and receptors for complement components (Gasque 2004; Haas et

al. 2007; Klos et al. 2009). It is noteworthy that the complement activation products C3a and C5a

cannot inform the cellular immunity about the type of pathogen because all the recognition

pathways lead to these same products - independently of the properties of the target surface. It

seems that C5a non-specifically enhances the proinflammatory response, while toll-like receptor

(TLR) and cytokine receptor activation on immune system cells determines the type of the response.

The interplay between the signaling pathways of C5a receptor (C5aR), TLRs, and cytokine receptors

has been recognized recently (Hajishengallis et al. 2010).

Figure 8. Complement in the initiation of innate immune response. Box 1. Activation of

complement on microbes opsonizes it and creates anaphylatoxin C5a. Box 2. C5a binds to C5aR on

the macrophages, monocytes, neutrophils, and DCs. C5aR activation attracts cells to the site of

infection and augments TLR signaling in macrophages and DCs. Box 3. TNF-α and IL-1 that are

synthesized mainly by macrophages activate endothelial cells. Box 4. C5a has synergistic effects on

TNF-α and IL-1 induced activation of endothelium and expression of selectins. Box 5. CR1, CR2, C1qR,

and FcγR bind to opsonins on the microbe that facilitates phagocytosis. Receptors and cells are

indicated with an oval and bolded circles, respectively. Soluble proteins are not circled. Arrows

schematically indicate diffusion of C5a or cytokines from the source of their production.


Review of the literature

1.5.1 Actions of anaphylatoxins

Complement activation leads to formation of C3adesArg and C5adesArg which induce leukocytosis in

20 minutes, some 30 minutes before cytokines take action, thus providing the first signal of an

infection (Menkin 1949; Rother 1972; Kajita et al. 1990; Jagels et al. 1994). At the infection focus,

C5a and C5adesArg act as powerful chemotactic agents for neutrophils, monocytes, macrophages,

and dendritic cells (DCs) to indicate the presence of an infection and to recruit them to the site of

activation, i.e. the site of invader (Figure 8, box 2) (Shin et al. 1968; Snyderman et al. 1970;

Snyderman et al. 1975; Fernandez et al. 1978). The importance of C3a or C3adesArg in inducing

chemotaxis has been controversial and poorly studied, but in the light of current information they

don’t induce neutrophil chemotaxis, but contribute to eosinophil chemotaxis (Daffern et al. 1995).

The physiological reason for the lack of this activity on neutrophils may be that C3a is also generated

during removal of late apoptotic/necrotic cells, where excess inflammation and recruitment of

neutrophils should be avoided.

Tissue-resident macrophages are important in phagocytosis and in sensing infection. The important

early response to infection by macrophages is to generate pro-inflammatory TNF-α and IL-1 via

activation of the NF-kappa B pathway that leads to activation of the cellular innate immune

responses. Complement activation and generation of C5a is triggered by e.g. yeast cell wall zymosan

and bacterial LPS that also bind to, respectively, TLR2/6 and TLR4 on the macrophage surface.

Triggering of C5aR and TLRs 2/6 or TLR4 in mice have been shown synergistically to induce the NFkappa

B pathway and the production of pro-inflammatory cytokines TNF-α, IL-1, IL-6 and IL-12

(Zhang et al. 2007). C5a has also been shown to induce production and release of TNF-α and IL-1

from LPS-stimulated and non-stimulated human macrophages (Goodman et al. 1982; Cavaillon et al.

1990; Pushparaj et al. 2009) and to activate macrophages dependent on NF-kappa B (Kastl et al.

2006) (Figure 8, box 3). It seems that C5a contributes very early in infection to enhance cellular

immunity together with TLRs (Hajishengallis et al. 2010).

The effects of TNF-α and IL-1 on the endothelium is to activate it via the NF-kappa B pathway

(Schleef et al. 1988) (Figure 8, box 4). C5a acts synergistically with cytokines to activate endothelium

to express neutrophil ligand P-selectin on its surface (Geng et al. 1990; Foreman et al. 1994; Mulligan

et al. 1997), and later in infection leukocyte ligand E-selectin (Springer 1994). Neutrophils that are

attached to the endothelium may be activated to release hydrolytic enzymes that can cause damage

to the endothelium (Varani et al. 1989; Hardy et al. 1994).

1.5.2 Opsonization of microbes

While complement has attracted inflammatory cells to the site of infection, it has also opsonized the

microbe to facilitate its phagocytosis. Professional phagocytes like macrophages, dendritic cells, and

neutrophils use an array of receptors to attach to the pathogen (Figure 8, box 5) (Stuart et al. 2005).

These include C1q receptors (C1qR), Fc gamma receptors (FcγR), integrins CR3 (CD11b/CD18) and

CR4 (CD11c/CD18), and complement receptor 1 (CD35) (Underhill et al. 2002). The most important

phagocytic receptors of the complement system are CR1 and CR3. CR1 binds C3b, iC3b, C3dg, C4b,

and iC4b, and also C1q and MBL on the target surface (Fearon 1979; Kinoshita et al. 1986; Klickstein

et al. 1997; Ghiran et al. 2000; Krych-Goldberg et al. 2001). CR3 recognizes iC3b and also microbial

components such as beta-glucan and LPS (Ross et al. 1985; Wright et al. 1989; Thornton et al. 1996).


Review of the literature

Complement receptors 1 and 3 potentiate FcγR mediated phagocytosis by clustering the receptors

that trigger common signaling pathways (Ehlenberger et al. 1977; Bobak et al. 1987; Fries et al. 1987;

Krauss et al. 1994; Zhou et al. 1995). The functions of CR4 and C1qRs are not well established, but

they seem to participate in recognizing the target and mediating signals to phagocytes (Myones et

al. 1988; Gasque 2004; Ghebrehiwet et al. 2004).

Phagocytes, especially DCs, sense the infection and type of pathogen by several TLRs. The deposition

of the complement components alone on the cells is not sufficient for phagocytosis (Wright et al.

1983), but inflammatory mediators like TNF-α and LPS can provide additional stimuli to induce CRdependent

phagocytosis by enhancing expression of the receptors (Berger et al. 1984; Wright et al.

1985). At least CR3 seems to act in concert with TLR4 and CD14 in regulating the NF-kappa B

pathway (Perera et al. 2001). Taken together, the activation of complement, opsonization of the

microbe, and production of C5a leads to phagocytosis and triggering of anti-microbial effector

mechanisms of the cells.

1.6 Complement in adaptive immunity

1.6.1 C3dg as an adjuvant in B-cell response

Generation of the antibody response against microbes is augmented by complement C3 (Pepys

1974). On complement activation, C3b is bound to microbes and is processed by the actions of CR1

and factor I to the C3dg fragment, which remains covalently bound to the antigen. B-lymphocytes

express complement receptor 2 (CR2, CD21) and the B-cell receptor (BCR) that recognize the C3dg

and the antigen, respectively. CR2 is part of the CD21/CD19/CD81 BCR co-receptor complex that is

able to augment BCR signaling (Carter et al. 1988; Carter et al. 1992). The B-cell response is thus

greatly enhanced when C3dg is bound to the co-receptor complex and antigen to the BCR (Fearon et

al. 2000).

T-cell dependent (TD) antigens (typically non-repetitive proteins) with one epitope for BCR, and one

C3dg attached to it, do not efficiently cluster receptors and trigger the B-cell response. The presence

of two or more C3dg molecules on the antigen clusters the BCR and its co-receptor, increasing the B-

cell response 10 000 fold (Dempsey et al. 1996). T-cell independent (TI) antigens such as

polysaccharides bear identical epitopes and are large enough to cluster receptors and activating B-

cells without T-cell help, but the binding of multiple C3dgs to TI antigens can still further increase the

B-cell activation.

TD (and TI) antigen presentation takes place in lymph nodes and in spleen where T-cell help and

antigen-presenting cells are available to provide second signals to B-cells. In tissues, DCs expressing

CRs capture antigens that may bear C3b, iC3b or C3dg and migrate into lymph nodes to present

antigen to T- and B-cells (Banchereau et al. 1998). Antigen does not need to be transported by DCs

to lymph nodes where resident macrophages, DCs, and follicular DCs can capture C3b/iC3b/C3dgantigen

complexes from lymph and present the antigen (Batista et al. 2009). The complement

receptors CR1, CR2, CR3 as well as FcγRs play critical roles in antigen capturing, trafficking and

presentation to B-cells in lymph nodes and spleen (Pozdnyakova et al. 2003; Ferguson et al. 2004;

Gonzalez et al. 2010).


Review of the literature

B-cells endocytose the antigen, cleave it to peptide fragments, and display some of these fragments

on MHC-II molecules and present the complex to helper T-cells that have been activated by DCs to

display CD40L. CD40-CD40L and TCR-MHCII interactions activate the B-cells that start the formation

of germinal centers. Follicular DCs in germinal centers bear CR1, CR2, and CR3 that retain the C3dgantigen

complex in the follicles and positively affect B-cell affinity maturation and memory B-cell

generation (Reynes et al. 1985).

1.6.2 Complement in T-cell responses

An emerging field in complement research is the role of complement components in T-cell

activation, regulation, and direction of the T-cell responses (Kemper et al. 2007). For example, there

is evidence that synthesis of C3 by DCs is required for full T-cell activation and T H 1 polarization (Peng

et al. 2006). In addition, a critical role for C3aR in DC antigen uptake and T-cell stimulation has been

suggested (Li et al. 2008; Peng et al. 2008) and also C3a and C5a that were produced by DCs were

found to provide costimulatory and survival signals to naïve CD4 cells (Strainic et al. 2008). All these

studies indicate an important role for C3 components, and C3aR and C5aR in the interplay between

DCs and T-cells. Taken together, the complement system is now becoming linked to T-cell biology as

well highlighting the importance of complement as an initiator and regulator of both the innate and

adaptive immune responses.

1.7 Complement in cell death

Uncontrolled complement activation on self cells produces sublytic and lytic depositions of MAC that

can induce apoptosis or cause necrosis (Mevorach et al. 1998; Cole et al. 2003). If MAC inserts into

the membrane it increases the influx of calcium to the cell, speeds up cell death and leads to

swelling of the cell and rupture of the membrane (Kim et al. 1987). These events lead to cell death

that has apoptotic and necrotic features (Shimizu et al. 2000). Importantly, for renal diseases and

aHUS, even sublytic MAC deposition has been shown to induce apoptosis in renal and endothelial

cells (Figure 9, box 1) (Sato et al. 1999; Hughes et al. 2000; Nauta et al. 2002).

1.7.1 Activation of complement

Dying cells present marker-molecules on the surface, like phospholipids and nucleic acids that are

recognized by soluble opsonins and on phagocytes by various receptors (Poon et al. 2010). The

soluble opsonins include MBL, C1q and ficolins that can bind to DNA and phospholipids on the

membrane. Also, IgM and the pentraxins serum amyloid protein (SAP) (Hicks et al. 1992), CRP (Mold

et al. 1999; Gershov et al. 2000), and long pentraxin 3 (PTX3) (Bottazzi et al. 1997) opsonize dying

cells and are recognized by C1q (Korb et al. 1997; Paidassi et al. 2008). C1q and CP activation are key

players in the clearance of dying cells (Gullstrand et al. 2009), and their dysfunction is associated

with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Botto et

al. 1998; Okroj et al. 2007).

CP is activated on late apoptotic and necrotic cells but not much on early apoptotic cells (Figure 9,

box 2) (Nauta et al. 2003). Complement activation on cells in late apoptosis is enhanced by the


Review of the literature

down-regulation of membrane-bound complement regulators DAF and CD59 or by shedding of MCP

and CD59 (Jones et al. 1995; Elward et al. 2005; Cole et al. 2006). If complement activation proceeds

to the terminal pathway, the activation of complement would lead to lysis of the cell and induction

of inflammation (Figure 9, box 3). However, the complement activation is restricted by simultaneous

acquisition of FH and C4bp that bind directly to the apoptotic or necrotic cells via e.g. DNA or by

utilizing opsonins such as CRP (Mold et al. 1984; Sjöberg et al. 2006; Trouw et al. 2007) or PTX3

(Deban et al. 2008). This restricts the launching of the terminal pathway and generation of highly

pro-inflammatory C5a, but not C3a (Figure 9, box 4). C3a has traditionally been considered to be proinflammatory,

but recent evidence suggests that C3a may in fact dampen the inflammatory response

that would be beneficial in clearing apoptotic or necrotic cells (Hawlisch et al. 2006). For example, it

has been shown that neutrophils possess theC3a receptor but C3a does not attract them (Martin et

al. 1997; Kemper et al. 2008). Also, C3a inhibits the production of proinflammatory IL-1β and TNF-α

production in non-adherent peripheral blood monocytes (Takabayashi et al. 1996), and IL-6 and TNFα

production in B-cells (Fischer et al. 1997).

Figure 9. Uncontrolled complement activation on endothelial cells can induce cell death. Box 1.

Complement activation on endothelial cells may induce apoptosis. Box 2. As apoptosis proceeds to

late phase complement is activated via classical pathway and cell surface regulation is downmodulated.

Box 3. If the complement activation is not held in check the cell may be lysed or become

necrotic. Box 4. Complement regulators like FH restrict the activation to C3-stage that prevents the

generation of pro-inflammatory C5a, but allows opsonization of the cells with C3b and iC3b.

1.7.2 Phagocytosis

The activation of complement on dying cells leads to opsonization of the cell with C1q, MBL, ficolins,

and iC3b, C3b, iC4b, and C4b that all act as ligands for phagocytes. For instance, iC3b on apoptotic

cells has been shown to enhance phagocytosis by macrophages (Takizawa et al. 1996). Phagocytes

mediate uptake of apoptotic cells by a variety of receptors. Particularly important are CR1 and CR3,

which recognize C3 and C4 fragments, and CD91/calreticulin receptors which recognize C1q, MBL,

and ficolins (Ogden et al. 2001; Honoré et al. 2007; Jensen et al. 2007). Uptake of apoptotic cells

induces anti-inflammatory cytokines such as TGF-β production in macrophages (Fadok et al. 1998). It

has been shown that CR3 and CR4 dependent uptake of apoptotic or necrotic cells bearing iC3b

dampens the inflammatory response by decreasing IL-12 production by macrophages (Mevorach et

al. 1998; Kim et al. 2004), and that the expression of costimulatory molecules in DCs is decreased

upon CR3 stimulation (Verbovetski et al. 2002; Morelli et al. 2003). Taken together, the activation of


Review of the literature

complement and other mechanisms in clearing late apoptotic or necrotic cells induces the type of

inflammation that is directed to tissue regeneration, while inhibiting the adaptive immune responses

to prevent generation of autoimmune diseases. The important difference in the clearance of late

apoptotic or necrotic cells vs microbes is that high amounts of C5a and MAC are generated only in

response against the microbes.

1.8 Crosstalk of complement with coagulation system

There are three major protease cascades in plasma - coagulation, fibrinolysis, and complement - that

are homologous in terms of protein structure and function (Krem et al. 2002). The complement and

coagulation systems work in an analogous fashion – both are quickly propagating enzyme cascades

that need to be activated only locally and controlled efficiently. These systems are interconnected

and triggered together in physiological and pathological conditions such as blood clotting and aHUS

that is characterized by microvascular thrombi and dysfunctional complement system (Markiewski

et al. 2007).

1.8.1 Initiation of coagulation by C5a and MAC

Coagulation can be initiated via an extrinsic pathway by damaged endothelium, leading to exposure

of subendothelial matrix bearing collagen, tissue factor (TF), von willebrand factor (vWf), and

fibrinogen, or by increased expression of TF and vWf by endothelium (Savage et al. 1996; Karpman

et al. 2006; Adams et al. 2009). C5a and MAC deposition have been shown to induce endothelial cell

damage, vWf release, and TF upregulation (Figure 10, box 1) (Ikeda et al. 1997; Tedesco et al. 1997).

Platelets bind to vWf on endothelium and provide negatively charged phophatidylserine surface and

receptors for binding of plasma coagulation factors (F) (Figure 10, box 2) (Hoffman et al. 2001). The

key protein in initiating the coagulation cascade is the endothelium-bound TF that binds factor VII

(FVII) and localizes the coagulation at the site (Figure 10, box 3). The TF:FVIIa complex (“a”

indicating activated protein) propagates the cascade to formation of prothrombinase FXa:FVa

complex. Prothrombinase cleaves prothrombin to thrombin that binds to negatively charged

surfaces on platelets and endothelium, and activates the platelets on the site of injury (Monroe et al.

1996; Diaz-Ricart et al. 2000).


Review of the literature

Figure 10 Simplified presentation of induction of coagulation system by complement and its feedback

mechanism. Box 1. Complement activation products C3a, C5a, and MAC can induce procoagulant

phenotype to endothelium that leads to up-regulation of TF and vWf. Box 2. Platelets

adhere to vWf and provide negatively charged surface for coagulation. Box 3. TF binds to FVIIa

initiating coagulation that generates thrombin. Thrombin binds to platelets and cleaves several

coagulation cascade enzymes and initiates clot formation. Box 4. Thrombin cleaves C3 and C5 that

initiates complement activation. Box 5. C3a, C5a, and MAC can further activate endothelium and

platelets thus creating a feed-back mechanism.

1.8.2 Activation of complement by coagulation

The amplification and propagation of blood clotting on platelet surfaces is initiated by the actions of

thrombin that leads to generation of the enzyme complexes FIXa:FVIIIa and FXa:FVa on the platelet

surface (Hoffman et al. 2001; Adams et al. 2009). These complexes generate massive amounts of

thrombin feeding back to the coagulation system. The platelets have high affinity binding sites for

thrombin, FIXa, FXa, and FXI that increases their local concentration (Greengard et al. 1986; Rawala-

Sheikh et al. 1990; Cirino et al. 1997). At high concentrations thrombin, FIXa, FXa, and FXIa can

cleave C3 and C5 and thus initiate complement activation at the site of coagulation (Figure 10, box 4)

(Spath et al. 1976; Huber-Lang et al. 2006; Amara et al. 2010). C3a, C5a and sublytic amounts of MAC

can further activate platelets and endothelium creating a feed-back loop (Figure 10, box 5)

(Grossklaus et al. 1976; Polley et al. 1983; Damerau et al. 1986; Wiedmer et al. 1986).

The endpoint of the coagulation cascade is the formation of a blood clot. Thrombin cleaves

fibrinogen to fibrin that forms a soft clot that is further covalently linked by FXIIIa to form hard clots.

In aHUS complement activation takes place ubiquitously on cell surfaces, but the renal vessels in the

kidney glomeruli are primarily occluded. Microvasculature in glomeruli forms a capillary bed where

the diameter of a capillary is only 3-10 µm whereas the average diameter of an erythrocyte is 7-8 µm

and that of a platelet is about 2 µm (Weinbaum et al. 2007) and thus the formation of thrombi easily

occludes these narrow capillaries.


Review of the literature

1.8.3 Interplay of complement and coagulation regulators

The spreading of the local blood clotting is controlled and down regulated by the plasma protease

inhibitors (e.g. anti-thrombin III) and the intact endothelium surrounding the clot. Thrombomodulin

(THBD) is expressed especially abundantly on endothelium in microvasculature (Cadroy et al. 1997)

and captures active thrombin that drifts from the site of coagulation. Upon binding to THBD,

thrombin changes its specificity from fibrinogen to protein C and activates it (Ye et al. 1991).

Activated protein C makes a complex with protein S that inactivates FVa and FVIIIa deposited on

bystander endothelial surfaces. Thus the action of thrombin on intact endothelium is antithrombotic.

THBD also enhances thrombin-mediated activation of pro-carboxypeptidase B, an

inhibitor of fibrinolysis that is also able to inactivate C3a and C5a to their desArg forms (Bajzar et al.

1995; Campbell et al. 2002). Recently, THBD was connected to aHUS and found to regulate

complement by binding FH and C3b, leading to enhanced C3b inactivation (Delvaeye et al. 2009).

Coagulation system protein S has also been shown to participate in complement regulation by

binding C4bp. In complex with C4bp, protein S cannot inactivate FVa and FVIIIa but can still mediate

binding of C4bp to negatively charged glycolipids (Rezende et al. 2004). This interaction thus

promotes coagulation while controlling complement CP and LP activation.

Blood clots are removed by the fibrinolysis system. The main enzyme of this process is plasmin that

cleaves fibrin clots. Plasmin is also able to cleave C3 and C5, suggesting that complement activation

is also kept on at the resolution stage of the clotting (Goldberger et al. 1981; Amara et al. 2010). The

clotting and the complement systems are interconnected and complement activation can induce a

pro-coagulant state (Shebuski et al. 2002; Esmon 2004). This is well exemplified in atypical hemolytic

uremic syndrome where complement and coagulation systems are both triggered.

1.9 Atypical hemolytic uremic syndrome

Hemolytic uremic syndrome is a disease that belongs to a class of diseases called thrombotic

microangiopathies and is characterized by hemolytic anemia, thrombocytopenia, and impairment of

renal function (Ruggenenti et al. 2001). The typical form of HUS usually presents with diarrhea and is

secondary to infections by shiga-toxin associated bacteria such as Escherichia coli serotype O157:H7

(Noris et al. 2005). The atypical form is not associated with diarrhea caused by shiga-toxin producing

bacteria, but has usually a genetic background. Both forms of HUS have similar histologic lesions and

are characterized by thrombotic and inflammatory state of small arteries and capillaries (Ruggenenti

et al. 2001).

1.9.1 Clinical findings

Typical clinical findings in aHUS patients are hemolysis and hemolytic anemia, thrombi in kidney

microcirculation, endothelial swelling and detachment, subendothelial accumulation of protein and

cell debris, and thickening of the arterioles and capillaries (Noris et al. 2009). The glomeruli are

heavily inflamed and occluded leading to impairment of renal function that is the most common

cause of death in aHUS. Renal function is quite well restored in patients with typical HUS, but

atypical form develops to end stage renal disease in about 50 % of the patients (Caprioli et al. 2006).

In 20 % of aHUS patients extrarenal complications, like neurological or cardiovascular, have been

described (Jalanko et al. 2008; Loirat et al. 2008; Sallee et al. 2010).


Review of the literature

1.9.2 Incidence and genetic background

The incidence of HUS is about 6 / 100 000 children under 5 years of which 10 % are classified as

atypical (Noris et al. 2009). Overall, 67 % of aHUS cases with known genetic background are

presented during childhood, but the disease may be triggered also at later age. Thus the disease has

incomplete penetrance and the onset may need mutations in more than one gene or an

environmental trigger like pregnancy or infection (Kavanagh et al. 2008).

The genetic background for all the aHUS patients has not been described, but in 50-60% of aHUS

patients one or more point mutations are found in genes encoding complement proteins FB (1-4 %

of patients), C3 (2-10 %), FH (20-30 %), MCP (5-15 %), and FI (4-10 %) (Noris et al. 2009; Maga et al.

2010). Mutations have also been described in complement and coagulation regulator THBD in 3-5 %

of the patients (Delvaeye et al. 2009; Maga et al. 2010). These patients are often heterozygous for

mutation, but still present the disease, i.e. the mutations have a dominant negative effect.

In 3-5 % of the aHUS patients a recombination event between FH and FHR-1 has occurred that leads

to an abnormal FH gene (Noris et al. 2009; Maga et al. 2010). In addition, the lack of FHR-1 and/or

FHR-3 predisposes to the development of auto-antibodies against FH that have been described in 6-

10 % of patients (Józsi et al. 2008; Noris et al. 2009).

In Finland three patients have been described having aHUS mutation (R1215Q) in factor H gene

((Jalanko et al. 2008); Jokiranta, personal communication). Two of the patients were from a family

that also had asymptomatic carriers indicating incomplete penetrance. One of the aHUS patients had

also factor V Leiden mutation that slightly increases the thrombogenicity of blood.

1.9.3 Molecular pathogenesis of aHUS

Pathogenesis of aHUS has been associated with defective regulation of AP activation and

amplification. The C3bBb complex that initiates the amplification of complement activation is

transient, but some aHUS associated mutations in the interface between the Bb and C3b increase

the half life of C3bBb, leading to uncontrolled deposition of C3b (Goicoechea de Jorge et al. 2007;

Roumenina et al. 2009). It would be expected that aHUS mutations could also disturb DAA and in

this way increase C3b deposition, but apparently this is not the case since DAF dysfunction has not

been associated with aHUS (Kavanagh et al. 2007).

Atypical HUS can also be caused by impaired inactivation of C3b that is normally inactivated on self

cells by FH or MCP that act as cofactors for factor I in cleaving C3b to iC3b. Upon binding of FH or

MCP to C3b, a cleavage site for factor I is formed on C3b (Wu et al. 2009). Factor I then cleaves two

bonds in the C3b releasing C3f and leaving iC3b on the surface in a conformation that is no longer

able to bind FB. Mutations in MCP, FH1-4, or FI can impair the inactivation of C3b (Noris et al. 2003;

Richards et al. 2003; Kavanagh et al. 2005).

In FH most aHUS mutations are not segregated to FH1-4 but to FH19-20. FH19-20 is crucial for selfnon-self

discrimination function of FH and it has been shown that patient-derived full-length FH

proteins bearing mutations W1183L, V1197A, and R1210C are unable to protect non-activator sheep

erythrocytes from lysis (Sánchez-Corral et al. 2004). Domain 19 mutations D1119G, V1134G, E1135D,

Y1142C/D, W1157R, C1163W, and V1168E, and domain 20 mutations R1182S, W1183L/R, T1184R,


Review of the literature

L1189F/P/R, S1191L/W, G1194D, V1197A, E1198A/K, F1199S, R1203W, R1206C, R1210C, S1211P,

R1215G/Q, and P1226S have been described in aHUS patients (see study IV for detailed references

or (Warwicker et al. 1998; Ying et al. 1999; Pérez-Caballero et al. 2001; Richards et

al. 2001; Sánchez-Corral et al. 2002; Neumann et al. 2003; Ståhl et al. 2008; Maga et al. 2010;

Sullivan et al. 2010; Westra et al. 2010).

1.9.4 Treatment

Atypical HUS has usually been treated with plasmapheresis, transplantation, or recently with

monoclonal antibody against C5 (Kavanagh et al. 2010; Köse et al. 2010; Loirat et al. 2010). The

severity of aHUS is dependent on the affected protein that has also an effect on the choice of

treatment. Plasmapheresis is used to treat acute episodes of aHUS, but in some patients this

treatment is only transiently beneficial. Patients carrying MCP mutations have a good prognosis after

kidney transplantation, since the defective protein in the kidney is replaced with functional

molecules in the transplant. For the soluble proteins factor H, factor B, factor I, and C3 kidney

transplantation is not a good option since these proteins are mainly produced in the liver. An option

for these patients is combined liver-kidney transplantation after extensive plasma exchange. This

procedure has been successfully done in Finland to three patients, of which all are still alive, and

abroad to a few patients also with good results (Jalanko et al. 2008; Kavanagh et al. 2010). An

alternative to transplantation has recently been shown to be the anti-C5 monoclonal antibody

Eculizumab, which prevents generation of C5a and propagation of the complement cascade to the

terminal pathway (Gruppo et al. 2009; Nürnberger et al. 2009; Zimmerhackl et al. 2010). This

antibody seems to be a very promising option in the treatment of aHUS, but knowledge of the

molecular pathogenesis of aHUS is critical in developing further treatment options for aHUS



Aims of the study

2 Aims of the study

I. To study the basis of rational mutagenesis to SCR domains and FH19-20.



To analyze the molecular mechanism of the FH19-20 mediated self non-self discrimination

by FH.

To elucidate the pathogenic mechanism of aHUS that is caused by mutations in FH19-20.


Materials and methods

3 Materials and Methods

3.1 Materials

3.1.1 Proteins and carbohydrates

In study II the wild-type FH19-20 without expression tags was cloned by MSc Maria Pärepalo

(Pärepalo 2002). FH19 Del -20 (Q1137A/ Q1139A/ Y1142A) and FH19-20 Del (T1184G/ K1202A/ R1203A/

Y1205A) used in study IV were cloned, expressed and purified by MSc Satu Hyvärinen (Study IV).

C3d that was used in the crystallization in study IV and in functional assays had surface exposed

cysteine (C17, part of the reactive thioester) mutated to alanine. C3dg wild-type, and the C3dg

mutant proteins D36A, E37A, E117A, D122A, E160A, D163A, and K291A were used in binding assays

of study IV. C3d and C3dg proteins were expressed in E. coli without tags and were received from

Professor David Isenman and Elisa Leung (Nagar et al. 1998; Isenman et al. 2010).

Rabbit polyclonal antibodies against FH19-20 and factor H (K2), and monoclonal anti FH19-20

antibody VIG8 (Prodinger et al. 1998) were received from Dr. Jens Hellwage. Commercial polyclonal

anti-FH antibody was from Calbiochem (Merck).

Heparin from porcine intestinal mucosa and dextran were from Sigma-Aldrich and heparin decamer

from Neoparin Ltd.

3.1.2 Microbial strains and cells

Cloning of the FH19-20 mutant constructs was done in E. coli XL10-Gold® cells (Stratagene) and the

expression of FH19-20 proteins in Pichia pastoris X-33 strain (Invitrogen). Conditionally immortalized

mouse glomerular endothelial cells (mGEnC-1) that were used in binding assays have been described

by Dr. Angelique Rops (Rops et al. 2004a) and were used in the Nijmegen Centre for Molecular Life

Sciences, The Netherlands, under supervision of adjunct professor Johan van der Vlag.

3.1.3 Bioinformatical databases

The following web resources were used to obtain sequence and structure information: European

Bioinformatics Institute ( (McWilliam et al. 2009), National Center for Biotechnology

Information ( (Benson et al. 1997), Protein Data Bank (

(Berman et al. 2000).

3.2 Methods

3.2.1 General laboratory methods for proteins

The proteins were resolved based on their size using standard SDS-PAGE techniques and visualized

using Coomassie Brilliant blue, silver staining, or by performing Western blotting where detection

was done with primary antibodies and horseradish peroxidase-conjugated secondary antibodies


Materials and methods

(Jackson Immunoresearch). Concentrations of the proteins were measured using absorbance at 280

nm wavelength, BCA assay (Thermo Scientific), and comparing intensity of the protein bands with a

standard in the gel using ImageJ program ( Buffer exchanges were

performed using dialysis membranes or PD-10 buffer exchange columns (GE Healthcare).

3.2.2 Protein iodination

Proteins were iodinated using the Iodogen method in a hood of a class B radioactive work area

(Fraker et al. 1978). Iodogen tubes were prepared by mixing Iodogen to chloroform and dried.

Iodination was performed by incubating 20-100 µg of protein with 0.2-1.0 mCi of 125-I for 10-20

minutes in PBS. After the reaction the mixture was transferred to a glass tube for unreacted iodine

ions to form molecular iodine I 2 . The free iodine was separated from proteins using PD-10 buffer

exchange columns. The specific activity of the proteins was typically 1.0 - 4.0 x 10 6 cpm/μg (16.7 –

66.7 kBq/μg).

3.2.3 Mutagenesis, cloning, and screening of FH19-20 mutant proteins

Mutagenesis was performed using QuikChange Multi site-directed mutagenesis kit (Stratagene). The

mutagenesis primers were designed based on the structure of the FH19-20 protein and criteria

presented by the manufacturer of the mutagenesis kit. The mutagenesis primers were obtained

from Sigma-Aldrich and are presented in studies II and III. These were used to induce mutations

D1119G, D1119G/Q1139A, Q1139A, W1157L, R1182A, W1183L, T1184R, K1186A, K1188A, E1198A,

R1203A, R1206A, R1210A, and R1215Q to FH19-20. The pPICZαB vector (Invitrogen) with the

inserted FH19-20 sequence was used as a template in mutagenesis PCR reactions. E. coli cells were

transformed with a PCR product using a heat shock transformation as instructed by the

manufacturer. The clones were selected using Zeocin® (InvivoGen) antibiotic selection on Luria-

Bertani medium and then sequencing from 3’ and 5’ ends over the FH19-20 region in the Haartman

Institute sequencing core facility. The plasmids harboring the mutation(s) were transferred to Pichia

pastoris for expression using electroporation according to manufacturer’s instructions (BioRad). The

transformed cells were selected using Zeocin® and small scale culture expression in 10 ml. The

media was screened for the presence of FH19-20 proteins using Western blotting with polyclonal

anti-FH19-20 antibody. The clones that were recognized by the antibody and had the highest

expression level were selected for large scale expression.

3.2.4 Expression and purification of FH19-20 mutant proteins

The FH19-20 proteins were expressed using the Pichia pastoris expression system as described by

the manufacturer (Stratagene). All the incubation steps were performed at 28 °C under continuous

shaking (225 rpm). Briefly, 5 ml of BMGY (Buffered Methanol, Glycerol, Yeast extract) medium with

100 µg/ml of Zeocin® was inoculated and incubated for approximately 17 hours. The culture was

used to inoculate 50 ml of pre-tempered BMGY that was incubated for 16 hours. The cells were

harvested by 5 min centrifugation at 3000 g at 22 °C. The cells were suspended to 1 liter of BMM

(Buffered Methanol Medium) medium for expression. 10 ml of methanol was added to the culture

after 24 hrs and 48 hrs of expression. The expression was stopped after 72 hrs and the subsequent


Materials and methods

steps were performed at 4 °C. The supernatant was collected by centrifuging the cells twice at 5000

g for 5-10 min. The supernatant was filtered using 0.8 µm/0.2 µm filters (Pall). The pH of the filtrate

(pH < 5) was adjusted to 6.0-6.5 with Na 2 HPO 4 due to stability of the Hi-Trap heparin column (pH

should be >5) and diluted 1+1 with H 2 O. To prevent microbial growth Na-azide (0.02% final

concentration) was added. The proteins were purified from the filtrate using 1 ml Hi-Trap heparin

columns (GE Healthcare) and eluted with 1 M NaCl in phosphate buffered saline (PBS: 137 mM NaCl,

2.8 mM KCl, 9.7 mM phosphate, pH 7.4). The protein preparations were further purified with

Sephadex 75 (10/300 GL) gel filtration column using the ÄKTA® purifier system (GE Healthcare).

3.2.5 Generation of C3b

The C3b protein was prepared from plasma purified C3 (kindly provided by MSc Karita Haapasalo)

using trypsin cleavage. 1 mg /ml of C3 in PBS was warmed in a water bath to 37 °C. Trypsin (Sigma-

Aldrich) was added to the solution to make it 1 % (w/v), mixed, and incubated for 3 min. Soy bean

trypsin inhibitor (SBTI)(Sigma-Aldrich) was added to the solution to make it 2 % (w/v) and mixed to

stop the reaction. The result was monitored with SDS-PAGE gels, where a shift in the alpha chain of

C3 indicated cleavage of C3a from C3 and generation of alpha’-chain. Trypsin, SBTI, C3a, and C3b

were separated with HiLoad 16/60 Sephadex 200 gel filtration column attached to an ÄKTA® purifier

system (GE Healthcare).

3.2.6 Radioligand assays

The proteins were attached to Nunc Polysorp Break Apart plates at 5-20 μg/ml in ½ PBS (68.5 mM

NaCl, 1.4 mM KCl, 4.85 mM phosphate, pH 7.4) by incubating for 17 hours at 4 °C. The wells were

blocked with 0.5 - 2.0 % bovine serum albumin (BSA, Sigma-Aldrich) in ½ or full PBS for 2 hours at 22

°C and washed 1-2 times with ½ or full PBS. In inhibition experiments the radioligand and inhibitor

were usually premixed. In direct binding experiments the radioligand was pipetted onto the wells

bearing the ligand. The plates were incubated for 2 - 3 hours at 22 °C and washed with ice cold ½ or

full PBS once or twice. The radioactivity of the separated wells was measured with a Wizard gamma

counter (Perkin-Elmer).

3.2.7 Surface plasmon resonance

Surface plasmon resonance assays were performed on a Biacore 2000 equipment using

carboxymethylated dextran chips CM4 and CM5 (GE Healthcare). For the amine coupling procedure

the proteins were dialyzed against 20 mM sodiumacetate buffer (pH 4.5). The optimal amount of

protein coupled to the chip in kinetic assays was decided based on the surface density calculator

( The reference flow cell was activated and deactivated using the amine coupling

procedure. The assays were performed at 22° C in ½ or full PBS using a flow rate of 30 µl /min in

kinetic experiments.


Materials and methods

3.2.8 Heparin affinity chromatography

Heparin affinity chromatography was performed with the ÄKTA® purifier system (GE Healthcare) and

Hitachi LaChrom HPLC (Merck). The proteins were injected into a 1 ml Hi-Trap Heparin column (GE

Healthcare) in ½ PBS and eluted using a linear salt gradient up to 0.5 - 1.0 M NaCl in PBS. The elution

was monitored using absorbance at 280 nm and by Western blotting the elution fractions with anti-

FH19-20 antibodies. The ionic strength of the elution buffer was monitored in real time with a

conductivity meter in ÄKTA® or measured from the elution fractions using a conductivity meter

CDM210 (Radiometer Analytical SAS).

3.2.9 Enzyme immunoassay on mGEnC-1

The mGEnC-1 were grown to confluence at 37° C under 5.0 % CO 2 atmosphere. The cells were

detached, washed with PBS and activated with 10 ng/ml of TNF-α (Peprotech, Rocky Hill, NJ, USA) on

96 well plates for 18 hours. The cells were washed with PBS and the FH19-20 proteins were added in

2 % BSA/PBS onto the cells and incubated for 2 hrs at 37° C under 5.0 % CO 2 atmosphere. The

proteins were detected with polyclonal rabbit anti-FH19-20 antibody and horse radish peroxidase

(HRP) conjugated donkey anti-rabbit IgG F(ab’) 2 (Jackson Immunoresearch). Antibody incubations

were performed in 2 % BSA/PBS and the cells were washed with 0.05 % Tween 20 in PBS between

the incubations. The HRP-conjugated antibodies were detected using 100 µl of tetramethylbenzidine

(TMB) substrate solution (SFRI Laboratories) for 15 min at 22° C after which the reaction was

stopped with 100 µl of 2 M H 2 SO 4 . The absorbance (at 450 nm) of the wells was measured

immediately after stopping the reaction.

3.2.10 General bioinformatical tools

Molecular modeling was performed at Finnish IT Center for Science ( using Insight II

(Accelrys Inc.). Sequences were analyzed using tools of Expert Protein Analysis System

( (Gasteiger et al. 2003), Baylor College of Medicine Search Launcher

( (Smith et al. 1996), and PFAM (Finn et al. 2009). The

sequences were aligned mainly with BLAST at NCBI or EBI (Altschul et al. 1990), and ClustalW at EBI

(Thompson et al. 1994) and resulting multiple sequence alignments visualized and analyzed using

GeneDoc program (Nicholas et al. 1997). The molecular images were created with PDBviewer (Guex

et al. 1997).

3.2.11 Determination of SCR consensus sequences

All the SCR containing proteins in the SwissProt-database release 40 (24.10.2001) (

were obtained using SRS Sequence Retrieval System. The proteins and the corresponding SCRs were

divided into three datasets RCA1: (CFAH (P08603), FHR1 (Q03591), FHR2 (P36980), FHR3 (Q02985),

FHR4 (Q92496), and FHR5 (Q9BXR6)), RCA2: (C4BPA (P04003), C4BPB (P20851), CR1 (P17927), CR2

(P20023), DAF (P08174), and MCP (P15529)), and non-RCA: (APOH (P02749), C1S (P09871), C1R

(P00736), CFAB (P00751), CO2 (P06681), CO6 (P13671), CO7 (P10643), MASP1 (P48740), F13B

(P05160), IL2RA (P01589), LYAM1 (P14151) , LYA2 (P16581), LYAM3 (P16109), MASP2 (O00187),

PAPP1 (Q13219), PGCA (P16112), CSPG2 (P13611), SRPX (P78539), and SE6L1 (Q9BYH1)). The codes


Materials and methods

present the UniProt (previously SwissProt) entry names in the form ABBREVIATION_HUMAN

(accession code). Representative experimental structures of the SCR domains (CR1 domains 15, 16

(PDB code 1GKG); CR1 17 (1GKN); CR2 1, 2 (1GHQ); DAF 2 (1NWV); DAF 3, 4 (1H03); FH 15 (1HFI); FH

16 (1HCC); MCP 1, 2 (1CKL)) coded by the RCA cluster were obtained from Protein Data Bank (Barlow

et al. 1993; Casasnovas et al. 1999; Smith et al. 2002; Uhrinova et al. 2003; Williams et al. 2003).

Multiple sequence alignment was created using three-dimensional structures of the SCRs that were

aligned using Combinatorial Extension (CE) method (Shindyalov et al. 1998). For a pair of structures

CE determines aligned fragment pairs that are used heuristically to create final optimal alignment

between the structures. Pairwise alignments from CE were used to generate multiple sequence

alignment of experimentally solved SCR domains with emphasis on the conservation of the extended

beta-strand regions denoted in PDB. Based on the degree of amino acid conservation, the columns in

the alignment were scored from 1 to 9 (12/12 hits yielded score of 9 ((X/12)*9 = score)). This

sequence profile was loaded to ClustalX-program (Thompson et al. 1997), where SCR domain

sequences without known structure were heuristically added one by one to the profile to create a

full alignment. Multiple sequence alignment was manually refined with emphasis on the conserved

cysteines and the secondary structure elements. The consensus sequences were produced from the

multiple sequence alignment using a GeneDoc program (Nicholas et al. 1997).

3.2.12 Analysis of interdomain interactions

The interdomain contact regions were calculated from experimentally-solved SCR domain pairs

deposited in RCSB Protein Data Bank (FH 15-16 (1HFH); MCP 1-2 (1CKL); VCP 2-3 and 3-4 (1VVC);

APOH 1-2, 2-3, and 3-4 (1C1Z); CR1 15-16 and 16-17 (1GKN, and 1GKG)) (Barlow et al. 1993;

Casasnovas et al. 1999; Schwarzenbacher et al. 1999; Szakonyi et al. 2001; Smith et al. 2002). Sting

Millenium suite (Neshich et al. 2003) was used to calculate the contacts between the SCR domain

pairs. The interdomain angles were measured using coordinates imported from modeling program

package Insight II (Accelrys Inc) to Microsoft Excel, where data were processed. The conserved

cysteines were used as reference points to calculate the tilt, twist, and skew angles that define the

interdomain orientation. Tilt defined the depth of the angle between the consecutive domains. Skew

defined the angle in which the C-terminal domain was tilted relative to the N-terminal domain. Twist

defined how much the C-terminal domain was rotated relative to the N-terminal domain. The

interdomain contact regions were extracted from SCR domain sequences in SwissProt-database

release 40 (24.10.2001) ( and their properties calculated using self made Perl

scripts (See study I for further details).

3.2.13 X-ray crystallography

The FH19-20 and FH19-20 D1119G/Q1139A , proteins were purified to 99 % purity and used in

crystallization experiments. The crystallographic experiments and structure determination were

performed in Professor Adrian Goldman’s laboratory by MSc Arnab Bhattacharjee, PhD Veli-Pekka

Jaakola, and PhD Tommi Kajander.


Materials and methods

3.2.14 Curve fitting and statistical analyses

Curve fitting was done using non-linear regression models implemented in GraphPad Prism®. For

HPLC data Unicorn® program of ÄKTA® system was used to analyze the elution curves (GE


The statistical analyzes were performed using Microsoft Excel® and GraphPad Prism®. The

comparison of mean values was done using combination of F-tests and t-tests, and ANOVA. The

results were presented with mean using standard deviation to indicate the error.



4 Results

4.1 Rational mutagenesis of FH19-20

4.1.1 Identification of conserved amino acids in SCRs

The SCR domains have a similar fold with each other but they mediate a variety of functions in

different biological systems. This indicates that the conserved amino acids in the SCR domain family

are important for the fold of the domain but not necessarily for their function per se. By avoiding

mutagenesis of the conserved structural amino acids it is more likely that the fold of the SCR is

conserved upon mutant protein expression and that the mutation has only local effect in the binding

interface. To identify the structurally important amino acids, consensus sequence of SCR domains

and SCR-SCR interdomain contact regions (ICRs) were identified and analyzed.

Sequence data of 177 SCR domains and 13 experimentally solved SCR structures were obtained from

publicly available databases to elucidate the structurally important amino acids in the SCR domain

family (Lehtinen et al. 2003; Lehtinen et al. 2004). Based on the location of the genes coding SCR

domains, the SCR-containing proteins were further divided to regulators of complement activation

group 1 (RCA1) at locus 1q31.3 (48 SCRs of FH, FHL-1 and FHRs 1 to 5), RCA2 group at 1q32.2 (64

SCRs of CR1, CR2, C4bp, DAF, and MCP), and to non-RCA group of domains whose genes are

scattered in the genome (total of 65 SCR domains). Multiple sequence alignment of the SCRs based

on the experimental SCR structures was created and analyzed. The consensus sequences of the SCR

domains in RCA1, RCA2, and non-RCA groups had some clearly identifiable features (Figure 11).

The four characteristic cysteines that make the disulphide bridges in the SCRs were the only fully

conserved amino acids in this analysis (sites 1, 42, 76, 102 in Figure 11). Some of the SCR domains in

the non-RCA group had two additional cysteines (sites 16 and 38) that were located in the beginning

and at the end of the functionally important hypervariable loop. Most of the semi-conserved sites

correlated with the location of the beta-strands in the SCR structures except the proline rich areas at

the beginning (sites 2-5) and at the end (sites 97-100) of the consensus sequences. The differences

between the RCA1 and RCA2 group consensus sequences were mainly due to insertions after the Cys

at site 42 and between Gly at site 80 and Trp at site 86. Factor H domains 19 and 20 that were

subjected to mutagenesis in the further studies had typical sequences for SCR domains, except the

N-terminus of FH20 that was found to have some unusual features. It lacked the typical proline rich

region after the first cysteine (sites 2-5) and the NG motif at sites 11-12 in the hypervariable loop

(Figure 11).



Figure 11. Structure based consensus sequences of SCR domains. Consensus sequences of all SCR

domains (first line) and the subgroups of RCA1, RCA2, and non-RCA are shown with FH19 and FH20

sequences. Fully conserved cysteines are marked with white font and black background. Amino acids

marked with white and grey background are conserved in 25-50 % and in 50-99 % of SCR domains

within the group, respectively. Dash indicates a gap and a dot indicates occurrence of at least one

amino acid on that position in the multiple sequence alignment. Numbers above the alignment

indicate the site of the amino acid in the multiple sequence alignment.

4.1.2 Identification of interdomain contact regions between SCRs

One goal of study I was to identify ICRs, characterize them and to examine their impact on

orientation between the neighbouring SCR domains. SCR domains in proteins are arranged like

beads-on-a-string connected by short linker peptides. This conformation allows adjacent domains to

interact with each other and to form contiguous binding sites. The amino acids that mediate the

interdomain interactions should not be subjected to mutagenesis in binding site mapping studies

because disruption of the interface may lead to loss-of-function due to change in the interdomain

orientation instead of disruption of the interface. Most of the experimental SCR structures have

been solved for pairs of SCR domains that made it possible to study the interactions of neighbouring

domains in silico. Previously published experimental structures of the SCR domain pairs were

analyzed using Sting Millenium suite (Neshich et al. 2003) in order to detect the amino acid residues

that made contacts to adjacent domains and formed the linker area between the SCRs. Four

sequence stretches N#1, C#1, C#2 and the interdomain linker were identified in conserved locations

that formed the ICRs. These locations can be used to predict ICRs in SCR domain sequences, as was

done for FH19-20 (Figure 12). Sequence analyses of the ICRs revealed that they were more

hydrophilic than human protein sequence on average and that the increasing interdomain linker

length correlated with increasing hydrophilicity. It was concluded in study I that in general the ICRs

are probably flexible and that the short linkers form more compact and rigid interface between the

adjacent domains, as would be expected.

To analyze the impact of ICRs to orientations of the neighboring SCR domains, tilt, twist, and skew

angles were calculated from experimental structures that describe the orientation between the

domains (See methods for explanation of the angles). The twist and skew angles were found to



depend on each other due to steric restriction by the linker and the N#1 region. The interdomain

angles were also compared to amino acid sequences of the ICRs but no correlation was found.

Figure 12. Predicted and experimental interdomain contact regions in FH19-20. The predicted ICRS

are shown with curved arrows above the FH19 and below the FH20 sequences and are mapped to

FH19-20 structure on the left (PDB ID: 2G7I). The experimental ICRs are shown with grey background

of the amino acids in the alignment and mapped to FH19-20 structure on the right. N#1-regions are

colored yellow in the structures, linkers green, C#1-regions red, and C#2-regions blue.

4.1.3 Crystal structure of FH19-20

Computer based predictions are a valuable aid in designing point mutations to functionally

important amino acids, however, the understanding of the impact of the mutations to the structure

of the protein requires a detailed atomic structure. In study II the crystal structure of FH19-20 (FH

amino acids G1109 - K1230) was solved at 1.8 Å resolution (Figure 13). The structure in the crystal of

FH19-20 was similar to previously solved SCR domain pair structures with two bead-like domains

arranged head-to-tail.The interdomain orientation was also typical. However, FH20 had two

exceptional features, a short alpha helix (R1171 - Y1177) in the hypervariable loop and a buried

water molecule that interacted with C1167, K1188 and S1191. Interestingly, the alpha helix site was

located in the non-typical hypervariable loop of FH20 identified in the SCR consensus sequence

analysis (Figure 11). In the crystal, the FH19-20 monomers formed a tight D2 tetramer structure (A,

B, C, and D monomers) with two dimerization interfaces (AxB and AxC) in one monomer (Figure



4.1.3) (Table 1). The heparin binding sites of FH19-20 monomers A and D, and monomers B and C

point to same direction, but these pairs do not have dimerization interfaces in the crystal.

Dimerization (and oligomerization) of FH19-20 has been shown in vitro and suggested to be

enhanced by polyanions, but the physiological meaning of this has remained unclear (Perkins et al.

1991; Pangburn et al. 2009). Based on the structure, polyanion binding should not enhance

dimerization but could be involved in stabilizing tetramers.

Figure 13. The crystal structure of FH19-20. A. Monomer of FH19-20 with otherwise typical

appearance of SCR domain pair except the alpha helix on SCR20. B. The tetramer of FH19-20 as it

was crystallized. One monomer (e.g. A) contacts two monomers (B and C) that are upside down

compared to A. This means that heparin binding to FH19-20 cannot stabilize dimers but may

stabilize tetramers.

Table 1. Dimerization interfaces in the crystal structure of FH19-20. AxB interface (880 Å 2 ) is

composed of interfacing residues: K1108, D1119-A1129, Q1137-Y1142, A1185-Y1190, R1192, and

E1195. AxC interface (982Å 2 ) is composed of interfacing residues: K1108-I1120, F1123-V1127,

Q1139-Y1142, G1155, Q1156, H1165-I1169, R1171, E1172, and Q1187-Y1190. H = hydrogen bond,

SB = salt bridge.

Interface AxB

Interface AxC

Residue in A Bond Residue in B Residue in A Bond Residue in C

R1192 H T1121 N1117 H N1117

S1126 2H A1185 N1117 H D1116

V1127 H A1185 Q1139 H Q1139

Q1156 H E1172

H1165 H, SB D1116

Y1190 H P1112



4.1.4 Crystal structure of FH19-20 and C3d complex

In study IV, the complex structure of FH19-20 with C3d was solved at 2.3 Å resolution (Figure 14).

The wild-type FH19-20 did not form crystals with C3d but crystallized easily on its own as a tetramer.

The mutant FH19-20 D1119G/Q1139A protein was used in crystallization procedure because it was found

to have decreased potential to form the tetramer. Overall, the structures of FH19-20 and C3d were

almost identical with the previously solved structures of FH19-20 or C3d alone, only FH19-20 had

some local changes upon binding to C3d. Also, there were no significant changes in the interdomain

orientation of FH19-20 D1119G/Q1139A in the FH19-20:C3d complex compared to that found in the FH19-

20 tetramer. The D1119G and Q1139A mutations were modeled back to original residues on the

complex structure and found to fit well into the interface, and thus FH19-20 D1119G/Q1139A will be later,

and was in the study IV, referred to as FH19-20. The existence of the two binding sites was

confirmed by analyzing binding of the C3dg point mutations in FH19 and FH20 interfaces in study IV.

In the FH19-20:C3d complex, importantly, two different interfaces were found between FH19-20 and

C3d in contrast to previous reports that assumed 1:1 binding (Figure 14). The sites were called the

FH19 site and the FH20 site. The FH19 site on FH19-20 (FH19-20 FH19 ) stretched from domain 19 to

the linker region and to the hypervariable loop of domain 20, and covered a buried surface area of

620 Å 2 . The FH19-20 FH19 consisted of four peptides on FH19-20: the hypervariable loop of FH19

(P1112-P1124), the stretch around the second cysteine and the loop after it (Q1137-Y1142), the

linker region (H1165-V1168) and the C-terminal part of the hypervariable loop of FH20 (K1188-

Y1190). On C3d, the FH19 site (C3d FH19 ) is located at the side of the α/α-barrel. The interface mainly

consisted of four peptides: the N-terminus of helix 4 (K112-K118), the loop around P121 (Q119-

Q126), the loop between helix 6 and 7 (E167-S171) and a stretch in the middle of helix 7 (K178-F182)

(C3d helices and numbering as in Nagar et al. 1998).

The FH20 site on FH19-20 (FH19-20 FH20 ) was located exclusively on domain 20 and covered a buried

surface area of 490 Å 2 . The binding site consisted of three peptides: the N-terminus of hypervariable

loop of FH20 (R1171-T1184), the stretch around the second cysteine and the loop after it (V1200-

Y1205), and the C-terminus of domain 20 (K1230-R1231). On C3d, the FH20 site (C3d FH20 ) was

formed around a negatively-charged pocket on the concave top of the α/α-barrel. The binding site

consisted of four peptides: seven amino acids in helix 1 - turn - helix 2 region (H33-L53), helix 3 and

the loop after it (S94-I102), the turn between helices 5 and 6 (E160-D163), and the C-terminus of

helix 12 (Q284-D292). The FH20 interface had many charged residues and was observed to be highly

electrostatic. The crystal structures of the FH19-20 homotetramer and the FH19-20:C3d complex

were the basis for designing mutagenesis on FH19-20 and allowed the structural interpretation of

the functional effects of the mutations and intriguingly revealed the molecular basis of the target

discrimination by factor H.



Figure 14. The crystal structure of FH19-20:C3d complex revealed two binding sites. A. FH19

binding site. B. FH20 binding site. In A and B FH19-20 is shown with cartoon presentation. The

residues that make bonds to C3d are shown with sticks. Green indicates SCR19, gold indicates

SCR20, and red indicates linker residues between SCR19 and SCR20. C3d is shown with surface

presentation. Blue color indicates the FH19 site, green color the FH20 site, and yellow color the

location of thioester that mediates covalent binding to surfaces. C. FH19 and FH20 binding sites on

C3d structure. C3d fold forms inner and outer α-barrel (α/α-barrel) that is clearly visible in the lower

panel. FH19 binding site is located on the side of the barrel (blue coloring) and the FH20 site on the

concave top (green coloring). C3d is shown with ribbon presentation and the residues in the FH19

site and in the FH20 site with sticks.

4.1.5 Mutagenesis of FH19-20

For analyzing the physiological function of FH19-20 and functional consequences of aHUS mutations,

a total of 17 mutant FH19-20 proteins were created using PCR and Pichia pastoris expression system

in studies II, III, and IV (Figure 15 and Table 2). The selection of the point mutations was based

mostly on crystallographic structures and the association of the mutation with aHUS, but also on the

previously published biochemical data on FH function, like the known involvement of arginines and



lysines in heparin binding (Table 2). The mutations were also compared to interdomain contact

regions and conserved amino acids of SCRs to avoid global changes in the structure. In studies II and

III mutations D1119G, D1119G/Q1139A, Q1139A and W1157L were induced to domain 19 and

R1182A, W1183L, K1186A, K1188A, E1198A, R1203A, R1206A, R1210A, and R1215Q to domain 20 to

study their impact on C3d, C3b, heparin, and cell binding. In study IV a Q1137A/ Q1139A/ Y1142A

triple mutation protein was designed to knock-out the FH19 site (FH19 Del -20) and a T1184G/

K1202A/ R1203A/ Y1205A quadruple mutation was designed to knock-out the FH20 site (FH19-20 Del ).

All the FH19-20 mutant proteins bound polyclonal anti-FH and anti-FH19-20, heparin (except FH19-

20 Del ), C3d, and C3b (Studies II, III, and IV) suggesting that the antigenicity of the FH19 or FH20 had

remained intact.

Figure 15. Mutations induced to FH19-20. The residues that were mutated are shown with sticks

and CPK coloring.



Table 2. Mutations in the generated mutant proteins used in studies II, III, IV and their role in

structure of FH19-20. The mutations were selected based on their association with aHUS (aHUS),

their positive charge that indicates a heparin binding site (+ charge), the location in the dimerization

interface (dimerization), or in the C3d binding site (FH19 Del -20 or FH19-20 Del ). None of the mutations

were located in the interdomain contact regions except Y1142A in FH19 Del -20.

Mutation aHUS Conserved Secondary Side chain Selection criteria


structure accessible

D1119G D1119G No Beta Yes aHUS, dimerization

Q1137A No Yes FH19 Del -20

Q1139A No Yes FH19 Del -20, dimerization

W1157L W1157R Yes Beta No aHUS

Y1142A Yes Only tip FH19 Del -20

R1182A R1182S No Yes aHUS, + charge

W1183L W1183L/R No Yes aHUS

T1184R T1184R No Turn Yes aHUS

T1184G No Turn Yes FH19-20 Del

K1186A No Yes + charge

K1188A No Yes + charge

L1189R L1189F/R/P No Yes aHUS

E1198A E1198A/K Yes Beta Only tip aHUS

K1202A No Yes FH19-20 Del

R1203A R1203W No Turn Yes aHUS, + charge, FH19-20 Del

Y1205A No Yes FH19-20 Del

R1206A R1206C No Beta Yes aHUS, + charge

R1210A R1210C No Turn Yes aHUS, + charge

R1215Q R1215G/Q No Beta Yes aHUS, + charge

4.1.6 Structure function analysis of FH19-20 mutations on C3d binding

In studies II and III binding of the FH19-20 mutant proteins to C3d and C3b was tested using

radioligand assays (RLA) and surface plasmon resonance (SPR) technique to map the binding site for

C3d/C3b without knowledge about the actual binding site (Table 3). In study IV, the FH19-20:C3d

structure was solved allowing interpretation of the results of the functional analyses in studies II and

III on the basis of the real interaction in FH19-20:C3d structure (Figure 16). The mutations D1119G,

Q1139A, T1184R, K1188A, and R1203A lead to loss of bonds between C3d and FH19-20 indicating

that these mutations should lower the affinity in the experiments as well. However, the mutant

proteins FH19-20 D1119G and FH19-20 T1184R did not have statistically-decreased binding in all assays

(Table 3). The FH19-20 binding to C3d is mediated by two low affinity binding sites that possibly

mask the effect of a single point mutation in one site.

Six mutations W1157L, R1182A, W1183L, E1198A, R1206A, and R1210A were not directly in the

FH19-20:C3d interfaces but caused decreased C3d or C3b binding by FH19-20. The analysis of the

structure showed that W1157L and W1183L probably destabilize the fold of SCR19 or SCR20,

respectively. R1182A, E1198A, R1206A, and R1210A lead to change of charge on SCR20 and probably

disrupt the electrostatics-based orientational steering that can significantly effect the affinity of

protein-protein interactions (Janin 1997; Sinha et al. 2002; Persson et al. 2009). The mutant proteins



FH19-20 R1215Q , FH19-20 L1189R , and FH19-20 K1186A had wild-type C3d/C3b binding, consistent with the

location of the mutations far away from the C3d binding sites in the complex structure.

Figure 16. Effects of FH19-20 mutations on binding to C3d/C3b in functional assays and their

location on FH19-20:C3d structure. FH19-20 shown in ribbon and two C3d’s in surface presentation.

Color scheme is as in Figure 14. The mutations that were analyzed in functional assays are shown in

sticks. The interpreted mechanical effects of the mutations on loss of FH19-20-C3d interaction: blue

destabilization of SCR, red loss of a bond in the interface, purple loss of electrostatic

complementarity in FH19-20-C3d interface. Residues marked with grey had no effect on binding to

C3d in functional assays.



Table 3. Functional consequences of FH19-20 mutations on binding to C3d/C3b and interpretation

of the mechanism on the basis of the FH19-20:C3d structure. All the results published in studies II,

III, and IV, except *(Lehtinen et al. 2008), # (Bhattacharjee et al. 2010), and () unpublished. ↓

decreased binding, ↑increased binding, and ↔ wild-type like binding.





Bond in













D1119G D1119G FH19 ↔ ↓* ↔ Loss of H-bond


FH19 ↓ ↓* ↓ Loss of 2 H-bonds


Q1139A FH19 ↓ ↓* ↓ Loss of H-bond

W1157L W1157R ↓ ↓* ↓ Destabilization

R1182A R1182S (↓) ↓ ↓ Possibly loss of



W1183L W1183L/R (↓) ↓ ↓ Possibly disruption of

loop V1168-S1191

T1184R T1184R FH20 ↔ ↔* ↓ Loss of 2 H-bonds

K1186A (↔) n.d. ↔ None

K1188A FH19 (↓) ↓ ↓ Loss of salt bridge

L1189R L1189F/R/P ↔ ↔* ↔ None

E1198A E1198A/K (↓) ↔ ↓ None or possibly loss of



R1203A R1203W FH20 ↓# ↓* ↓# Loss of 2 H-bonds and 3

salt bridges

R1206A R1206C ↓ ↓* ↓ Possibly loss of



R1210A R1210C ↓ ↓* ↓ Possibly loss of



R1215Q R1215G/Q ↔ ↔* ↔ None

4.2 Self - non-self discrimination by FH19-20

4.2.1 FH19 site is exposed and FH20 site is partially occluded on C3b

In study IV, the FH19-20:C3d structure revealed that there were two binding interfaces between

FH19-20 and C3d. The affinities of FH19 Del -20 and FH19-20 Del to C3d and C3b were measured using

SPR. It was found that the FH19 site had similar affinities to both (Table 4), but the FH20 binding site

had roughly four times higher affinity to C3d than to C3b. This discrepancy was structurally explained

by analyzing the availability of FH19 and FH20 sites on the C3b and C3b:FH1-4 structures (PDB IDs:

2I07 and 2WII). The FH20 site in the C3d part of C3b (D1007-Q1021) was occluded by the MG1

domain belonging to the C3c part of C3b (residues F40-K43; R80-V98; Q631-A636). Although the

FH20 site is partially buried in the C3b crystal it is available on soluble C3b, since FH19 Del -20 clearly

bound to it (Table 4). Thus it seems that C3c might act as a competitive inhibitor for FH19-20 on

FH20 site binding.



Wu and co-workers compared the contacts between C3d and C3c in C3b and FH1-4:C3b structures

and found that the FH1-4 binding tightens up the C3d-C3c interface by introducing hydrogen bonds

to the interface that also leads to change in orientation of C3d (12 degree rotation) (Wu et al. 2009).

In addition, FH4 was bound partially onto the FH20 site on C3d in the FH1-4:C3b complex that could

further block the FH19-20 binding. These findings indicated that FH1-4 binding to C3b favors binding

of FH19-20 to the FH19 site since the C3c binding to FH20 is enhanced.

Table 4. Relative affinities of FH19 and FH20 sites on C3d and C3b binding. C3d and C3b columns:

The numbers present relative decrease in affinity compared to FH19-20 that is 1.0. Fold change

column: For each protein the number presents decrease of affinity on C3b binding compared to C3d


Protein C3d Fold change C3b

FH19-20 1.0 3.0 3.0

FH19 Del -20 2.3 4.4 10.2

FH19-20 Del 6.3 1.3 8.2

4.2.2 FH20 mediates binding to heparin and mGEnCs

The heparin binding site of FH19-20 was mapped to FH20 in studies II and III using heparin affinity

chromatography and the mutant proteins (Table 5). Heparin binding was found to be mediated by

residues R1182, K1186, K1188, R1203, R1206, R1210, and R1215. These results were corroborated

by the results of Herbert and co-workers, who analyzed the perturbations in the NMR-spectra upon

addition of heparin tetrasaccharide to FH19-20 solution (Herbert et al. 2006). In that study, the

heparin binding site was also mapped to lysine and arginine residues on FH20 (Table 5), but in

addition, the residues E1145, L1164, H1165 and T1193 in the hinge between FH19 and FH20 were

affected by heparin binding. The authors suggested that these changes were most probably

structural and might have an effect on the interdomain orientation. The heparin binding site was

also confirmed by Ferreira and co-workers who used FH19-20 mutant proteins in heparin affinity

chromatography and gel mobility shift assays (Table 5) (Ferreira et al. 2009). These data were

collectively used in study IV to map the heparin binding site on FH19-20 of the FH19-20:C3d complex

structure (Figure 17). The heparin binding site was found to extensively overlap with the FH20

binding site (Table 5), but not with the FH19 site that shared only the K1188A residue with the

heparin site. These findings were analyzed experimentally using the FH19 Del -20 and FH19-20 Del

mutant proteins in a radioligand assay where binding of only FH19 Del -20 to C3d was inhibited by

heparin. Importantly, this result showed that the FH19 site is not affected by heparin binding and

can bind simultaneously to C3d or C3b while heparin is bound to FH20.

Heparin binding has been used to model FH binding to HS glycosaminoglycans that are present also

on glomerular endothelial cells and glomerular basement membrane. Binding of the FH19-20 mutant

proteins to mouse glomerular endothelial cells (mGEnCs) was analyzed in study III, and it was found

that the binding site of FH19-20 to mGEnCs and was almost identical with the heparin binding site on

FH20 (Figure 17 and Table 5). The only differences were that the mutation K1186A increased binding

to mGEnC-1 but decreased binding to heparin and that the mutation R1215Q had no effect on

mGEnC binding but decreased binding to heparin.



Figure 17. Heparin and mGEnC binding sites on FH20. FH19-20 shown in ribbons bind to surface

contoured C3d via FH19 site (Coloring as in Figure 14). The view is from the surface that is indicated

by the thioester shown in yellow in the left lower corner. Red residues shown in sticks indicate

heparin binding sites and blue residues indicate mutations that increase affinity to heparin. The bold

labels indicate residues that similarly affected binding to mGEnC and heparin.



Table 5. Comparison of functional consequences of FH19-20 binding to heparin and cells. ↓

decreased binding, ↑increased binding, ↔ wild-type like binding, + perturbation in NMR spectra,

and # (Bhattacharjee et al. 2010).

Residue or




(Study III)



(Ferreira et

al. 2009)

Heparin binding

by NMR

(Herbert et al.




(Study III)

Overlap with

C3d binding

sites (Study IV)

E1145 +

L1164 +

H1165 +


R1182A ↓ + ↓

R1182S ↓ +

W1183R ↑ FH20

T1184R ↑ ↑ ↑ FH20

K1186A ↓ + ↑

K1188A ↓ + ↓ FH19

K1188Q ↓ + FH19


L1189R ↑ ↑ ↑

T1193 +

E1198A ↑ ↑


K1202 + FH20

R1203A ↓# + FH20

R1203S ↓ + FH20

R1206A ↓ ↓

R1210A ↓ ↓



R1215G ↓ +

R1215Q ↓ + ↔

K1230A ↓ + FH20

R1231A + FH20

4.2.3 Simultaneous binding of FH19 site to C3b and FH20 to anionic self cell structures

explains target recognition by FH

Based on FH19-20:C3d complex, its superimpositions onto C3b and FH1-4:C3b structures, and

biochemical data, it was deduced that FH19-20 binding via FH19 site to C3d/C3b left the FH20 free to

bind heparin or mGEnC (Figure 18). This indicated that FH19 site and FH20 are needed for self

surface recognition. This was confirmed experimentally by analyzing the binding of FH19-20 to C3bcoated

sheep erythrocytes (Esh-C3b) that have been widely used to model self surfaces. FH19 Del -20

and FH19-20 Del proteins had decreased binding to Esh-C3b, as expected. In addition, the binding of

FH19-20 to the FH19 site on FH1-4:C3b complex did not interfere with FH1-4 binding that mediates

the CA and DAA functions of FH. This was also experimentally confirmed by performing CA and DAA

assays with FH19 Del -20 and FH19-20 Del mutant proteins in study IV.

It was shown in study IV by structural analyses that C3b (via FH19 site) and C3d (via FH20 site) could

bind simultaneously to a single FH19-20 molecule (Figure 18) and in studies III and IV that C3d and



heparin binding sites on FH20 were overlapping. The FH20 site on C3d is anionic like heparin

suggesting that FH19-20 could bind to self surface bound C3d similarly to polyanions.

The dual interaction of FH19 site to C3b and FH20 to heparin or cells is likely to present the

physiological complex that recognizes self surfaces. These results are in very good agreement with

all the previously published biochemical data about target recognition that had indicated

simultaneous binding of FH to C3b and to polyanions on self surfaces (Pangburn et al. 2008).

Figure 18. Molecular basis of self – non-self discrimination by FH. A. Binding of FH19-20 to C3b:FH1-

4 via FH19 site does not interfere with FH1-4 binding and allows simultaneous binding to heparin or

cells. B. C3d binds to the same region site on FH20 that mediates binding to heparin and cells. The

SCR domains are shown in ribbon and are colored blue in FH1-4, green in FH19, and gold in FH20.

The residues that mediate binding to heparin are presented with red space fill on FH20. C3b is

presented in surface contour with dark grey indicating C3d and light grey C3c parts of C3b. In B the

C3d binding to heparin or cell binding site is shown with ribbons and in light blue color.

4.3 aHUS mutations disturb target recognition function of FH19-20 by

various mechanisms

4.3.1 Disruption of the FH19 site

Mutations D1119G, Y1142C/D, and V1168E cause loss of at least one bond in the interface and

clearly must disrupt or weaken the binding to C3d (Figure 19). In vitro FH19-20 D1119G bound weakly to

C3d/C3b in some of the experiments (Table 3)(Ferreira et al. 2009). The mutations V1134G, E1135D,

W1157R, and C1163W in domain 19 probably destabilize the fold leading to impaired binding of C3d

(Figure 19), but this has not been proven experimentally.



4.3.2 Disruption of the FH20 site

Mutations T1184R and R1203W lead to loss of bonds in the FH20 site interface and thus should

decrease the affinity of FH19-20 to C3d (Figure 20). Experimental results on the R1203A mutation

clearly support this, but the experimental result on T1184R was controversial (Table 3). T1184R had

no effect on binding of FH19-20 to C3d but decreased binding to C3b.

Mutations R1182S, E1198A/K, R1206C and R1210C or alanine mutations in these residues have been

shown to decrease affinity to C3d/C3b (Table 3) (Sánchez-Corral et al. 2002; Ferreira et al. 2009).

However, these mutations are not located in the FH20 binding site, but close to it. These mutations

lead to loss of positive charge in FH19-20 that may disturb long-range electrostatic interactions

between positively charged FH19-20 FH20 and negatively charged C3d FH20 , possibly explaining the loss

of C3d/C3b binding in vitro.

4.3.3 Destabilization of FH20 may impair binding to C3d FH19 , C3d FH20 , or polyanions

The mutations L1189F/R/P, W1183L/R, S1191L/W, G1194D, V1197A, F1199S, S1211P, and P1226S in

FH20 are more or less buried and probably disturb the fold of the SCR20 and thus may disturb

heparin or cell binding, or the FH19 or FH20 site. Some aHUS mutations in SCR20 are close to the

FH19 site since it also expands to SCR20. For example, the residues K1188 and Y1190 that make

contacts to C3d FH19 are located next to L1189F/R/P and S1191L/W and could disturb the FH19 site,

but this has not been experimentally confirmed.

W1183L was shown to disturb only C3d/C3b binding but not heparin or mGEnC binding (Table 3 and

5). Patient-derived full length FH with W1183L and V1197A have been shown by others to decrease

affinity to C3b (Sánchez-Corral et al. 2002).

4.3.4 Disruption of heparin or cell binding

The mutations R1182S, R1203W, R1206C, R1210C, and R1215G/Q probably disturb binding to

heparin and cells. FH19-20 mutant proteins R1182A/S, R1203A/S, R1206A, R1210A/S/C, and

R1215G/Q have been shown to impair mGEnC and heparin binding by us (Table 5) and others

(Manuelian et al. 2003; Sánchez-Corral et al. 2004; Ferreira et al. 2009). Exceptionally, R1215Q had

only impaired heparin binding, but not mGEnC binding (Table 5).

4.3.5 Function of FH19 site and FH20 site is disturbed by enhanced heparin binding

In studies II and III, the aHUS associated mutations T1184R, L1189R and E1198A were found to

increase the affinity of FH19-20 to heparin and mGEnC (Table 5). Mutation W1183R has also been

shown to increase affinity to heparin by others (Ferreira et al. 2009). In study III these mutations

were observed to extend the positively charged heparin binding site on FH19-20 by either adding

positive charge or by taking away negative charge (Figure 21). Furthermore, it was shown in study IV

that enhanced heparin binding by the mutant T1184R lead to impaired binding of FH19-20 to C3d.

Mutations W1183R, L1189R, and E1198A/K may have similar effects on the FH19-20 function. Taken

together, the location of the mutations on the structure explains the increased affinity to heparin

and decreased affinity to C3d that were observed experimentally.



4.3.6 All aHUS mutations disturb FH19 site binding to C3b or FH20 binding to

polyanions or cells

In conclusion, aHUS mutations may cause (i) loss of bond(s) in FH19-20 - C3d and/or heparin

interfaces, (ii) defective long-range electrostatic interactions between C3d and FH19-20, (iii)

enhanced binding to heparin that can inhibit binding of FH19-20 to C3d, or (iv) destabilization of the

structure of the domains 19 or 20 that leads to disturbed binding to C3d and/or heparin. In addition,

mutations may disturb binding of FH19-20 to the FH20 site on C3d that was suggested to participate

in down-regulation of complement activation on self surfaces in study IV. It is clear that the aHUS

mutations have various combinations of effects on C3d and heparin or cell binding, but the common

denominator for the aHUS mutations seems to be impaired binding of the FH19 site to C3b and FH20

to heparin or cells that are crucial interactions for self surface recognition by factor H.

Figure 19. Atypical HUS mutations on FH19 site. FH19-20 is shown with ribbons and C3d with

surface contour. FH19 is indicated with green and FH20 with golden color. The aHUS mutations are

shown with sticks. Mutations to residues marked with red cause loss of bond(s) in the FH19 site and

those marked with blue may cause destabilization of the SCR fold.



Figure 20. Atypical HUS mutations on FH20 site. FH19-20 is shown with ribbons and C3d with

surface contour. FH19 is indicated with green and FH20 with golden color. The aHUS mutations are

shown with sticks. Mutations in residues marked with red cause loss of bond(s) in the FH20 site and

those marked with blue may cause destabilization of the SCR fold. Mutations in residues marked

with green probably disturb the electrostatic complementarity of the FH20 site.

Figure 21. Atypical HUS mutations on heparin or cell binding. FH19-20 shown with ribbons is

binding to the FH19 site on C3d that is shown with surface contour. FH19 is indicated with green and

FH20 with golden color. The aHUS mutations are shown with sticks. Mutations in residues marked

with red decrease affinity to heparin and those marked with blue increase affinity to heparin that

interferes with FH19 site and FH20 site binding to C3d.



5 Discussion

5.1 Sequence and structure analysis in mutagenesis of FH19-20

In this thesis, structure-based multiple sequence alignment of SCRs was employed to identify

conserved and structurally important amino acids of SCR family. Mutations in these residues would

likely lead to destabilization of the SCR fold and thus not be informative in binding site mapping or

functional studies. For example, W1157 was located in the core of SCR19 and in the SCR consensus

sequence it was highly conserved (Figure 11 site 86). The mutation W1157L was shown to decrease

binding to C3b (Table 3), but it was not located in the FH19 binding site (Figure 16). W1157L

probably destabilizes the fold of the SCR and thus the experimental result on W1157L binding was

misleading for mapping the C3d binding site on FH19-20 in study III.

Mutations to surface-exposed residues are more informative in terms of binding site mapping, since

they are more likely to change structure only locally. This has been shown crystallographically for

Q1139A and R1203A mutations on FH19-20 (Bhattacharjee et al. 2010) and with NMR assays for

many others mutations in FH19-20 (Herbert et al. 2006; Ferreira et al. 2009). Q1139A and R1203A

mutations also reduced binding to C3d in functional assays and were located in the FH19 and FH20

sites, respectively. On the other hand, some mutations outside the binding interface, like R1206 and

R1210, also decreased affinity of FH19-20 to C3d in functional assays, indicating that there is always

uncertainty in interpreting the results of these assays.

In study I the interdomain contact regions between SCR domain pairs were characterized based on

the experimental SCR structures and found to be located in conserved sites. These sites were used in

study I to design a model that could be used to predict ICRs. In this thesis, the ICRs were predicted

for FH19-20 and found to be nearly the same compared to the actual ICRs in the crystal structure of

FH19-20 (Figure 12) indicating that the model has true predictive value. Mutations to amino acids

located in the ICRs were avoided in designing mutagenesis to FH19-20 in studies II, III, and IV to

prevent possible changes in orientations between the two SCR domains. For example, the FH19

binding site was found to extend over the ICR between SCR19 and SCR20 in study IV and thus a

change in the orientation between the SCRs would also lead to disruption of the FH19 site.

Taken together, bioinformatical analyses have clearly predictive value in designing mutagenesis to

SCR domains that are not experimentally solved. This was shown by comparing the predicted ICRs

and conserved amino acids to experimental structures and the results of functional assays.

5.2 Self – non-self discrimination by FH19-20

The molecular basis of the FH-mediated target discrimination has been a puzzling question in the

complement field for decades. It has been shown already in the late 1970’s that negatively-charged

surfaces bearing sialic acids and heparin were critical for protection of erythrocytes from

complement attack by FH (Fearon 1978; Pangburn et al. 1978; Kazatchkine et al. 1979b). In 1990 the

self - non-self discrimination of C3b deposited surfaces was found to be mediated by the polyanion

binding site on FH (Meri et al. 1990b). Later, C3d/C3b and heparin binding sites were mapped on the

FH19-20 region (Gordon et al. 1995; Sharma et al. 1996; Blackmore et al. 1998; Jokiranta et al. 2000)



and FH19-20 was found to be critical for factor H activity on self surfaces (Pangburn 2002). However,

at the same time, the C3d binding and heparin sites on FH19-20 were shown to be overlapping

(Hellwage et al. 2002). This observation was enigmatic since previous results have suggested

simultaneous binding of FH to C3b and heparin, which would strengthen the avidity of FH to C3b on

self surfaces.

5.2.1 Molecular basis of self – non-self discrimination

In study IV, it was found by crystallography that FH19-20 had two distinct binding sites for C3d, the

FH19 site and the FH20 site. The validity of the crystal structure was confirmed by analyzing the

binding of the FH19-20 wild-type and point mutant proteins to C3d and C3b, and binding of C3dg

wild-type and point mutant proteins to FH19-20. The results of the functional assays were consistent

with the binding interfaces in the crystal structure indicating that the interactions were not

crystallographic artifacts. In addition, aHUS mutations have been described in the FH19 and FH20

site in both FH19-20 and C3d (Frémeaux-Bacchi et al. 2008; Maga et al. 2010).

When the FH19-20:C3d complex was superimposed on experimentally solved C3b and C3b:FH1-4

structures, it was found that only the FH19 site was fully available and that this conformation left

FH20 free to bind heparin, mGEnC or C3d. Importantly, heparin did not inhibit binding of the FH19

site to C3d, and the FH19 and FH20 site were both found to be necessary in recognizing Esh-C3b.

These results indicated that the FH19 site can bind to C3b and FH20 on self surfaces simultaneously.

FH19-20 binding via FH19 site did not overlap with the FH1-4 binding on FH1-4:C3b structure and did

not interfere with CA and DAA functions of FH1-4 in vitro. Thus it can be concluded that this complex

is likely to present the physiological target recognition complex between FH and C3b (Figure 18) that

would explain the increased affinity of FH to C3b on self surfaces (Carreno et al. 1989; Meri et al.

1990b). Affinity of FH19-20 to C3b via the FH19 site probably remains the same in self and non-self

surfaces if hypothetical conformational changes in C3b on different surfaces are excluded. Thus, the

determining factor in self- non- self discrimination by FH is the affinity of FH20 to anionic structures

on self surfaces.

5.2.2 The role of C3dg as marker of self

In study IV, structural analyses showed that FH19-20 could bind to C3b via the FH19 site and to an

additional C3d via the FH20 site (Figure 18) that has interesting implications for complement

regulation on surfaces by FH. C3b is converted to C3dg on self surfaces that have membrane bound

CR1 catalyzing the cleavage from iC3b to C3dg, and on those surfaces which are in close contact with

erythrocytes that have abundantly CR1 on the surface. The surface bound C3dg could compensate

for the loss or unavailability of polyanion binding sites or could even create additional binding sites

for FH. Thus C3d on the self surface may enhance binding of FH to self surfaces and lead to

complement inactivation through amplified down-regulation of AP attack. Interestingly, DNA has

also been shown to bind FH19-20 (Leffler et al. 2010) that indicates that GAGs, sialic acids, C3d, and

other anionic cell surface molecules could utilize the same binding site on FH20 in downregulation of

complement activation.



5.3 Molecular pathogenesis of aHUS

The aHUS mutations on FH19-20 caused various combinations of dysfunction. Mutations were

shown to cause loss of bond(s) in the FH19 site and FH20 site, to disrupt heparin or cell binding sites,

or to inhibit the FH19 site and FH20 site binding to C3d by enhancing binding to heparin. The

mutations that were not located in the binding interfaces probably lead to destabilization of the fold

and thereby cause disruption of FH19-20 function. Despite all the different effects of the aHUS

mutations on FH19-20 structure they disrupt the same function of FH - self surface recognition - by

interfering with simultaneous binding of the FH19 site to C3b and the FH20 site to polyanions or


The decreased binding of the FH20 site to C3d may also play a role in the pathogenesis of aHUS if the

binding of FH to the FH20 site on C3d will be shown to amplify downregulation of complement on

self cells as was suggested in study IV. This model is supported by two aHUS patient mutations, R49L

and A101V in the FH20 site on C3d (Frémeaux-Bacchi et al. 2008; Maga et al. 2010).

Ferreira and co-workers found that aHUS associated mutations W1183R, T1184R, L1189F/R, S1191L,

and S1191L/V1197A enhanced binding of FH19-20 to C3b (Ferreira et al. 2009). These results are

contradictory to the results presented in this thesis. They studied the interaction by using only SPR

binding assays that may be sensitive to charge changes on proteins due to the high negative charge

of carboxymethylated dextran matrix. FH19-20 may have some affinity on the matrix since it

recognizes highly negative dextran sulfate.

Taken together, the aHUS-associated mutations disrupt FH19 binding to C3b and FH20 binding to

anionic self surface structures by different mechanisms. The consequence of the aHUS mutations on

FH19-20 function is however the same - decreased avidity of FH-C3b interaction on the self surfaces

that is the basic pathogenic mechanism of aHUS.

5.4 Dysfunction of FH19-20 in aHUS

C3b that is deposited on self or non-self surfaces does not seem to discriminate between them (Meri

et al. 1990b) and thus it is probable that FH19-20 binding affinity to the exposed FH19 site on C3b is

the same on all surfaces. Thus, the binding affinity of FH20 to self surfaces is crucial for inhibiting

complement activation. This means that FH20 needs to recognize all the different types of cell

surfaces in contact with plasma like erythrocytes, platelets and glomerular endothelial cells that are

damaged in aHUS.

5.4.1 Complement activation on CS-A rich platelets causes thrombocytopenia

It has been noted that thrombi in glomerular capillaries cannot alone explain thrombocytopenia in

aHUS, indicating that platelets are also activated also in extra-renal circulation (Walters et al. 1988).

Chondroitin sulfate A is the main GAG on platelets that seem to be devoid of HS (Ward et al. 1979;

Okayama et al. 1986). It has been shown that FH binds to washed human platelets with FH15-20

(Vaziri-Sani et al. 2005) and that FH with misfolded FH20 has impaired binding to platelets (Ståhl et

al. 2008). In addition, FH15-20 binding to platelets can be partially blocked with heparin (Vaziri-Sani



et al. 2005) and FH15-20 binding to heparin can be inhibited with CS-A (Jokiranta et al. 2005). Since

FH15-20 has only one heparin binding site it is likely that CS-A binding is mediated by the heparin

site on FH20 that would then also be important for recognizing platelets. Ståhl et al. studied platelets

from aHUS patients with the V1168E, E1198K, and E1198Stop mutations on FH20 and found that C3

and C9 were deposited on their surface (Ståhl et al. 2008). Platelets from aHUS patients form

aggregates and agglutinate, indicating their activation in the extra-renal circulation (Ståhl et al. 2008;

Licht et al. 2009). It can be concluded that dysfunctional FH19-20 cannot control complement

deposition on the platelets, inducing adherence and aggregation, thus explaining the resultant


5.4.2 Glomerular endothelial cell damage and thrombus formation

In studies III-IV it was shown that the mGEnC and heparin binding sites were nearly identical on

FH20. Although the mouse GEnC binding results cannot be directly applied to humans, it has been

shown that (i) mouse GEnCs have features of human GEnC like fenestrae, also in vitro (Rops et al.

2004a), (ii) the HS moieties are well conserved in mammalian cells (Esko et al. 2002), and (iii) the C-

terminal domains 18-20 of human and mouse FH bind similarily to heparin and HUVECs (Cheng et al.

2006). In GBM, HS accounts for up to 80 % of total GAGs (Kanwar et al. 1981; van den Heuvel et al.

1988) and is probably the main ligand for FH20. Glomerular endothelial cell glycocalyx has about 50 -

90 % HS and 10-20 % CS side chains on proteoglycans (Oohira et al. 1983) that are probably

important for FH20 binding.

Defective binding of FH to glomerular endothelium leads to generation of C3a, C5a, and MAC that

activate the endothelium leading to a pro-coagulant phenotype of the GEnCs (Ikeda et al. 1997;

Tedesco et al. 1997). Activated endothelium expresses TF that initiates coagulation and release of

vWf that can act as a receptor for platelets (Hattori et al. 1989; Saadi et al. 1995; Savage et al. 1996).

Platelets are activated by released C3a and C5a and thereafter attach more easily to activated

glomerular endothelium (Polley et al. 1983). Platelet adherence to the endothelial matrix leads to

propagation of the coagulation cascade and to thrombus formation in the glomeruli. Thrombin and

several other coagulation cascade enzymes cleave C3 and C5 that further activates complement on

the platelets and on endothelium creating a vicious cycle (Figure 10) (Huber-Lang et al. 2006). The

actions of complement and coagulation systems can therefore lead to the formation of thrombi and

endothelial swelling in the glomeruli - the typical histological findings in aHUS patients.

5.4.3 Complement activation on sialic acid rich erythrocytes causes hemolysis

Hemolysis in aHUS probably occurs mechanically in occluded capillaries as well as non-mechanically

in the circulation (Söderholm et al. 2009). Erythrocytes surfaces are rich in sialic acids and FH16-20

has been shown to bind to sialic acids (Ram et al. 1998). Although direct interactions of the FH20

domain with sialic acids have not been proven, results of functional assays suggest that heparin and

sialic acid sites overlap. Ferreira et al. studied the effect of the R1215G and K1230A mutations on

FH19-20 binding to Esh-C3b (Ferreira et al. 2009). They found that these mutations do not impair

C3b binding and decrease the affinity of FH19-20 to Esh-C3b. Based on the FH19-20:C3d structure,

these surface exposed mutations are not in the C3d binding site, indicating that these mutations

probably interact with sialic acids on erythrocytes. Although the presence of other receptors on



erythrocytes cannot be excluded, it is known that FH binding is dependent on sialic acids on

erythrocytes and it can be postulated that the FH20 site probably mediates this interaction. It has

been shown that FH19-20 is needed to protect erythrocytes from complement mediated lysis

(Ferreira et al. 2006) and that the aHUS mutations in FH19 or FH20 cause hemolysis of human

erythrocytes (Ferreira et al. 2009). Also, FH from aHUS patients with theE1172Stop, W1183L,

V1197A, E1198K, and R1210C mutations in FH20 areunable to protect non-activator sheep

erythrocytes from lysis (Sánchez-Corral et al. 2004; Vaziri-Sani et al. 2006; Heinen et al. 2007). Taken

together, it is likely that FH20 also recognizes sialic acids and that dysfunctional FH in aHUS cannot

protect erythrocytes from complement attack.

5.4.4 Pathogenesis model of aHUS

In conclusion, the aHUS-associated mutations in FH19-20 decrease the avidity of FH to C3b

deposited on self surfaces, leading to decreased efficiency in inactivation of C3b. C3b deposition on

GBM and GEnCs leads to complement amplification and generation of C5a and MAC. These

synergistically damage and activate the endothelium known to lead to pro-coagulant and proinflammatory

phenotypes. Platelets are activated in this manner and adhere to the glomerular

vessel walls and form thrombi. Thrombi and swelling of the endothelium lead to occluded capillaries

also contributing to hemolysis. In the systemic circulation complement attack against RBCs and

platelets leads to hemolysis and platelet activation and aggregation. Complement activation induces

inflammation, coagulation and cell damage that each feed-back to activate more complement and

coagulation, explaining the acute attacks often seen in aHUS patients.



6 Acknowledgments

This work was done almost completely in Haartman Institute, University of Helsinki, between 2001

and 2011. I want to thank the administration of the Medical Faculty and the Haartman Institute for

providing me the facilities to do the research, and the Academy of Finland, Biomedicum Helsinki

Foundation, Emil Aaltonen Foundation, Finnish Cultural Foundation, Maud Kuistila Memorial

Foundation, and Sigrid Juselius Foundation for financial support.

I am thankful and honored that Anna Blom accepted the invitation to be my opponent. I want to

express my gratitude to the reviewers of my thesis, Sarah Butcher and Olli Lassila, for their expert


I am most indebted to my supervisor Sakari Jokiranta who has believed in me as a scientist from the

beginning. His endless optimism, encouraging attitude, support and vast expertise have been the

driving forces in the making of this thesis. Sakari has taught me how to do science – what more could

a PhD student want?

I am very thankful for Seppo Meri who gave me the opportunity to enter the world of science. He

has shared his deep understanding in immunology with me and helped on many matters along the


True friends are a rarity – I want to very warmly thank Taru Meri for her friendship and support in

matters of science and life. She has been invaluable.

I am grateful for all the past and present members of the Jokiranta-group for keeping up the spirit

and giving support in the daily work. I am very thankful for Hanne Amdahl, Arnab Bhattacharjee,

Zhu-Zhu Cheng, Karita Haapasalo-Tuomainen, Satu Hyvärinen, Harriet Hägglund, Aino Koskinen,

Tanja Pasanen, Elina Pusa, Hannah Söderholm, and Maria Pärepalo for their contributions and nice

moments in the lab.

This work was in great part based on high quality structural biology work done in the Institute of

Biotechnology. I want to thank Adrian Goldman, Tommi Kajander, and Veli-Pekka Jaakola for very

fruitful collaboration and sharing their expertise.

I am thankful for Johan van der Vlag for the opportunity to work in his group at Nijmegen, the

Netherlands, and sharing his deep understanding on glycobiology and endothelial cells. I want to

thank Angelique Rops for collaboration and her support during my three-months stay in the lab. I

and my family were very warmly welcome to the Netherlands. I am (and we are) indebted to Johan

and his family, Angelique, Marinka Bakker and her family, Casandra van Bavel, Jürgen Dieker, Martijn

Kramer, Tom Nijenhuis, and Wim Tamboer.

I am honored and grateful that I had a chance to collaborate with David Isenman from the University

of Toronto who gave excellent advice on studies III and IV and provided together with Elisa Leung

the C3dg mutant proteins.



Mathematician Simopekka Vänskä is acknowledged for his expert advice on interdomain angle

calculations and Derek Ho for checking my thesis for the correctness of English language.

I want to sincerely thank the “more experienced” generation of the Meri-group Hanna Jarva, Sami

Junnikkala, Antti Lavikainen, Antti Sommarhem, and Jorma Tissari for all the help, collaborations,

scientific discussions and nice moments. I also wish to thank Antti Lavikainensis for our productive

collaboration that has certainly helped me to fund this thesis project. I have shared many

memorable scientific and non-scientific moments with, and I am thankful for the help I got from

many members of the Meri-group: Antti Alitalo, Tobias Freitag, Nathalie Friberg, Juha Hakulinen,

Mikko Holmberg, Ayman Khattab, Vesa Kirjavainen, Vesa Koistinen, Nina Lindeman, Sami

Nikoskelainen, Jaana Panelius, Riina Richardson, Rauna Riva, Jari Suvilehto, Jasse Tiainen, Antti

Väkevä, and Jinghui Yang.

The early “koppi” years were hilarious and most informative, and I want to thank Laura Kerosuo-

Palmberg for her friendship and sharing the booth with me and Taru. I wish to thank Jussi Hepojoki

and Tomas Strandin for helpful advice and great moments in the depths of the peptide lab.

I want to thank Livija Deban for our collaboration and nice discussions. I am honored that I have had

a chance to know many foreign colleagues like Kyra Gelderman, David Gordon, Florian Eberle, Jens

Hellwage, Mihaly Jozsi, Rebecca Ormsby, Tanya Sadlon, Leendert Trouw, and Valentina la Verde with

whom I have shared good moments and scientific discussions.

I want to very warmly thank Tiina Kasanen for her support, friendship, and all the cheerful coffee

breaks with Laura Kalin, Laura Mattinen, and Jukka-Pekka Palomäki. In addition I want to

acknowledge Marta Biedzka-Sarek and Mikael Skurnik for their positive spirit.

I have shared an office with many members of the Arstila-group and I wish to thank Eliisa

Kekäläinen, Tuisku-Tuulia Laurinolli, Anni Lehtoviita, Pirkka Pekkarinen, Laura Rossi, and Heli

Tuovinen for their positive spirit and T-cell help.

I want to warmly thank Kirsi Udueze for her support, cheerful discussions, and all the help with so

many practical matters. I would like to thank Marjatta Ahonen, Marko Hietavuo, Pirkko Kokkonen,

Ilkka Vanhatalo and Kirsti Widing for good moments, practical and technical support, and keeping

the lab, computers, and equipment running.

Very nice and professional people have worked with me in the department of bacteriology and

immunology. I want to thank Petteri Arstila, Mia Eholuoto, Anu Kantele, Hilkka Lankinen, Sari

Pakkanen, Nora Pöntynen, Heikki Repo, Heidi Sillanpää, Eine Virolainen, and Anni Virolainen-

Julkunen for all the discussions and good moments. I wish to acknowledge Christophe Roos who

gave me the inspiration to learn bioinformatics and helped me in the beginning of my career. My

fellow engineers at Haartman institute Samu Myllymaa and Tuomas Raitila are thanked for their


Many wonderful persons and personalities have enriched my life in science and I have shared good

moments and scientific discussions with them – I am grateful for also those that were not mentioned




I want to acknowledge all my friends and relatives for their support, good moments, and bringing

balance to my life.

I am deeply indebted and thankful beyond words to my wife Minna for all her unselfish support

during these years. It has been quite an effort to stand by me, but you always did – I love you.

Helsinki, 2011



7 References

Adams RL and Bird RJ. Review article: Coagulation cascade and therapeutics update: relevance to

nephrology. Part 1: Overview of coagulation, thrombophilias and history of anticoagulants.

Nephrology (Carlton). 14: 462-70. 2009.

Agnello V, Winchester RJ, and Kunkel HG. Precipitin reactions of the C1q component of complement

with aggregated gamma-globulin and immune complexes in gel diffusion. Immunology. 19: 909-19.


Alsenz J, Lambris JD, Schulz TF, and Dierich MP. Localization of the complement-component-C3bbinding

site and the cofactor activity for factor I in the 38kDa tryptic fragment of factor H. Biochem J.

224: 389-98. 1984.

Altschul SF, Gish W, Miller W, Myers EW, and Lipman DJ. Basic local alignment search tool. J Mol

Biol. 215: 403-10. 1990.

Amara U, Flierl MA, Rittirsch D, Klos A, Chen H, Acker B, Bruckner UB, Nilsson B, Gebhard F, Lambris

JD, and Huber-Lang M. Molecular intercommunication between the complement and coagulation

systems. J Immunol. 185: 5628-36. 2010.

Angata T and Varki A. Chemical diversity in the sialic acids and related alpha-keto acids: an

evolutionary perspective. Chem Rev. 102: 439-69. 2002.

Aslam M and Perkins SJ. Folded-back solution structure of monomeric factor H of human

complement by synchrotron X-ray and neutron scattering, analytical ultracentrifugation and

constrained molecular modelling. J Mol Biol. 309: 1117-38. 2001.

Bajzar L, Manuel R, and Nesheim ME. Purification and characterization of TAFI, a thrombin-activable

fibrinolysis inhibitor. J Biol Chem. 270: 14477-84. 1995.

Banchereau J and Steinman RM. Dendritic cells and the control of immunity. Nature. 392: 245-52.


Barlow PN, Steinkasserer A, Norman DG, Kieffer B, Wiles AP, Sim RB, and Campbell ID. Solution

structure of a pair of complement modules by nuclear magnetic resonance. J Mol Biol. 232: 268-84.


Batista FD and Harwood NE. The who, how and where of antigen presentation to B cells. Nat Rev

Immunol. 9: 15-27. 2009.

Benson DA, Boguski MS, Lipman DJ, and Ostell J. GenBank. Nucleic Acids Res. 25: 1-6. 1997.

Berger M, O'Shea J, Cross AS, Folks TM, Chused TM, Brown EJ, and Frank MM. Human neutrophils

increase expression of C3bi as well as C3b receptors upon activation. J Clin Invest. 74: 1566-71. 1984.

Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, and Bourne PE. The

Protein Data Bank. Nucleic Acids Res. 28: 235-42. 2000.



Bhakdi S, Kaflein R, Halstensen TS, Hugo F, Preissner KT, and Mollnes TE. Complement S-protein

(vitronectin) is associated with cytolytic membrane-bound C5b-9 complexes. Clin Exp Immunol. 74:

459-64. 1988.

Bhattacharjee A, Lehtinen MJ, Kajander T, Goldman A, and Jokiranta TS. Both domain 19 and domain

20 of factor H are involved in binding to complement C3b and C3d. Mol Immunol. 47: 1686-91. 2010.

Blackmore TK, Hellwage J, Sadlon TA, Higgs N, Zipfel PF, Ward HM, and Gordon DL. Identification of

the second heparin-binding domain in human complement factor H. J Immunol. 160: 3342-8. 1998.

Blom AM, Kask L, and Dahlbäck B. Structural requirements for the complement regulatory activities

of C4bp. J Biol Chem. 276: 27136-44. 2001.

Blom AM, Webb J, Villoutreix BO, and Dahlbäck B. A cluster of positively charged amino acids in the

C4BP alpha-chain is crucial for C4b binding and factor I cofactor function. J Biol Chem. 274: 19237-45.


Bobak DA, Gaither TA, Frank MM, and Tenner AJ. Modulation of FcR function by complement:

subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and

culture-derived macrophages. J Immunol. 138: 1150-6. 1987.

Bokisch VA, Muller-Eberhard HJ, and Cochrane CG. Isolation of a fragment (C3a) of the third

component of human complement containing anaphylatoxin and chemotactic activity and

description of an anaphylatoxin inactivator of human serum. J Exp Med. 129: 1109-30. 1969.

Bottazzi B, Vouret-Craviari V, Bastone A, De Gioia L, Matteucci C, Peri G, Spreafico F, Pausa M,

D'Ettorre C, Gianazza E, Tagliabue A, Salmona M, Tedesco F, Introna M, and Mantovani A. Multimer

formation and ligand recognition by the long pentraxin PTX3. Similarities and differences with the

short pentraxins C-reactive protein and serum amyloid P component. J Biol Chem. 272: 32817-23.


Botto M, Dell'Agnola C, Bygrave AE, Thompson EM, Cook HT, Petry F, Loos M, Pandolfi PP, and

Walport MJ. Homozygous C1q deficiency causes glomerulonephritis associated with multiple

apoptotic bodies. Nat Genet. 19: 56-9. 1998.

Brouwer N, Dolman KM, van Zwieten R, Nieuwenhuys E, Hart M, Aarden LA, Roos D, and Kuijpers

TW. Mannan-binding lectin (MBL)-mediated opsonization is enhanced by the alternative pathway

amplification loop. Mol Immunol. 43: 2051-60. 2006.

Cadroy Y, Diquelou A, Dupouy D, Bossavy JP, Sakariassen KS, Sie P, and Boneu B. The

thrombomodulin/protein C/protein S anticoagulant pathway modulates the thrombogenic

properties of the normal resting and stimulated endothelium. Arterioscler Thromb Vasc Biol. 17: 520-

7. 1997.

Campbell WD, Lazoura E, Okada N, and Okada H. Inactivation of C3a and C5a octapeptides by

carboxypeptidase R and carboxypeptidase N. Microbiol Immunol. 46: 131-4. 2002.

Caprioli J, Noris M, Brioschi S, Pianetti G, Castelletti F, Bettinaglio P, Mele C, Bresin E, Cassis L,

Gamba S, Porrati F, Bucchioni S, Monteferrante G, Fang CJ, Liszewski MK, Kavanagh D, Atkinson JP,

and Remuzzi G. Genetics of HUS: the impact of MCP, CFH, and IF mutations on clinical presentation,

response to treatment, and outcome. Blood. 108: 1267-79. 2006.



Carreno MP, Labarre D, Maillet F, Jozefowicz M, and Kazatchkine MD. Regulation of the human

alternative complement pathway: formation of a ternary complex between factor H, surface-bound

C3b and chemical groups on nonactivating surfaces. Eur J Immunol. 19: 2145-50. 1989.

Carroll MC, Alicot EM, Katzman PJ, Klickstein LB, Smith JA, and Fearon DT. Organization of the genes

encoding complement receptors type 1 and 2, decay-accelerating factor, and C4-binding protein in

the RCA locus on human chromosome 1. J Exp Med. 167: 1271-80. 1988.

Carter RH and Fearon DT. CD19: lowering the threshold for antigen receptor stimulation of B

lymphocytes. Science. 256: 105-7. 1992.

Carter RH, Spycher MO, Ng YC, Hoffman R, and Fearon DT. Synergistic interaction between

complement receptor type 2 and membrane IgM on B lymphocytes. J Immunol. 141: 457-63. 1988.

Casasnovas JM, Larvie M, and Stehle T. Crystal structure of two CD46 domains reveals an extended

measles virus-binding surface. EMBO J. 18: 2911-22. 1999.

Cavaillon JM, Fitting C, and Haeffner-Cavaillon N. Recombinant C5a enhances interleukin 1 and

tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages. Eur J

Immunol. 20: 253-7. 1990.

Chen CB and Wallis R. Two mechanisms for mannose-binding protein modulation of the activity of its

associated serine proteases. J Biol Chem. 279: 26058-65. 2004.

Cheng ZZ, Hellwage J, Seeberger H, Zipfel PF, Meri S, and Jokiranta TS. Comparison of surface

recognition and C3b binding properties of mouse and human complement factor H. Mol Immunol.

43: 972-9. 2006.

Cirino G, Cicala C, Bucci M, Sorrentino L, Ambrosini G, DeDominicis G, and Altieri DC. Factor Xa as an

interface between coagulation and inflammation. Molecular mimicry of factor Xa association with

effector cell protease receptor-1 induces acute inflammation in vivo. J Clin Invest. 99: 2446-51. 1997.

Claus DR, Siegel J, Petras K, Osmand AP, and Gewurz H. Interactions of C-reactive protein with the

first component of human complement. J Immunol. 119: 187-92. 1977.

Cochrane CG and Muller-Eberhard HJ. The derivation of two distinct anaphylatoxin activities from

the third and fifth components of human complement. J Exp Med. 127: 371-86. 1968.

Cole DS, Hughes TR, Gasque P, and Morgan BP. Complement regulator loss on apoptotic neuronal

cells causes increased complement activation and promotes both phagocytosis and cell lysis. Mol

Immunol. 43: 1953-64. 2006.

Cole DS and Morgan BP. Beyond lysis: how complement influences cell fate. Clin Sci (Lond). 104: 455-

66. 2003.

Cole JL, Housley GA, Jr., Dykman TR, MacDermott RP, and Atkinson JP. Identification of an additional

class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines. Proc

Natl Acad Sci U S A. 82: 859-63. 1985.



Cooper NR. Enzymatic activity of the second component of complement. Biochemistry. 14: 4245-51.


Daffern PJ, Pfeifer PH, Ember JA, and Hugli TE. C3a is a chemotaxin for human eosinophils but not for

neutrophils. I. C3a stimulation of neutrophils is secondary to eosinophil activation. J Exp Med. 181:

2119-27. 1995.

Dahl MR, Thiel S, Matsushita M, Fujita T, Willis AC, Christensen T, Vorup-Jensen T, and Jensenius JC.

MASP-3 and its association with distinct complexes of the mannan-binding lectin complement

activation pathway. Immunity. 15: 127-35. 2001.

Damerau B, Zimmermann B, Czorniak K, Wustefeld H, and Vogt W. Role of the N-terminal regions of

hog C3a, C5a and C5a-desArg in their biological activities. Mol Immunol. 23: 433-40. 1986.

Deban L, Jarva H, Lehtinen MJ, Bottazzi B, Bastone A, Doni A, Jokiranta TS, Mantovani A, and Meri S.

Binding of the long pentraxin PTX3 to factor H: interacting domains and function in the regulation of

complement activation. J Immunol. 181: 8433-40. 2008.

Delvaeye M, Noris M, De Vriese A, Esmon CT, Esmon NL, Ferrell G, Del-Favero J, Plaisance S, Claes B,

Lambrechts D, Zoja C, Remuzzi G, and Conway EM. Thrombomodulin mutations in atypical

hemolytic-uremic syndrome. N Engl J Med. 361: 345-57. 2009.

Dempsey PW, Allison ME, Akkaraju S, Goodnow CC, and Fearon DT. C3d of complement as a

molecular adjuvant: bridging innate and acquired immunity. Science. 271: 348-50. 1996.

Devine DV and Rosse WF. Regulation of the activity of platelet-bound C3 convertase of the

alternative pathway of complement by platelet factor H. Proc Natl Acad Sci U S A. 84: 5873-7. 1987.

Diaz-Ricart M, Estebanell E, Lozano M, Aznar-Salatti J, White JG, Ordinas A, and Escolar G. Thrombin

facilitates primary platelet adhesion onto vascular surfaces in the absence of plasma adhesive

proteins: studies under flow conditions. Haematologica. 85: 280-8. 2000.

Dodds AW, Ren XD, Willis AC, and Law SK. The reaction mechanism of the internal thioester in the

human complement component C4. Nature. 379: 177-9. 1996.

Ehlenberger AG and Nussenzweig V. The role of membrane receptors for C3b and C3d in

phagocytosis. J Exp Med. 145: 357-71. 1977.

Elward K, Griffiths M, Mizuno M, Harris CL, Neal JW, Morgan BP, and Gasque P. CD46 plays a key role

in tailoring innate immune recognition of apoptotic and necrotic cells. J Biol Chem. 280: 36342-54.


Erdos EG and Sloane EM. An enzyme in human blood plasma that inactivates bradykinin and

kallidins. Biochem Pharmacol. 11: 585-92. 1962.

Esko JD and Selleck SB. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu

Rev Biochem. 71: 435-71. 2002.

Esmon CT. Interactions between the innate immune and blood coagulation systems. Trends

Immunol. 25: 536-42. 2004.



Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, and Henson PM. Macrophages that have

ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through

autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest. 101: 890-8. 1998.

Farries TC, Seya T, Harrison RA, and Atkinson JP. Competition for binding sites on C3b by CR1, CR2,

MCP, factor B and factor H. Complement Inflamm. 7: 30-41. 1990.

Fearon DT. Regulation by membrane sialic acid of beta1H-dependent decay-dissociation of

amplification C3 convertase of the alternative complement pathway. Proc Natl Acad Sci U S A. 75:

1971-5. 1978.

Fearon DT. Regulation of the amplification C3 convertase of human complement by an inhibitory

protein isolated from human erythrocyte membrane. Proc Natl Acad Sci U S A. 76: 5867-71. 1979.

Fearon DT and Austen KF. Properdin: binding to C3b and stabilization of the C3b-dependent C3

convertase. J Exp Med. 142: 856-63. 1975.

Fearon DT and Carroll MC. Regulation of B lymphocyte responses to foreign and self-antigens by the

CD19/CD21 complex. Annu Rev Immunol. 18: 393-422. 2000.

Ferguson AR, Youd ME, and Corley RB. Marginal zone B cells transport and deposit IgM-containing

immune complexes onto follicular dendritic cells. Int Immunol. 16: 1411-22. 2004.

Fernandez HN, Henson PM, Otani A, and Hugli TE. Chemotactic response to human C3a and C5a

anaphylatoxins. I. Evaluation of C3a and C5a leukotaxis in vitro and under stimulated in vivo

conditions. J Immunol. 120: 109-15. 1978.

Ferreira VP, Herbert AP, Cortes C, McKee KA, Blaum BS, Esswein ST, Uhrin D, Barlow PN, Pangburn

MK, and Kavanagh D. The binding of factor H to a complex of physiological polyanions and C3b on

cells is impaired in atypical hemolytic uremic syndrome. J Immunol. 182: 7009-18. 2009.

Ferreira VP, Herbert AP, Hocking HG, Barlow PN, and Pangburn MK. Critical role of the C-terminal

domains of factor H in regulating complement activation at cell surfaces. J Immunol. 177: 6308-16.


Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund

K, Holm L, Sonnhammer EL, Eddy SR, and Bateman A. The Pfam protein families database. Nucleic

Acids Res. 38: D211-22. 2009.

Fischer WH and Hugli TE. Regulation of B cell functions by C3a and C3a(desArg): suppression of TNFalpha,

IL-6, and the polyclonal immune response. J Immunol. 159: 4279-86. 1997.

Foreman KE, Vaporciyan AA, Bonish BK, Jones ML, Johnson KJ, Glovsky MM, Eddy SM, and Ward PA.

C5a-induced expression of P-selectin in endothelial cells. J Clin Invest. 94: 1147-55. 1994.

Fraker PJ and Speck JC, Jr. Protein and cell membrane iodinations with a sparingly soluble

chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 80: 849-

57. 1978.

Frémeaux-Bacchi V, Miller EC, Liszewski MK, Strain L, Blouin J, Brown AL, Moghal N, Kaplan BS, Weiss

RA, Lhotta K, Kapur G, Mattoo T, Nivet H, Wong W, Gie S, Hurault de Ligny B, Fischbach M, Gupta R,



Hauhart R, Meunier V, Loirat C, Dragon-Durey MA, Fridman WH, Janssen BJ, Goodship TH, and

Atkinson JP. Mutations in complement C3 predispose to development of atypical hemolytic uremic

syndrome. Blood. 112: 4948-52. 2008.

Fries LF, Siwik SA, Malbran A, and Frank MM. Phagocytosis of target particles bearing C3b-IgG

covalent complexes by human monocytes and polymorphonuclear leucocytes. Immunology. 62: 45-

51. 1987.

Gasque P. Complement: a unique innate immune sensor for danger signals. Mol Immunol. 41: 1089-

98. 2004.

Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, and Bairoch A. ExPASy: The proteomics server

for in-depth protein knowledge and analysis. Nucleic Acids Res. 31: 3784-8. 2003.

Geng JG, Bevilacqua MP, Moore KL, McIntyre TM, Prescott SM, Kim JM, Bliss GA, Zimmerman GA,

and McEver RP. Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature.

343: 757-60. 1990.

Gershov D, Kim S, Brot N, and Elkon KB. C-Reactive protein binds to apoptotic cells, protects the cells

from assembly of the terminal complement components, and sustains an antiinflammatory innate

immune response: implications for systemic autoimmunity. J Exp Med. 192: 1353-64. 2000.

Gewurz H, Shin HS, and Mergenhagen SE. Interactions of the complement system with endotoxic

lipopolysaccharide: consumption of each of the six terminal complement components. J Exp Med.

128: 1049-57. 1968.

Ghebrehiwet B and Peerschke EI. cC1q-R (calreticulin) and gC1q-R/p33: ubiquitously expressed

multi-ligand binding cellular proteins involved in inflammation and infection. Mol Immunol. 41: 173-

83. 2004.

Ghiran I, Barbashov SF, Klickstein LB, Tas SW, Jensenius JC, and Nicholson-Weller A. Complement

receptor 1/CD35 is a receptor for mannan-binding lectin. J Exp Med. 192: 1797-808. 2000.

Gigli I, Fujita T, and Nussenzweig V. Modulation of the classical pathway C3 convertase by plasma

proteins C4 binding protein and C3b inactivator. Proc Natl Acad Sci U S A. 76: 6596-600. 1979.

Gigli I, Porter RR, and Sim RB. The unactivated form of the first component of human complement,

C1. Biochem J. 157: 541-8. 1976.

Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, Carreras L, Arranz EA, Garrido CA, López-

Trascasa M, Sánchez-Corral P, Morgan BP, and Rodríguez de Córdoba S. Gain-of-function mutations

in complement factor B are associated with atypical hemolytic uremic syndrome. Proc Natl Acad Sci

U S A. 104: 240-5. 2007.

Goldberger G, Thomas ML, Tack BF, Williams J, Colten HR, and Abraham GN. NH2-terminal structure

and cleavage of guinea pig pro-C3, the precursor of the third complement component. J Biol Chem.

256: 12617-9. 1981.

Gonzalez SF, Kuligowski MP, Pitcher LA, Roozendaal R, and Carroll MC. The role of innate immunity

in B cell acquisition of antigen within LNs. Adv Immunol. 106: 1-19. 2010.



Goodman MG, Chenoweth DE, and Weigle WO. Induction of interleukin 1 secretion and

enhancement of humoral immunity by binding of human C5a to macrophage surface C5a receptors.

J Exp Med. 156: 912-7. 1982.

Gordon DL, Kaufman RM, Blackmore TK, Kwong J, and Lublin DM. Identification of complement

regulatory domains in human factor H. J Immunol. 155: 348-56. 1995.

Gorski JP, Hugli TE, and Muller-Eberhard HJ. C4a: the third anaphylatoxin of the human complement

system. Proc Natl Acad Sci U S A. 76: 5299-302. 1979.

Greengard JS, Heeb MJ, Ersdal E, Walsh PN, and Griffin JH. Binding of coagulation factor XI to washed

human platelets. Biochemistry. 25: 3884-90. 1986.

Grossklaus C, Damerau B, Lemgo E, and Vogt W. Induction of platelet aggregation by the

complement-derived peptides C3a and C5a. Naunyn-Schmiedebergs Archives of Pharmacology. 295:

71-6. 1976.

Gruppo RA and Rother RP. Eculizumab for congenital atypical hemolytic-uremic syndrome. N Engl J

Med. 360: 544-6. 2009.

Guex N and Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative

protein modeling. Electrophoresis. 18: 2714-23. 1997.

Gullstrand B, Martensson U, Sturfelt G, Bengtsson AA, and Truedsson L. Complement classical

pathway components are all important in clearance of apoptotic and secondary necrotic cells. Clin

Exp Immunol. 156: 303-11. 2009.

Haas PJ and van Strijp J. Anaphylatoxins: their role in bacterial infection and inflammation. Immunol

Res. 37: 161-75. 2007.

Hajishengallis G and Lambris JD. Crosstalk pathways between Toll-like receptors and the

complement system. Trends Immunol. 31: 154-63. 2010.

Hammer CH, Nicholson A, and Mayer MM. On the mechanism of cytolysis by complement: evidence

on insertion of C5b and C7 subunits of the C5b,6,7 complex into phospholipid bilayers of erythrocyte

membranes. Proc Natl Acad Sci U S A. 72: 5076-80. 1975.

Harboe M and Mollnes TE. The alternative complement pathway revisited. J Cell Mol Med. 12: 1074-

84. 2008.

Harboe M, Ulvund G, Vien L, Fung M, and Mollnes TE. The quantitative role of alternative pathway

amplification in classical pathway induced terminal complement activation. Clin Exp Immunol. 138:

439-46. 2004.

Hardy MM, Flickinger AG, Riley DP, Weiss RH, and Ryan US. Superoxide dismutase mimetics inhibit

neutrophil-mediated human aortic endothelial cell injury in vitro. J Biol Chem. 269: 18535-40. 1994.

Harmat V, Gal P, Kardos J, Szilagyi K, Ambrus G, Vegh B, Naray-Szabo G, and Zavodszky P. The

structure of MBL-associated serine protease-2 reveals that identical substrate specificities of C1s and

MASP-2 are realized through different sets of enzyme-substrate interactions. J Mol Biol. 342: 1533-

46. 2004.



Harrison RA and Lachmann PJ. Novel cleavage products of the third component of human

complement. Mol Immunol. 17: 219-28. 1980a.

Harrison RA and Lachmann PJ. The physiological breakdown of the third component of human

complement. Mol Immunol. 17: 9-20. 1980b.

Hattori R, Hamilton KK, McEver RP, and Sims PJ. Complement proteins C5b-9 induce secretion of

high molecular weight multimers of endothelial von Willebrand factor and translocation of granule

membrane protein GMP-140 to the cell surface. J Biol Chem. 264: 9053-60. 1989.

Hawlisch H and Köhl J. Complement and Toll-like receptors: key regulators of adaptive immune

responses. Mol Immunol. 43: 13-21. 2006.

Heinen S, Hartmann A, Lauer N, Wiehl U, Dahse HM, Schirmer S, Gropp K, Enghardt T, Wallich R,

Hälbich S, Mihlan M, Schlötzer-Schrehardt U, Zipfel PF, and Skerka C. Factor H-related protein 1

(CFHR-1) inhibits complement C5 convertase activity and terminal complex formation. Blood. 114:

2439-47. 2009.

Heinen S, Józsi M, Hartmann A, Noris M, Remuzzi G, Skerka C, and Zipfel PF. Hemolytic uremic

syndrome: a factor H mutation (E1172Stop) causes defective complement control at the surface of

endothelial cells. J Am Soc Nephrol. 18: 506-14. 2007.

Hellwage J, Jokiranta TS, Friese MA, Wolk TU, Kampen E, Zipfel PF, and Meri S. Complement C3b/C3d

and cell surface polyanions are recognized by overlapping binding sites on the most carboxylterminal

domain of complement factor H. J Immunol. 169: 6935-44. 2002.

Herbert AP, Uhrin D, Lyon M, Pangburn MK, and Barlow PN. Disease-associated sequence variations

congregate in a polyanion recognition patch on human factor H revealed in three-dimensional

structure. J Biol Chem. 281: 16512-20. 2006.

Hicks PS, Saunero-Nava L, Du Clos TW, and Mold C. Serum amyloid P component binds to histones

and activates the classical complement pathway. J Immunol. 149: 3689-94. 1992.

Hoffman M and Monroe DM, 3rd. A cell-based model of hemostasis. Thromb Haemost. 85: 958-65.


Honoré C, Hummelshøj T, Hansen BE, Madsen HO, Eggleton P, and Garred P. The innate immune

component ficolin 3 (Hakata antigen) mediates the clearance of late apoptotic cells. Arthritis Rheum.

56: 1598-607. 2007.

Hourcade DE. The role of properdin in the assembly of the alternative pathway C3 convertases of

complement. J Biol Chem. 281: 2128-32. 2006.

Huber-Lang M, Sarma JV, Zetoune FS, Rittirsch D, Neff TA, McGuire SR, Lambris JD, Warner RL, Flierl

MA, Hoesel LM, Gebhard F, Younger JG, Drouin SM, Wetsel RA, and Ward PA. Generation of C5a in

the absence of C3: a new complement activation pathway. Nat Med. 12: 682-7. 2006.

Hughes J, Nangaku M, Alpers CE, Shankland SJ, Couser WG, and Johnson RJ. C5b-9 membrane attack

complex mediates endothelial cell apoptosis in experimental glomerulonephritis. Am J Physiol Renal

Physiol. 278: F747-57. 2000.



Hugli TE and Müller-Eberhard HJ. Anaphylatoxins: C3a and C5a. Adv Immunol. 26: 1-53. 1978.

Iida K and Nussenzweig V. Functional properties of membrane-associated complement receptor CR1.

J Immunol. 130: 1876-80. 1983.

Ikeda K, Nagasawa K, Horiuchi T, Tsuru T, Nishizaka H, and Niho Y. C5a induces tissue factor activity

on endothelial cells. Thromb Haemost. 77: 394-8. 1997.

Ikeda K, Sannoh T, Kawasaki N, Kawasaki T, and Yamashina I. Serum lectin with known structure

activates complement through the classical pathway. J Biol Chem. 262: 7451-4. 1987.

Isenman DE, Leung E, Mackay JD, Bagby S, and van den Elsen JM. Mutational analyses reveal that the

staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping

contact residues on C3d: implications for the controversy regarding the CR2/C3d cocrystal structure.

J Immunol. 184: 1946-55. 2010.

Jagels MA and Hugli TE. Mechanisms and mediators of neutrophilic leukocytosis.

Immunopharmacology. 28: 1-18. 1994.

Jalanko H, Peltonen S, Koskinen A, Puntila J, Isoniemi H, Holmberg C, Pinomäki A, Armstrong E,

Koivusalo A, Tukiainen E, Mäkisalo H, Saland J, Remuzzi G, Rodriguez de Córdoba S, Lassila R, Meri S,

and Jokiranta TS. Successful liver-kidney transplantation in two children with aHUS caused by a

mutation in complement factor H. Am J Transplant. 8: 216-21. 2008.

Janin J. The kinetics of protein-protein recognition. Proteins. 28: 153-61. 1997.

Jenne DE and Tschopp J. Molecular structure and functional characterization of a human

complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein

2, a constituent of rat testis fluid. Proc Natl Acad Sci U S A. 86: 7123-7. 1989.

Jensen ML, Honoré C, Hummelshøj T, Hansen BE, Madsen HO, and Garred P. Ficolin-2 recognizes

DNA and participates in the clearance of dying host cells. Mol Immunol. 44: 856-65. 2007.

Jokiranta TS, Cheng ZZ, Seeberger H, Jozsi M, Heinen S, Noris M, Remuzzi G, Ormsby R, Gordon DL,

Meri S, Hellwage J, and Zipfel PF. Binding of complement factor H to endothelial cells is mediated by

the carboxy-terminal glycosaminoglycan binding site. Am J Path. 167: 1173-81. 2005.

Jokiranta TS, Hellwage J, Koistinen V, Zipfel PF, and Meri S. Each of the three binding sites on

complement factor H interacts with a distinct site on C3b. J Biol Chem. 275: 27657-62. 2000.

Jones J and Morgan BP. Apoptosis is associated with reduced expression of complement regulatory

molecules, adhesion molecules and other receptors on polymorphonuclear leucocytes: functional

relevance and role in inflammation. Immunology. 86: 651-60. 1995.

Józsi M, Licht C, Strobel S, Zipfel SL, Richter H, Heinen S, Zipfel PF, and Skerka C. Factor H

autoantibodies in atypical hemolytic uremic syndrome correlate with CFHR1/CFHR3 deficiency.

Blood. 111: 1512-4. 2008.

Kajita T and Hugli TE. C5a-induced neutrophilia. A primary humoral mechanism for recruitment of

neutrophils. Am J Pathol. 137: 467-77. 1990.



Kanwar YS, Hascall VC, and Farquhar MG. Partial characterization of newly synthesized

proteoglycans isolated from the glomerular basement membrane. J Cell Biol. 90: 527-32. 1981.

Karpman D, Manea M, Vaziri-Sani F, Ståhl AL, and Kristoffersson AC. Platelet activation in hemolytic

uremic syndrome. Semin Thromb Hemost. 32: 128-45. 2006.

Kastl SP, Speidl WS, Kaun C, Rega G, Assadian A, Weiss TW, Valent P, Hagmueller GW, Maurer G,

Huber K, and Wojta J. The complement component C5a induces the expression of plasminogen

activator inhibitor-1 in human macrophages via NF-kappaB activation. J Thromb Haemost. 4: 1790-7.


Kavanagh D, Burgess R, Spitzer D, Richards A, Diaz-Torres ML, Goodship JA, Hourcade DE, Atkinson

JP, and Goodship TH. The decay accelerating factor mutation I197V found in hemolytic uraemic

syndrome does not impair complement regulation. Mol Immunol. 44: 3162-7. 2007.

Kavanagh D, Kemp EJ, Mayland E, Winney RJ, Duffield JS, Warwick G, Richards A, Ward R, Goodship

JA, and Goodship TH. Mutations in complement factor I predispose to development of atypical

hemolytic uremic syndrome. J Am Soc Nephrol. 16: 2150-5. 2005.

Kavanagh D, Richards A, and Atkinson J. Complement regulatory genes and hemolytic uremic

syndromes. Annu Rev Med. 59: 293-309. 2008.

Kavanagh D, Richards A, Goodship T, and Jalanko H. Transplantation in atypical hemolytic uremic

syndrome. Semin Thromb Hemost. 36: 653-9. 2010.

Kazatchkine MD, Fearon DT, and Austen KF. Human alternative complement pathway: membraneassociated

sialic acid regulates the competition between B and beta1 H for cell-bound C3b. J

Immunol. 122: 75-81. 1979a.

Kazatchkine MD, Fearon DT, Silbert JE, and Austen KF. Surface-associated heparin inhibits zymosaninduced

activation of the human alternative complement pathway by augmenting the regulatory

action of the control proteins on particle-bound C3b. J Exp Med. 150: 1202-15. 1979b.

Kemper C and Atkinson JP. T-cell regulation: with complements from innate immunity. Nat Rev

Immunol. 7: 9-18. 2007.

Kemper C and Hourcade DE. Properdin: New roles in pattern recognition and target clearance. Mol

Immunol. 45: 4048-56. 2008.

Kim S, Elkon KB, and Ma X. Transcriptional suppression of interleukin-12 gene expression following

phagocytosis of apoptotic cells. Immunity. 21: 643-53. 2004.

Kim SH, Carney DF, Hammer CH, and Shin ML. Nucleated cell killing by complement: effects of C5b-9

channel size and extracellular Ca2+ on the lytic process. J Immunol. 138: 1530-6. 1987.

Kinoshita T, Medof ME, Hong K, and Nussenzweig V. Membrane-bound C4b interacts endogenously

with complement receptor CR1 of human red cells. J Exp Med. 164: 1377-88. 1986.



Kinoshita T, Takata Y, Kozono H, Takeda J, Hong KS, and Inoue K. C5 convertase of the alternative

complement pathway: covalent linkage between two C3b molecules within the trimolecular complex

enzyme. J Immunol. 141: 3895-901. 1988.

Klickstein LB, Barbashov SF, Liu T, Jack RM, and Nicholson-Weller A. Complement receptor type 1

(CR1, CD35) is a receptor for C1q. Immunity. 7: 345-55. 1997.

Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, and Köhl J. The role of the anaphylatoxins in health

and disease. Mol Immunol. 46: 2753-66. 2009.

Korb LC and Ahearn JM. C1q binds directly and specifically to surface blebs of apoptotic human

keratinocytes: complement deficiency and systemic lupus erythematosus revisited. J Immunol. 158:

4525-8. 1997.

Krauss JC, PooH, Xue W, Mayo-Bond L, Todd RFr, and Petty HR. Reconstitution of antibodydependent

phagocytosis in fibroblasts expressing Fc gamma receptor IIIB and the complement

receptor type 3. J Immunol. 153: 1769-77. 1994.

Krem MM and Di Cera E. Evolution of enzyme cascades from embryonic development to blood

coagulation. Trends Biochem Sci. 27: 67-74. 2002.

Krushkal J, Bat O, and Gigli I. Evolutionary relationships among proteins encoded by the regulator of

complement activation gene cluster. Mol Biol Evol. 17: 1718-30. 2000.

Krych-Goldberg M and Atkinson JP. Structure-function relationships of complement receptor type 1.

Immunol Rev. 180: 112-22. 2001.

Kühn S, Skerka C, and Zipfel PF. Mapping of the complement regulatory domains in the human factor

H-like protein 1 and in factor H. J Immunol. 155: 5663-70. 1995a.

Kühn S, Skerka C, and Zipfel PF. Mapping of the complement regulatory domains in the human factor

H-like protein 1 and in factor H1. J Immunol. 155: 5663-70. 1995b.

Kühn S and Zipfel PF. The baculovirus expression vector pBSV-8His directs secretion of histidinetagged

proteins. Gene. 162: 225-9. 1995c.

Kühn S and Zipfel PF. Mapping of the domains required for decay acceleration activity of the human

factor H-like protein 1 and factor H. Eur J Immunol. 26: 2383-7. 1996a.

Kühn S and Zipfel PF. Mapping of the domains required for decay acceleration activity of the human

factor H-like protein 1 and factor H. Eur J Immunol. 26: 2383-7. 1996b.

Köhl J. Self, non-self, and danger: a complementary view. Adv Exp Med Biol. 586: 71-94. 2006.

Köse O, Zimmerhackl LB, Jungraithmayr T, Mache C, and Nürnberger J. New treatment options for

atypical hemolytic uremic syndrome with the complement inhibitor eculizumab. Semin Thromb

Hemost. 36: 669-72. 2010.

Lachmann PJ and Halbwachs L. The influence of C3b inactivator (KAF) concentration on the ability of

serum to support complement activation. Clin Exp Immunol. 21: 109-14. 1975.



Laurell AB, Johnson U, Mårtensson U, and Sjöholm AG. Formation of complexes composed of C1r,

C1s, and C1 inactivator in human serum on activation of C1. Acta Pathol Microbiol Scand C. 86C: 299-

306. 1978.

Law SK and Levine RP. Interaction between the third complement protein and cell surface

macromolecules. Proc Natl Acad Sci U S A. 74: 2701-5. 1977.

Law SK, Lichtenberg NA, Holcombe FH, and Levine RP. Interaction between the labile binding sites of

the fourth (C4) and fifth (C5) human complement proteins and erythrocyte cell membranes. J

Immunol. 125: 634-9. 1980.

Leffler J, Herbert AP, Norström E, Schmidt CQ, Barlow PN, Blom AM, and Martin M. Annexin-II, DNA,

and histones serve as factor H ligands on the surface of apoptotic cells. J Biol Chem. 285: 3766-76.


Lehtinen MJ and Jokiranta TS. Mapping and characterisation of the C3d binding site on factor H

domains 19-20. Mol Immunol. 45: P89. 2008.

Lehtinen MJ, Jokiranta TS, and Meri S. Prediction of functional sites in human complement

regulators by motif profile analysis. Mol Immunol. 40: 204. 2003.

Lehtinen MJ, Jokiranta TS, and Meri S. Sequence and motif analysis of SCR domains. Mol Immunol.

41: 143. 2004.

Lesavre PH, Hugli TE, Esser AF, and Müller-Eberhard HJ. The alternative pathway C3/C5 convertase:

chemical basis of factor B activation. J Immunol. 123: 529-34. 1979.

Li K, Anderson KJ, Peng Q, Noble A, Lu B, Kelly AP, Wang N, Sacks SH, and Zhou W. Cyclic AMP plays a

critical role in C3a-receptor-mediated regulation of dendritic cells in antigen uptake and T-cell

stimulation. Blood. 112: 5084-94. 2008.

Licht C, Pluthero FG, Li L, Christensen H, Habbig S, Hoppe B, Geary DF, Zipfel PF, and Kahr WH.

Platelet-associated complement factor H in healthy persons and patients with atypical HUS. Blood.

114: 4538-45. 2009.

Loirat C, Garnier A, Sellier-Leclerc AL, and Kwon T. Plasmatherapy in atypical hemolytic uremic

syndrome. Semin Thromb Hemost. 36: 673-81. 2010.

Loirat C, Noris M, and Frémeaux-Bacchi V. Complement and the atypical hemolytic uremic syndrome

in children. Pediatr Nephrol. 23: 1957-72. 2008.

Loos M, Bitter-Suermann D, and Dierich M. Interaction of the first (C1), the second (C2) and the

fourth (C4) component of complement with different preparations of bacterial lipopolysaccharides

and with lipid A. J Immunol. 112: 935-40. 1974.

Lukacik P, Roversi P, White J, Esser D, Smith GP, Billington J, Williams PA, Rudd PM, Wormald MR,

Harvey DJ, Crispin MD, Radcliffe CM, Dwek RA, Evans DJ, Morgan BP, Smith RA, and Lea SM.

Complement regulation at the molecular level: the structure of decay-accelerating factor. Proc Natl

Acad Sci U S A. 101: 1279-84. 2004.



Lynch NJ, Roscher S, Hartung T, Morath S, Matsushita M, Maennel DN, Kuraya M, Fujita T, and

Schwaeble WJ. L-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of Gram-positive

bacteria, and activates the lectin pathway of complement. J Immunol. 172: 1198-202. 2004.

MacPherson GG. Synthesis and localization of sulphated mucopolysaccharide in megakaryocytes and

platelets of the rat, an anlysis by electron-microscope autoradiography. J Cell Sci. 10: 705-17. 1972.

Maga TK, Nishimura CJ, Weaver AE, Frees KL, and Smith RJ. Mutations in alternative pathway

complement proteins in American patients with atypical hemolytic uremic syndrome. Hum Mutat.

31: E1445-60. 2010.

Manuelian T, Hellwage J, Meri S, Caprioli J, Noris M, Heinen S, Józsi M, Neumann HP, Remuzzi G, and

Zipfel PF. Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to

endothelial cells in hemolytic uremic syndrome. J Clin Invest. 111: 1181-90. 2003.

Markiewski MM, Nilsson B, Ekdahl KN, Mollnes TE, and Lambris JD. Complement and coagulation:

strangers or partners in crime? Trends Immunol. 28: 184-92. 2007.

Martin U, Bock D, Arseniev L, Tornetta MA, Ames RS, Bautsch W, Köhl J, Ganser A, and Klos A. The

human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes. J

Exp Med. 186: 199-207. 1997.

Matsushita M. Ficolins: complement-activating lectins involved in innate immunity. J Innate Immun.

2: 24-32. 2009.

Matsushita M, Endo Y, and Fujita T. Cutting edge: complement-activating complex of ficolin and

mannose-binding lectin-associated serine protease. J Immunol. 164: 2281-4. 2000a.

Matsushita M, Thiel S, Jensenius JC, Terai I, and Fujita T. Proteolytic activities of two types of

mannose-binding lectin-associated serine protease. J Immunol. 165: 2637-42. 2000b.

McWilliam H, Valentin F, Goujon M, Li W, Narayanasamy M, Martin J, Miyar T, and Lopez R. Web

services at the European Bioinformatics Institute-2009. Nucleic Acids Res. 37: W6-10. 2009.

Medicus RG, Götze O, and Müller-Eberhard HJ. Alternative pathway of complement: recruitment of

precursor properdin by the labile C3/C5 convertase and the potentiation of the pathway. J Exp Med.

144: 1076-93. 1976.

Medicus RG, Melamed J, and Arnaout MA. Role of human factor I and C3b receptor in the cleavage

of surface-bound C3bi molecules. Eur J Immunol. 13: 465-70. 1983.

Menkin V. The determination of the level of leukocytes in the blood stream with inflammation; a

thermostable component concerned in the mechanism of leukocytosis. Blood. 4: 1323-37. 1949.

Meri S, Morgan BP, Davies A, Daniels RH, Olavesen MG, Waldmann H, and Lachmann PJ. Human

protectin (CD59), an 18,000-20,000 MW complement lysis restricting factor, inhibits C5b-8 catalysed

insertion of C9 into lipid bilayers. Immunology. 71: 1-9. 1990a.

Meri S and Pangburn MK. Discrimination between activators and nonactivators of the alternative

pathway of complement: regulation via a sialic acid/polyanion binding site on factor H. Proc Natl

Acad Sci U S A. 87: 3982-6. 1990b.



Meri S and Pangburn MK. A mechanism of activation of the alternative complement pathway by the

classical pathway: protection of C3b from inactivation by covalent attachment to C4b. Eur J Immunol.

20: 2555-61. 1990c.

Meuer S, Hugli TE, Andreatta RH, Hadding U, and Bitter-Suermann D. Comparative study on

biological activities of various anaphylatoxins (C4a, C3a, C5a). Investigations on their ability to induce

platelet secretion. Inflammation. 5: 263-73. 1981.

Mevorach D, Mascarenhas JO, Gershov D, and Elkon KB. Complement-dependent clearance of

apoptotic cells by human macrophages. J Exp Med. 188: 2313-20. 1998.

Misasi R, Huemer HP, Schwaeble W, Solder E, Larcher C, and Dierich MP. Human complement factor

H: an additional gene product of 43 kDa isolated from human plasma shows cofactor activity for the

cleavage of the third component of complement. Eur J Immunol. 19: 1765-8. 1989.

Mold C, Gewurz H, and Du Clos TW. Regulation of complement activation by C-reactive protein.

Immunopharmacology. 42: 23-30. 1999.

Mold C, Kingzette M, and Gewurz H. C-reactive protein inhibits pneumococcal activation of the

alternative pathway by increasing the interaction between factor H and C3b. J Immunol. 133: 882-5.


Monroe DM, Hoffman M, and Roberts HR. Transmission of a procoagulant signal from tissue factorbearing

cell to platelets. Blood Coagul Fibrinolysis. 7: 459-64. 1996.

Morelli AE, Larregina AT, Shufesky WJ, Zahorchak AF, Logar AJ, Papworth GD, Wang Z, Watkins SC,

Falo LD, Jr., and Thomson AW. Internalization of circulating apoptotic cells by splenic marginal zone

dendritic cells: dependence on complement receptors and effect on cytokine production. Blood. 101:

611-20. 2003.

Mulligan MS, Schmid E, Till GO, Hugli TE, Friedl HP, Roth RA, and Ward PA. C5a-dependent upregulation

in vivo of lung vascular P-selectin. J Immunol. 158: 1857-61. 1997.

Müller-Eberhard HJ. Molecular organization and function of the complement system. Annu Rev

Biochem. 57: 321-47. 1988.

Myones BL, Dalzell JG, Hogg N, and Ross GD. Neutrophil and monocyte cell surface p150,95 has iC3breceptor

(CR4) activity resembling CR3. J Clin Invest. 82: 640-51. 1988.

Nagar B, Jones RG, Diefenbach RJ, Isenman DE, and Rini JM. X-ray crystal structure of C3d: a C3

fragment and ligand for complement receptor 2. Science. 280: 1277-81. 1998.

Nagasawa S and Stroud RM. Mechanism of action of the C3b inactivator: requirement for a high

molecular weight cofactor (C3b-C4bINA cofactor) and production of a new C3b derivative (C3b').

Immunochemistry. 14: 749-56. 1977.

Nauta AJ, Daha MR, Tijsma O, van de Water B, Tedesco F, and Roos A. The membrane attack

complex of complement induces caspase activation and apoptosis. Eur J Immunol. 32: 783-92. 2002.



Nauta AJ, Raaschou-Jensen N, Roos A, Daha MR, Madsen HO, Borrias-Essers MC, Ryder LP, Koch C,

and Garred P. Mannose-binding lectin engagement with late apoptotic and necrotic cells. Eur J

Immunol. 33: 2853-63. 2003.

Neshich G, Togawa RC, Mancini AL, Kuser PR, Yamagishi ME, Pappas G, Jr., Torres WV, Fonseca e

Campos T, Ferreira LL, Luna FM, Oliveira AG, Miura RT, Inoue MK, Horita LG, de Souza DF,

Dominiquini F, Alvaro A, Lima CS, Ogawa FO, Gomes GB, Palandrani JF, dos Santos GF, de Freitas EM,

Mattiuz AR, Costa IC, de Almeida CL, Souza S, Baudet C, and Higa RH. STING Millennium: A webbased

suite of programs for comprehensive and simultaneous analysis of protein structure and

sequence. Nucleic Acids Res. 31: 3386-92. 2003.

Neumann HP, Salzmann M, Bohnert-Iwan B, Mannuelian T, Skerka C, Lenk D, Bender BU, Cybulla M,

Riegler P, Konigsrainer A, Neyer U, Bock A, Widmer U, Male DA, Franke G, and Zipfel PF. Haemolytic

uraemic syndrome and mutations of the factor H gene: a registry-based study of German speaking

countries. Journal of Medical Genetics. 40: 676-81. 2003.

Ng PM, Le Saux A, Lee CM, Tan NS, Lu J, Thiel S, Ho B, and Ding JL. C-reactive protein collaborates

with plasma lectins to boost immune response against bacteria. EMBO J. 26: 3431-40. 2007.

Nicholas KB and Nicholas H.B. J. GeneDoc: Analysis and Visualization of Genetic Variation. 1997.

Nicholson-Weller A, Burge J, Fearon DT, Weller PF, and Austen KF. Isolation of a human erythrocyte

membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement

system. J Immunol. 129: 184-9. 1982.

Noris M, Brioschi S, Caprioli J, Todeschini M, Bresin E, Porrati F, Gamba S, Remuzzi G, International

Registry of R, and Familial HT. Familial haemolytic uraemic syndrome and an MCP mutation.[see

comment]. Lancet. 362: 1542-7. 2003.

Noris M and Remuzzi G. Hemolytic uremic syndrome. J Am Soc Nephrol. 16: 1035-50. 2005.

Noris M and Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med. 361: 1676-87. 2009.

Norman DG, Barlow PN, Baron M, Day AJ, Sim RB, and Campbell ID. Three-dimensional structure of a

complement control protein module in solution. J Mol Biol. 219: 717-25. 1991.

Nürnberger J, Philipp T, Witzke O, Opazo Saez A, Vester U, Baba HA, Kribben A, Zimmerhackl LB,

Janecke AR, Nagel M, and Kirschfink M. Eculizumab for atypical hemolytic-uremic syndrome. N Engl J

Med. 360: 542-4. 2009.

Ogden CA, deCathelineau A, Hoffmann PR, Bratton D, Ghebrehiwet B, Fadok VA, and Henson PM.

C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates

macropinocytosis and uptake of apoptotic cells. J Exp Med. 194: 781-95. 2001.

Okayama M, Oguri K, Fujiwara Y, Nakanishi H, Yonekura H, Kondo T, and Ui N. Purification and

characterization of human platelet proteoglycan. Biochem J. 233: 73-81. 1986.

Okroj M, Heinegard D, Holmdahl R, and Blom AM. Rheumatoid arthritis and the complement system.

Ann Med. 39: 517-30. 2007.



Oohira A, Wight TN, and Bornstein P. Sulfated proteoglycans synthesized by vascular endothelial

cells in culture. J Biol Chem. 258: 2014-21. 1983.

Oppermann M, Manuelian T, Jozsi M, Brandt E, Jokiranta TS, Heinen S, Meri S, Skerka C, Götze O,

and Zipfel PF. The C-terminus of complement regulator Factor H mediates target recognition:

evidence for a compact conformation of the native protein. Clin Exp Immunol. 144: 342-52. 2006.

Ormsby RJ, Jokiranta TS, Duthy TG, Griggs KM, Sadlon TA, Giannakis E, and Gordon DL. Localization

of the third heparin-binding site in the human complement regulator factor H1. Mol Immunol. 43:

1624-32. 2006.

Paidassi H, Tacnet-Delorme P, Garlatti V, Darnault C, Ghebrehiwet B, Gaboriaud C, Arlaud GJ, and

Frachet P. C1q binds phosphatidylserine and likely acts as a multiligand-bridging molecule in

apoptotic cell recognition. J Immunol. 180: 2329-38. 2008.

Pangburn MK. Cutting edge: localization of the host recognition functions of complement factor H at

the carboxyl-terminal: implications for hemolytic uremic syndrome. J Immunol. 169: 4702-6. 2002.

Pangburn MK, Atkinson MA, and Meri S. Localization of the heparin-binding site on complement

factor H. J Biol Chem. 266: 16847-53. 1991.

Pangburn MK, Ferreira VP, and Cortés C. Discrimination between host and pathogens by the

complement system. Vaccine. 26 Suppl 8: I15-21. 2008.

Pangburn MK, Morrison DC, Schreiber RD, and Müller-Eberhard HJ. Activation of the alternative

complement pathway: recognition of surface structures on activators by bound C3b. J Immunol. 124:

977-82. 1980a.

Pangburn MK and Muller-Eberhard HJ. Relation of putative thioester bond in C3 to activation of the

alternative pathway and the binding of C3b to biological targets of complement. J Exp Med. 152:

1102-14. 1980b.

Pangburn MK and Muller-Eberhard HJ. Initiation of the alternative complement pathway due to

spontaneous hydrolysis of the thioester of C3. Ann N Y Acad Sci. 421: 291-8. 1983.

Pangburn MK and Muller-Eberhard HJ. The C3 convertase of the alternative pathway of human

complement. Enzymic properties of the bimolecular proteinase. Biochem J. 235: 723-30. 1986.

Pangburn MK and Müller-Eberhard HJ. Complement C3 convertase: cell surface restriction of beta1H

control and generation of restriction on neuraminidase-treated cells. Proc Natl Acad Sci U S A. 75:

2416-20. 1978.

Pangburn MK, Rawal N, Cortés C, Alam MN, Ferreira VP, and Atkinson MA. Polyanion-induced selfassociation

of complement factor H. J Immunol. 182: 1061-8. 2009.

Pangburn MK, Schreiber RD, and Muller-Eberhard HJ. Formation of the initial C3 convertase of the

alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the

putative thioester in native C3. J Exp Med. 154: 856-67. 1981.



Pangburn MK, Schreiber RD, and Müller-Eberhard HJ. Human complement C3b inactivator: isolation,

characterization, and demonstration of an absolute requirement for the serum protein beta1H for

cleavage of C3b and C4b in solution. J Exp Med. 146: 257-70. 1977.

Pangburn MK, Shreedhar M, Alam N, Rawal N, Herbert AP, and Haque A. Functional analysis of each

of the three C3b binding sites of factor H and comparison with full length factor H. Mol Immunol. 41:

196. 2004.

Pardo-Manuel F, Rey-Campos J, Hillarp A, Dahlbäck B, and Rodriguez de Córdoba S. Human genes for

the alpha and beta chains of complement C4b-binding protein are closely linked in a head-to-tail

arrangement. Proc Natl Acad Sci U S A. 87: 4529-32. 1990.

Peng Q, Li K, Anderson K, Farrar CA, Lu B, Smith RA, Sacks SH, and Zhou W. Local production and

activation of complement up-regulates the allostimulatory function of dendritic cells through C3a-

C3aR interaction. Blood. 111: 2452-61. 2008.

Peng Q, Li K, Patel H, Sacks SH, and Zhou W. Dendritic cell synthesis of C3 is required for full T cell

activation and development of a Th1 phenotype. J Immunol. 176: 3330-41. 2006.

Pepys MB. Role of complement in induction of antibody production in vivo. Effect of cobra factor

and other C3-reactive agents on thymus-dependent and thymus-independent antibody responses. J

Exp Med. 140: 126-45. 1974.

Perera PY, Mayadas TN, Takeuchi O, Akira S, Zaks-Zilberman M, Goyert SM, and Vogel SN.

CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide

and taxol-inducible gene expression. J Immunol. 166: 574-81. 2001.

Pérez-Caballero D, González-Rubio C, Gallardo ME, Vera M, López-Trascasa M, Rodríguez de Córdoba

S, and Sánchez-Corral P. Clustering of missense mutations in the C-terminal region of factor H in

atypical hemolytic uremic syndrome. Am J Hum Genet. 68: 478-84. 2001.

Perkins SJ, Nealis AS, and Sim RB. Oligomeric domain structure of human complement factor H by X-

ray and neutron solution scattering. Biochemistry. 30: 2847-57. 1991.

Persson BA, Jönsson B, and Lund M. Enhanced protein steering: cooperative electrostatic and van

der Waals forces in antigen-antibody complexes. J Phys Chem B. 113: 10459-64. 2009.

Pillemer L, Blum L, Lepow IH, Ross OA, Todd EW, and Wardlaw AC. The properdin system and

immunity. I. Demonstration and isolation of a new serum protein, properdin, and its role in immune

phenomena. Science. 120: 279-85. 1954.

Podack ER, Biesecker G, Kolb WP, and Müller-Eberhard HJ. The C5b-6 complex: reaction with C7, C8,

C9. J Immunol. 121: 484-90. 1978a.

Podack ER, Esser AF, Biesecker G, and Müller-Eberhard HJ. Membrane attack complex of

complement: a structural analysis of its assembly. J Exp Med. 151: 301-13. 1980.

Podack ER and Muller-Eberhard HJ. Isolation of human S-protein, an inhibitor of the membrane

attack complex of complement. J Biol Chem. 254: 9808-14. 1979.



Podack ER and Müller-Eberhard HJ. Binding of desoxycholate, phosphatidylcholine vesicles,

lipoprotein and of the S-protein to complexes of terminal complement components. J Immunol. 121:

1025-30. 1978b.

Podack ER, Preissner KT, and Müller-Eberhard HJ. Inhibition of C9 polymerization within the SC5b-9

complex of complement by S-protein. Acta Pathol Microbiol Immunol Scand Suppl. 284: 89-96.


Podack ER and Tschopp J. Membrane attack by complement. Mol Immunol. 21: 589-603. 1984b.

Podack ER, Tschopp J, and Müller-Eberhard HJ. Molecular organization of C9 within the membrane

attack complex of complement. Induction of circular C9 polymerization by the C5b-8 assembly. J Exp

Med. 156: 268-82. 1982.

Polley MJ and Nachman RL. Human platelet activation by C3a and C3a des-arg. J Exp Med. 158: 603-

15. 1983.

Poon IK, Hulett MD, and Parish CR. Molecular mechanisms of late apoptotic/necrotic cell clearance.

Cell Death Differ. 17: 381-97. 2010.

Pozdnyakova O, Guttormsen HK, Lalani FN, Carroll MC, and Kasper DL. Impaired antibody response

to group B streptococcal type III capsular polysaccharide in C3- and complement receptor 2-deficient

mice. J Immunol. 170: 84-90. 2003.

Presanis JS, Hajela K, Ambrus G, Gál P, and Sim RB. Differential substrate and inhibitor profiles for

human MASP-1 and MASP-2. Mol Immunol. 40: 921-9. 2004.

Prodinger WM, Hellwage J, Spruth M, Dierich MP, and Zipfel PF. The C-terminus of factor H:

monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor

H-related proteins. Biochem J. 331: 41-7. 1998.

Prosser BE, Johnson S, Roversi P, Herbert AP, Blaum BS, Tyrrell J, Jowitt TA, Clark SJ, Tarelli E, Uhrin

D, Barlow PN, Sim RB, Day AJ, and Lea SM. Structural basis for complement factor H linked agerelated

macular degeneration. J Exp Med. 204: 2277-83. 2007.

Pushparaj PN, Tay HK, H'Ng S C, Pitman N, Xu D, McKenzie A, Liew FY, and Melendez AJ. The cytokine

interleukin-33 mediates anaphylactic shock. Proc Natl Acad Sci U S A. 106: 9773-8. 2009.

Pärepalo M. Faktori H:n SCR domeenien 18-20 ekspressointi Pichia pastoris hiivassa. Tekniikka ja

liikenne. Helsingin ammattikorkeakoulu. 2002.

Ram S, Sharma AK, Simpson SD, Gulati S, McQuillen DP, Pangburn MK, and Rice PA. A novel sialic

acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae. J Exp

Med. 187: 743-52. 1998.

Ratnoff OD, Pensky J, Ogston D, and Naff GB. The inhibition of plasmin, plasma kallikrein, plasma

permeability factor, and the C'1r subcomponent of the first component of complement by serum C'1

esterase inhibitor. J Exp Med. 129: 315-31. 1969.

Rawal N and Pangburn MK. Structure/function of C5 convertases of complement. Int

Immunopharmacol. 1: 415-22. 2001.



Rawal N and Pangburn MK. Role of the C3b-binding site on C4b-binding protein in regulating classical

pathway C5 convertase. Mol Immunol. 44: 1105-14. 2007.

Rawala-Sheikh R, Ahmad SS, Ashby B, and Walsh PN. Kinetics of coagulation factor X activation by

platelet-bound factor IXa. Biochemistry. 29: 2606-11. 1990.

Reid KB and Day AJ. Structure-function relationships of the complement components. Immunol

Today. 10: 177-80. 1989.

Reynes M, Aubert JP, Cohen JH, Audouin J, Tricottet V, Diebold J, and Kazatchkine MD. Human

follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens. J Immunol. 135:

2687-94. 1985.

Rezende SM, Simmonds RE, and Lane DA. Coagulation, inflammation, and apoptosis: different roles

for protein S and the protein S-C4b binding protein complex. Blood. 103: 1192-201. 2004.

Richards A, Buddles MR, Donne RL, Kaplan BS, Kirk E, Venning MC, Tielemans CL, Goodship JA, and

Goodship TH. Factor H mutations in hemolytic uremic syndrome cluster in exons 18-20, a domain

important for host cell recognition. Am J Hum Genet. 68: 485-90. 2001.

Richards A, Kemp EJ, Liszewski MK, Goodship JA, Lampe AK, Decorte R, Muslumanoglu MH, Kavukcu

S, Filler G, Pirson Y, Wen LS, Atkinson JP, and Goodship TH. Mutations in human complement

regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic

uremic syndrome. Proc Natl Acad Sci U S A. 100: 12966-71. 2003.

Robey FA, Jones KD, and Steinberg AD. C-reactive protein mediates the solubilization of nuclear DNA

by complement in vitro. J Exp Med. 161: 1344-56. 1985.

Rodríguez de Córdoba S, Diaz-Guillen MA, and Heine-Suner D. An integrated map of the human

regulator of complement activation (RCA) gene cluster on 1q32. Mol Immunol. 36: 803-8. 1999.

Rops AL, van der Vlag J, Jacobs CW, Dijkman HB, Lensen JF, Wijnhoven TJ, van den Heuvel LP, van

Kuppevelt TH, and Berden JH. Isolation and characterization of conditionally immortalized mouse

glomerular endothelial cell lines. Kidney Int. 66: 2193-201. 2004a.

Rops AL, van der Vlag J, Lensen JF, Wijnhoven TJ, van den Heuvel LP, van Kuppevelt TH, and Berden

JH. Heparan sulfate proteoglycans in glomerular inflammation. Kidney Int. 65: 768-85. 2004b.

Ross GD, Cain JA, and Lachmann PJ. Membrane complement receptor type three (CR3) has lectin-like

properties analogous to bovine conglutinin as functions as a receptor for zymosan and rabbit

erythrocytes as well as a receptor for iC3b. J Immunol. 134: 3307-15. 1985.

Ross GD, Lambris JD, Cain JA, and Newman SL. Generation of three different fragments of bound C3

with purified factor I or serum. I. Requirements for factor H vs CR1 cofactor activity. J Immunol. 129:

2051-60. 1982.

Rother K. Leucocyte mobilizing factor: a new biological activity derived from the third component of

complement. Eur J Immunol. 2: 550-8. 1972.



Roumenina LT, Jablonski M, Hue C, Blouin J, Dimitrov JD, Dragon-Durey MA, Cayla M, Fridman WH,

Macher MA, Ribes D, Moulonguet L, Rostaing L, Satchell SC, Mathieson PW, Sautes-Fridman C, Loirat

C, Regnier CH, Halbwachs-Mecarelli L, and Frémeaux-Bacchi V. Hyperfunctional C3 convertase leads

to complement deposition on endothelial cells and contributes to atypical hemolytic uremic

syndrome. Blood. 114: 2837-45. 2009.

Ruggenenti P, Noris M, and Remuzzi G. Thrombotic microangiopathy, hemolytic uremic syndrome,

and thrombotic thrombocytopenic purpura. Kidney Int. 60: 831-46. 2001.

Saadi S, Holzknecht RA, Patte CP, Stern DM, and Platt JL. Complement-mediated regulation of tissue

factor activity in endothelium. J Exp Med. 182: 1807-14. 1995.

Sahu A, Kozel TR, and Pangburn MK. Specificity of the thioester-containing reactive site of human C3

and its significance to complement activation. Biochem J. 302 ( Pt 2): 429-36. 1994.

Sahu A and Pangburn MK. Tyrosine is a potential site for covalent attachment of activated

complement component C3. Mol Immunol. 32: 711-6. 1995.

Sallee M, Daniel L, Piercecchi MD, Jaubert D, Fremeaux-Bacchi V, Berland Y, and Burtey S.

Myocardial infarction is a complication of factor H-associated atypical HUS. Nephrol Dial Transplant.

25: 2028-32. 2010.

Sánchez-Corral P, González-Rubio C, Rodríguez de Córdoba S, and López-Trascasa M. Functional

analysis in serum from atypical Hemolytic Uremic Syndrome patients reveals impaired protection of

host cells associated with mutations in factor H. Mol Immunol. 41: 81-4. 2004.

Sánchez-Corral P, Pérez-Caballero D, Huarte O, Simckes AM, Goicoechea E, López-Trascasa M, and

Rodríguez de Córdoba S. Structural and functional characterization of factor H mutations associated

with atypical hemolytic uremic syndrome. Am J Hum Genet. 71: 1285-95. 2002.

Sato T, Van Dixhoorn MG, Prins FA, Mooney A, Verhagen N, Muizert Y, Savill J, Van Es LA, and Daha

MR. The terminal sequence of complement plays an essential role in antibody-mediated renal cell

apoptosis. J Am Soc Nephrol. 10: 1242-52. 1999.

Savage B, Saldivar E, and Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or

translocation on von Willebrand factor. Cell. 84: 289-97. 1996.

Scharfstein J, Ferreira A, Gigli I, and Nussenzweig V. Human C4-binding protein. I. Isolation and

characterization. J Exp Med. 148: 207-22. 1978.

Schifferli JA, Ng YC, and Peters DK. The role of complement and its receptor in the elimination of

immune complexes. N Engl J Med. 315: 488-95. 1986.

Schleef RR, Bevilacqua MP, Sawdey M, Gimbrone MA, Jr., and Loskutoff DJ. Cytokine activation of

vascular endothelium. Effects on tissue-type plasminogen activator and type 1 plasminogen

activator inhibitor. J Biol Chem. 263: 5797-803. 1988.

Schmidt CQ, Herbert AP, Kavanagh D, Gandy C, Fenton CJ, Blaum BS, Lyon M, Uhrin D, and Barlow

PN. A new map of glycosaminoglycan and C3b binding sites on factor H. J Immunol. 181: 2610-9.




Schreiber RD, Pangburn MK, Lesavre PH, and Muller-Eberhard HJ. Initiation of the alternative

pathway of complement: recognition of activators by bound C3b and assembly of the entire pathway

from six isolated proteins. Proc Natl Acad Sci U S A. 75: 3948-52. 1978.

Schwarzenbacher R, Zeth K, Diederichs K, Gries A, Kostner GM, Laggner P, and Prassl R. Crystal

structure of human beta2-glycoprotein I: implications for phospholipid binding and the

antiphospholipid syndrome. EMBO J. 18: 6228-39. 1999.

Scwaeble W, Zwirner J, Schulz TF, Linke RP, Dierich MP, and Weiss EH. Human complement factor H:

expression of an additional truncated gene product of 43 kDa in human liver. Eur J Immunol. 17:

1485-9. 1987.

Seya T, Holers VM, and Atkinson JP. Purification and functional analysis of the polymorphic variants

of the C3b/C4b receptor (CR1) and comparison with H, C4b-binding protein (C4bp), and decay

accelerating factor (DAF). J Immunol. 135: 2661-7. 1985.

Seya T, Nakamura K, Masaki T, Ichihara-Itoh C, Matsumoto M, and Nagasawa S. Human factor H and

C4b-binding protein serve as factor I-cofactors both encompassing inactivation of C3b and C4b. Mol

Immunol. 32: 355-60. 1995.

Seya T, Turner JR, and Atkinson JP. Purification and characterization of a membrane protein (gp45-

70) that is a cofactor for cleavage of C3b and C4b. J Exp Med. 163: 837-55. 1986.

Sharma AK and Pangburn MK. Identification of three physically and functionally distinct binding sites

for C3b in human complement factor H by deletion mutagenesis. Proc Natl Acad Sci U S A. 93:

10996-1001. 1996.

Shebuski RJ and Kilgore KS. Role of inflammatory mediators in thrombogenesis. J Pharmacol Exp

Ther. 300: 729-35. 2002.

Shimizu A, Masuda Y, Kitamura H, Ishizaki M, Ohashi R, Sugisaki Y, and Yamanaka N. Complementmediated

killing of mesangial cells in experimental glomerulonephritis: cell death by a combination

of apoptosis and necrosis. Nephron. 86: 152-60. 2000.

Shin HS, Snyderman R, Friedman E, Mellors A, and Mayer MM. Chemotactic and anaphylatoxic

fragment cleaved from the fifth component of guinea pig complement. Science. 162: 361-3. 1968.

Shindyalov IN and Bourne PE. Protein structure alignment by incremental combinatorial extension

(CE) of the optimal path. Protein Eng. 11: 739-47. 1998.

Sim RB, Porter RR, Reid KB, and Gigli I. The structure and enzymic activities of the C1r and C1s

subcomponents of C1, the first component of human serum complement. Biochem J. 163: 219-27.


Sim RB, Twose TM, Paterson DS, and Sim E. The covalent-binding reaction of complement

component C3. Biochem J. 193: 115-27. 1981.

Sinha N and Smith-Gill SJ. Electrostatics in protein binding and function. Curr Protein Pept Sci. 3: 601-

14. 2002.



Sjöberg AP, Trouw LA, McGrath FD, Hack CE, and Blom AM. Regulation of complement activation by

C-reactive protein: targeting of the inhibitory activity of C4b-binding protein. J Immunol. 176: 7612-

20. 2006.

Skjøedt MO, Hummelshøj T, Palarasah Y, Honoré C, Koch C, Skjødt K, and Garred P. A novel

mannose-binding lectin/ficolin-associated protein is highly expressed in heart and skeletal muscle

tissues and inhibits complement activation. J Biol Chem. 285: 8234-43. 2010.

Smith BO, Mallin RL, Krych-Goldberg M, Wang X, Hauhart RE, Bromek K, Uhrin D, Atkinson JP, and

Barlow PN. Structure of the C3b binding site of CR1 (CD35), the immune adherence receptor. Cell.

108: 769-80. 2002.

Smith RF, Wiese BA, Wojzynski MK, Davison DB, and Worley KC. BCM Search Launcher--an

integrated interface to molecular biology data base search and analysis services available on the

World Wide Web. Genome Res. 6: 454-62. 1996.

Smith RJ, Alexander J, Barlow PN, Botto M, Cassavant TL, Cook HT, de Cordoba SR, Hageman GS,

Jokiranta TS, Kimberling WJ, Lambris JD, Lanning LD, Levidiotis V, Licht C, Lutz HU, Meri S, Pickering

MC, Quigg RJ, Rops AL, Salant DJ, Sethi S, Thurman JM, Tully HF, Tully SP, van der Vlag J, Walker PD,

Wurzner R, and Zipfel PF. New approaches to the treatment of dense deposit disease. J Am Soc

Nephrol. 18: 2447-56. 2007.

Snyderman R, Phillips J, and Mergenhagen SE. Polymorphonuclear leukocyte chemotactic activity in

rabbit serum and Guinea pig serum treated with immune complexes: evidence for c5a as the major

chemotactic factor. Infect Immun. 1: 521-5. 1970.

Snyderman R, Pike MC, McCarley D, and Lang L. Quantification of mouse macrophage chemotaxis in

vitro: role of C5 for the production of chemotactic activity. Infect Immun. 11: 488-92. 1975.

Spath P and Gabl F. Critical role of the conversion of the third complement component C3 (beta

1C/beta 1A) for its immunochemical quantitation. Clin Chim Acta. 73: 171-5. 1976.

Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep

paradigm. Cell. 76: 301-14. 1994.

Strainic MG, Liu J, Huang D, An F, Lalli PN, Muqim N, Shapiro VS, Dubyak GR, Heeger PS, and Medof

ME. Locally produced complement fragments C5a and C3a provide both costimulatory and survival

signals to naive CD4+ T cells. Immunity. 28: 425-35. 2008.

Strang CJ, Siegel RC, Phillips ML, Poon PH, and Schumaker VN. Ultrastructure of the first component

of human complement: electron microscopy of the crosslinked complex. Proc Natl Acad Sci U S A. 79:

586-90. 1982.

Stuart LM and Ezekowitz RA. Phagocytosis: elegant complexity. Immunity. 22: 539-50. 2005.

Ståhl AL, Vaziri-Sani F, Heinen S, Kristoffersson AC, Gydell KH, Raafat R, Gutierrez A, Beringer O,

Zipfel PF, and Karpman D. Factor H dysfunction in patients with atypical hemolytic uremic syndrome

contributes to complement deposition on platelets and their activation. Blood. 111: 5307-15. 2008.



Suankratay C, Zhang XH, Zhang Y, Lint TF, and Gewurz H. Requirement for the alternative pathway as

well as C4 and C2 in complement-dependent hemolysis via the lectin pathway. J Immunol. 160:

3006-13. 1998.

Sullivan M, Erlic Z, Hoffmann MM, Arbeiter K, Patzer L, Budde K, Hoppe B, Zeier M, Lhotta K, Rybicki

LA, Bock A, Berisha G, and Neumann HP. Epidemiological approach to identifying genetic

predispositions for atypical hemolytic uremic syndrome. Ann Hum Genet. 74: 17-26. 2010.

Szakonyi G, Guthridge JM, Li D, Young K, Holers M, and Chen XS. Structure of complement receptor 2

in complex with its C3d ligand. Science. 292: 1725-8. 2001.

Söderholm H, Lehtinen MJ, Koskinen A, and Jokiranta TS. Impaired protection of erythrocytes from

complement attack in aHUS can be caused by C-terminal mutations of factor H. Mol Immunol. 46:

2868-. 2009.

Tack BF, Harrison RA, Janatova J, Thomas ML, and Prahl JW. Evidence for presence of an internal

thiolester bond in 3rd componenent of human complement. Proceedings of the National Academy of

Sciences of the United States of America-Biological Sciences. 77: 5764-8. 1980.

Takabayashi T, Vannier E, Clark BD, Margolis NH, Dinarello CA, Burke JF, and Gelfand JA. A new

biologic role for C3a and C3a desArg: regulation of TNF-alpha and IL-1 beta synthesis. J Immunol.

156: 3455-60. 1996.

Takahashi M, Iwaki D, Kanno K, Ishida Y, Xiong J, Matsushita M, Endo Y, Miura S, Ishii N, Sugamura K,

and Fujita T. Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to

activation of the lectin complement pathway. J Immunol. 180: 6132-8. 2008.

Takata Y, Kinoshita T, Kozono H, Takeda J, Tanaka E, Hong K, and Inoue K. Covalent association of

C3b with C4b within C5 convertase of the classical complement pathway. J Exp Med. 165: 1494-507.


Takayama Y, Takada F, Takahashi A, and Kawakami M. A 100-kDa protein in the C4-activating

component of Ra-reactive factor is a new serine protease having module organization similar to C1r

and C1s. J Immunol. 152: 2308-16. 1994.

Takizawa F, Tsuji S, and Nagasawa S. Enhancement of macrophage phagocytosis upon iC3b

deposition on apoptotic cells. FEBS Lett. 397: 269-72. 1996.

Tedesco F, Pausa M, Nardon E, Introna M, Mantovani A, and Dobrina A. The cytolytically inactive

terminal complement complex activates endothelial cells to express adhesion molecules and tissue

factor procoagulant activity. J Exp Med. 185: 1619-27. 1997.

Thiel S. Complement activating soluble pattern recognition molecules with collagen-like regions,

mannan-binding lectin, ficolins and associated proteins. Mol Immunol. 44: 3875-88. 2007.

Thiel S, Vorup-Jensen T, Stover CM, Schwaeble W, Laursen SB, Poulsen K, Willis AC, Eggleton P,

Hansen S, Holmskov U, Reid KB, and Jensenius JC. A second serine protease associated with mannanbinding

lectin that activates complement. Nature. 386: 506-10. 1997.



Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, and Higgins DG. The CLUSTAL_X windows

interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic

Acids Res. 25: 4876-82. 1997.

Thompson JD, Higgins DG, and Gibson TJ. CLUSTAL W: improving the sensitivity of progressive

multiple sequence alignment through sequence weighting, position-specific gap penalties and

weight matrix choice. Nucleic Acids Res. 22: 4673-80. 1994.

Thornton BP, Vetvicka V, Pitman M, Goldman RC, and Ross GD. Analysis of the sugar specificity and

molecular location of the beta-glucan-binding lectin site of complement receptor type 3

(CD11b/CD18). J Immunol. 156: 1235-46. 1996.

Trouw LA, Bengtsson AA, Gelderman KA, Dahlbäck B, Sturfelt G, and Blom AM. C4b-binding protein

and factor H compensate for the loss of membrane-bound complement inhibitors to protect

apoptotic cells against excessive complement attack. J Biol Chem. 282: 28540-8. 2007.

Tschopp J, Chonn A, Hertig S, and French LE. Clusterin, the human apolipoprotein and complement

inhibitor, binds to complement C7, C8 beta, and the b domain of C9. J Immunol. 151: 2159-65. 1993.

Tschopp J, Müller-Eberhard HJ, and Podack ER. Formation of transmembrane tubules by

spontaneous polymerization of the hydrophilic complement protein C9. Nature. 298: 534-8. 1982.

Uhrinova S, Lin F, Ball G, Bromek K, Uhrin D, Medof ME, and Barlow PN. Solution structure of a

functionally active fragment of decay-accelerating factor. Proc Natl Acad Sci U S A. 100: 4718-23.


Underhill DM and Ozinsky A. Phagocytosis of microbes: complexity in action. Annu Rev Immunol. 20:

825-52. 2002.

Wallis R, Mitchell DA, Schmid R, Schwaeble WJ, and Keeble AH. Paths reunited: Initiation of the

classical and lectin pathways of complement activation. Immunobiology. 215: 1-11. 2010.

Walters MD, Levin M, Smith C, Nokes TJ, Hardisty RM, Dillon MJ, and Barratt TM. Intravascular

platelet activation in the hemolytic uremic syndrome. Kidney Int. 33: 107-15. 1988.

van den Heuvel LP, Veerkamp JH, Monnens LA, and Schroder CH. Heparan sulfate proteoglycan from

human and equine glomeruli and tubules. Int J Biochem. 20: 1391-400. 1988.

Varani J, Ginsburg I, Schuger L, Gibbs DF, Bromberg J, Johnson KJ, Ryan US, and Ward PA. Endothelial

cell killing by neutrophils. Synergistic interaction of oxygen products and proteases. Am J Pathol.

135: 435-8. 1989.

Ward JV and Packham MA. Characterization of the sulfated glycosaminoglycan on the surface and in

the storage granules of rabbit platelets. Biochim Biophys Acta. 583: 196-207. 1979.

Varki A. Sialic acids in human health and disease. Trends Mol Med. 14: 351-60. 2008.

Warwicker P, Goodship TH, Donne RL, Pirson Y, Nicholls A, Ward RM, Turnpenny P, and Goodship JA.

Genetic studies into inherited and sporadic hemolytic uremic syndrome. Kidney Int. 53: 836-44.




Vaziri-Sani F, Hellwage J, Zipfel PF, Sjöholm AG, Iancu R, and Karpman D. Factor H binds to washed

human platelets. J Thromb Haemos. 3: 154-62. 2005.

Vaziri-Sani F, Holmberg L, Sjöholm AG, Kristoffersson AC, Manea M, Frémeaux-Bacchi V, Fehrman-

Ekholm I, Raafat R, and Karpman D. Phenotypic expression of factor H mutations in patients with

atypical hemolytic uremic syndrome. Kidney Int. 69: 981-8. 2006.

Weiler JM, Daha RM, Austen KF, and Fearon DT. Control of the amplification convertase of

complement by the plasma protein beta 1H. Proc Natl Acad Sci U S A. 73: 3268-72. 1976.

Weinbaum S, Tarbell JM, and Damiano ER. The structure and function of the endothelial glycocalyx

layer. Annu Rev Biomed Eng. 9: 121-67. 2007.

Verbovetski I, Bychkov H, Trahtemberg U, Shapira I, Hareuveni M, Ben-Tal O, Kutikov I, Gill O, and

Mevorach D. Opsonization of apoptotic cells by autologous iC3b facilitates clearance by immature

dendritic cells, down-regulates DR and CD86, and up-regulates CC chemokine receptor 7. J Exp Med.

196: 1553-61. 2002.

Westra D, Volokhina E, van der Heijden E, Vos A, Huigen M, Jansen J, van Kaauwen E, van der Velden

T, van de Kar N, and van den Heuvel L. Genetic disorders in complement (regulating) genes in

patients with atypical haemolytic uraemic syndrome (aHUS). Nephrol Dial Transplant. 25: 2195-202.


Whaley K and Ruddy S. Modulation of the alternative complement pathways by beta 1 H globulin. J

Exp Med. 144: 1147-63. 1976.

Wiedmer T, Esmon CT, and Sims PJ. Complement proteins C5b-9 stimulate procoagulant activity

through platelet prothrombinase. Blood. 68: 875-80. 1986.

Williams P, Chaudhry Y, Goodfellow IG, Billington J, Powell R, Spiller OB, Evans DJ, and Lea S.

Mapping CD55 function. The structure of two pathogen-binding domains at 1.7 A. J Biol Chem. 278:

10691-6. 2003.

Wright SD and Griffin FM, Jr. Activation of phagocytic cells' C3 receptors for phagocytosis. J Leukoc

Biol. 38: 327-39. 1985.

Wright SD, Levin SM, Jong MT, Chad Z, and Kabbash LG. CR3 (CD11b/CD18) expresses one binding

site for Arg-Gly-Asp-containing peptides and a second site for bacterial lipopolysaccharide. J Exp

Med. 169: 175-83. 1989.

Wright SD and Silverstein SC. Receptors for C3b and C3bi promote phagocytosis but not the release

of toxic oxygen from human phagocytes. J Exp Med. 158: 2016-23. 1983.

Wu J, Wu YQ, Ricklin D, Janssen BJ, Lambris JD, and Gros P. Structure of complement fragment C3bfactor

H and implications for host protection by complement regulators. Nat Immunol. 10: 728-33.


Ye J, Esmon NL, Esmon CT, and Johnson AE. The active site of thrombin is altered upon binding to

thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one

correlates with protein C activation. J Biol Chem. 266: 23016-21. 1991.



Ying L, Katz Y, Schlesinger M, Carmi R, Shalev H, Haider N, Beck G, Sheffield VC, and Landau D.

Complement factor H gene mutation associated with autosomal recessive atypical hemolytic uremic

syndrome. Am J Hum Genet. 65: 1538-46. 1999.

Zhang J, Koh J, Lu J, Thiel S, Leong BS, Sethi S, He CY, Ho B, and Ding JL. Local inflammation induces

complement crosstalk which amplifies the antimicrobial response. PLoS Pathog. 5: e1000282. 2009.

Zhang X, Kimura Y, Fang C, Zhou L, Sfyroera G, Lambris JD, Wetsel RA, Miwa T, and Song WC.

Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo. Blood. 110:

228-36. 2007.

Zhou MJ, Lublin DM, Link DC, and Brown EJ. Distinct tyrosine kinase activation and Triton X-100

insolubility upon Fc gamma RII or Fc gamma RIIIB ligation in human polymorphonuclear leukocytes.

Implications for immune complex activation of the respiratory burst. J Biol Chem. 270: 13553-60.


Ziccardi RJ and Cooper NR. Active disassembly of the first complement component, C-1, by C-1

inactivator. J Immunol. 123: 788-92. 1979.

Zimmerhackl LB, Hofer J, Cortina G, Mark W, Wurzner R, Jungraithmayr TC, Khursigara G, Kliche KO,

and Radauer W. Prophylactic eculizumab after renal transplantation in atypical hemolytic-uremic

syndrome. N Engl J Med. 362: 1746-8. 2010.

Zipfel PF and Skerka C. Complement regulators and inhibitory proteins. Nat Rev Immunol. 9: 729-40.


Zundel S, Cseh S, Lacroix M, Dahl MR, Matsushita M, Andrieu JP, Schwaeble WJ, Jensenius JC, Fujita

T, Arlaud GJ, and Thielens NM. Characterization of recombinant mannan-binding lectin-associated

serine protease (MASP)-3 suggests an activation mechanism different from that of MASP-1 and

MASP-2. J Immunol. 172: 4342-50. 2004.


More magazines by this user
Similar magazines