Mechanisms and principles of N-linked protein glycosylation - EPFL

Available online at www.sciencedirect.comMechanisms and principles of N-linked protein glycosylationFlavio Schwarz and Markus AebiN-linked glycosylation, a protein modification system present inall domains of life, is characterized by a high structural diversityof N-linked glycans found among different species and by alarge number of proteins that are glycosylated. Based onstructural, functional, and phylogenetic approaches, thisreview discusses the highly conserved processes that are atthe basis of this unique general protein modification system.AddressInstitute of Microbiology, Department of Biology, ETH Zurich, 8093Zurich, SwitzerlandCorresponding author: Aebi, Markus (aebi@micro.biol.ethz.ch)Current Opinion in Structural Biology 2011, 21:576–582This review comes from a themed issue onCarbohydrates and glucoconjugatesEdited by Robert S Haltiwanger and Ten Feizi0959-440X/$ – see front matter# 2011 Elsevier Ltd. All rights reserved.DOI 10.1016/j.sbi.2011.08.005IntroductionAsparagine (N)-linked glycosylation of proteins is a fundamentaland extensive post-translational modificationthat results in the covalent attachment of an oligosaccharideonto asparagine residues of polypeptide chains.This protein modification is found both in eukaryotes andin prokaryotes. Although a number of variations occur,studies in model organisms representing all threedomains of life reveal that three homologous processesrepresent the core of N-glycosylation:1. A glycan is assembled from nucleotide-activatedbuilding blocks on a lipid anchor through the stepwiseincorporation of monosaccharides by various glycosyltransferases.The lipid-linked oligosaccharide (LLO)is then re-oriented from the cytosolic to the luminalside of the eukaryotic ER membrane or of the plasmamembrane in prokaryotes, where it serves as donor forglycosylation. The LLO can be further extended inmany eukaryotes after flipping to the luminal side ofthe ER.2. Proteins with the consensus sequence for glycosylation(N-X-S/T) when translocated to the ER lumen (or tothe periplasm) serve as acceptors.3. The oligosaccharyltransferase (OST) catalyzes the enbloc transfer of the oligosaccharide to the asparagineside chain of the acceptor polypeptides.After being covalently linked to proteins, the N-glycancan be modified in eukaryotes. This sequential processingis coupled to the secretory pathway and results in aspecies-specific or even cell type-specific diversity ofN-linked glycans.Recently, an alternative type of N-glycosylation has beenidentified in a few bacteria [1 ,2,3 ,4]. This novel N-glycosylation pathway takes place in the cytoplasm and itis characterized by the addition of monosaccharides fromnucleotide activated donors to asparagine side chains.Notably, cytosolic N-glycosylation is performed by anenzyme structurally different from OST, but displayingthe same acceptor site specificity N-X-S/T, as in the casefor the conventional N-glycosylation process.This review describes the basic structural concepts of N-glycosylation and places them in a phylogenetic framework.Recent studies have uncovered a substantial diversityamong archaeal and bacterial N-glycans, while at thesame time making available novel features of OST. Thedetection of an alternative pathway for N-glycosylationtaking place in the cytoplasm of bacteria represents anunexpected turn in the field, and raises questions about thedriving forces leading to the N-X-S/T sequon as a generalacceptor for a post-translational modification of proteins.Structure of N-linked glycan precursors:diverse in prokaryotes, conserved ineukaryotesThe oligosaccharide substrate for N-glycosylation isassembled at the membrane of the endoplasmic reticulumin eukaryotes and at the plasma membrane in prokaryotes.Phosphorylated polyisoprenoid lipids ofdifferent chain length and saturation (dolicol or undecaprenol)are invariably used as a molecular dock for oligosaccharidebiosynthesis at membranes. The use of a lipidas a carrier for the activated oligosaccharide has severalconsequences. On the one hand, it leads to an increasedlocal concentration of substrate for the OST. At the sametime, the availability of the lipid carrier regulates the fluxthrough a pathway where the committing and the finalsteps are located in topologically distinct compartments[5,6]. Specific structural properties of the isoprenoidmolecule might also be functional in the LLO translocationprocess across membranes [7,8]. Finally, phosphorylatedlipids function as good leaving groups in the OSTreaction and remain bound to the membrane, where theycan be recycled for further rounds of biosynthesis.In contrast to the conserved lipid part of the LLO, there islittle similarity between the oligosaccharide portionCurrent Opinion in Structural Biology 2011, 21:576–582 www.sciencedirect.com

N-linked protein glycosylation Schwarz and Aebi 577generated in prokaryotes and in eukaryotes. The bacteriumCampylobacter jejuni decorates proteins with a heptasaccharideGlc-GalNAc 5 -Bac (Figure 1) [9], whereasC. lari produces a similar glycan GalNAc 5 -Bac missingthe branching glucose [10]. Helicobacter pullorum synthesizea linear pentasaccharide containing uncharacterizedmonosaccharides of 216 and 217 Da, which are alsodetected in the N-glycan of Wolinella succinogenes[11,12]. Though extensive studies are missing, currentknowledge reveals that an even larger structural diversityis found in the archaea, where glycans can be also modifiedby methylation, sulfation, and addition of amino acids(Figure 1). For instance, Methanococcus voltae builds atrisaccharide composed of ManNAcA6Thr-GlcNAc3-NAcA-GlcNAc [13]. M. maripaludis N-glycans are madeof the tetrasaccharide Sug-ManNAcNAm-GlcNAcNAcA-GalNAc, amidated with a threonine amino group [14].The diversity is additionally increased in Halobacteriumsalinarum, where two different oligosaccharides areassembled either on dolichyl pyrophosphate or dolichylphosphate,using N-acetylhexosamine or hexose as linkingunits, respectively [15]. Notably, dolichyl phosphatelinkedoligosaccharides have been detected exclusively inarchaea to date [15,16 ].By contrast, in the large majority of eukaryotes, a Glc 3 -Man 9 -GlcNAc 2 oligosaccharide is built on a dolichylpyrophosphate carrier by ordered action of glycosyltransferasesof the ALG pathway [5]. Remarkably, some organismsthat branched off early in eukaryotic evolutionproduce smaller versions of this glycan [17].Therefore, it appears that archaeal and bacterial glycanprecursors are heterogeneous, whereas eukaryotes producea conserved lipid-linked oligosaccharide structure(Figure 1). Archaea exhibit the greatest diversity inglycosylation pathways: they use a large pool of buildingblocks, they produce both dolichyl phosphate-linked andpyrophosphate-linked glycans, and they can synthesizemore than one type of LLO in the same cell. Bacteriadisplay a limited diversity, and N-glycosylation is confinedto a small number of species. By contrast, eukaryotespresent a conserved core unit Man 5 -GlcNAc 2 ,extended with luminal added units to Glc 3 -Man 9 -GlcNAc 2 . The remarkable diversity of N-glycans foundamong eukaryotes is a consequence of a successive elaborationtaking place in the late ER and in the Golgi,featuring a different evolutionary origin. The reason forthis convergent process in N-glycosylation is probablybound to the function of the glycan structures and thecompartment where they are localized. In prokaryotes,proteins are modified in the periplasm and maintainedoutside. Instead, the homologous process in eukaryotestakes place in the ER, where it is vitally bound to thefolding events of glycoproteins. The N-glycan is elaboratedonce the glycoprotein is destined to the outside ofthe cell. The resulting high diversity of extracellularglycans can be viewed as the result of similar selectionFigure 1Diversity2 SO 42-CH 3SCH 33 SO2- Thr4SO 3-QBBPPtoPPArchaeaBacteriaEukayotesDolicholUndecaprenolPhosphateGalacturonic acid (GalA)Iduronic acid (IdoA)Glucoronic acid (GlcA)Hexuronic acid (HexA)Galactose (Gal)Glucose (Glc)Mannose (Man)Hexose (Hex)BSQN-Acetylgalactosamine (GalNAc)N-Acetylglucosamine (GlcNAc)N-Acetylmannosamine (ManNAc)N-Acetylhexosamine (HexNAc)2,4-Diacetamido-2,4,6-trideoxy-glucose (Bacillosamine, Bac)2-Acetamido-2,4-dideoxy-5-O-methyl-erythro-hexos-5-ulo-1,5-pyranose (Sug)QuinovoseUncharacterized sugarUncharacterized sugarCurrent Opinion in Structural BiologyN-glycosylation occurs in all domains of life, but the glycan being transferred is different. Prokaryotes synthesize a variety of lipid-linkedoligosaccharides, whereas eukaryotes produce a conserved structure. Representative glycan structures are indicated with colored geometricsymbols, conform to those recommended by the Consortium for Functional Glycomics.www.sciencedirect.com Current Opinion in Structural Biology 2011, 21:576–582

578 Carbohydrates and glucoconjugatesmechanisms acting on two different processes of theN-linked protein glycosylation.The N-X-S/T sequon: a unique acceptorsequence conserved in N-glycosylationThe acceptor substrate of N-glycosylation is an asparagineresidue present within the consensus sequence N-X-S/T [18]. Site occupancy analysis of different modelglycoproteins shows a preference for N-X-T sites overN-X-S [19,20], and reveals that proline is not tolerated inthe second position. Based on these findings, it has beenhypothesized that a specific conformation of the acceptorpeptide is required to increase the nucleophilicity of theamide group of the asparagine [21,22]. These models linkthe generation of the reactive intermediates to the formationof a secondary structure and functionally dependon the presence of the hydroxyl amino acid at the +2position. However, recent experimental data questionthese ideas. Exchange of serine for valine, leucine orasparagine in the sequon N-A-S does not prevent glycosylationof the cell surface glycoprotein from the archaeaHalobacterium halobium [23]. The OST from the bacteriumC. lari is able to modify the N-S-G site of a protein[10]. Four non-consensus sequences are glycosylated inIgG Fc purified from CHO cells [24 ,25 ]. Lastly, N-glycosylations are detected at 24 N-G-X and 68 N-X-Vsites in the mouse glycoproteome [26 ]. Therefore, itappears that the sequence requirements of the acceptorsubstrate are less strict than previously stated. Serine orthreonine in the third position is important, but can bedispensable for the glycosyl transfer reaction mediated bythe OST.Nevertheless, N-glycosylation sequons have to adopt aspecific conformation during the reaction process. Therequirement of flexibility at the N-X-S/T has found amolecular description for the bacterial system, whichrepresents an optimal experimental model as glycosylationtakes place after protein folding [27]. NMR spectroscopyof a protein substrate demonstrates that abacterial glycosylation site is located on moderately flexibleregions, but does not adopt a predefined motif [28].Therefore, a defined secondary structure seems notessential for recognition of the substrate, but it mightbe imposed upon binding to the OST.It is remarkable that the bacterial cytoplasmic N-glycosyltransferase(NGT) HMW1Crecognizes the same consensussequence N-X-S/T [1 ,3 ]. Similar to OST,NGT is an inverting glycosyltransferase and can modifypeptides featuring sequences of eukaryotic glycoproteins(Schwarz, Fan, Schubert, Aebi, manuscript in preparation).Replacement of the asparagine to glutamineor presence of proline in the +1 position impairs glycosylation.NGT does not share sequence identity to theOST, and the two enzymes belong to structurally unrelatedclasses. Therefore, the same substrate requirementsemerged in two independent pathways resulting in analogousenzymatic activity. Considering the hypothesisthat the hydroxyl amino acid is not directly involved inthe catalytic mechanism of OST, it remains unexplainedwhy glycosylation at asparagine side chains, whichevolved independently (at least) twice, favored asparagineversus glutamine, and selected a consensus sequencewith a hydroxyl amino acid at the +2 position relative tothe asparagine.It is evident that a short recognition element is a prerequisitefor the observed evolutionary trends in N-glycanmultiplicity in specific sets of proteins [29,30]. Once theglycosylation machinery is in place, emergence of newsequons on proteins leads to additional N-glycosylations,which are then subject to selection. Given a potentialbeneficial effect of the N-glycan modification, a pathwaywith a short recognition motif is likely to develop into ageneral modification system of proteins. Indeed, analysisof mutations in a set of human genes encoding proteinstransiting trough the secretory pathway illustrates thatmutations resulting in a gain-of-glycosylation phenotypeare over-represented [31].OST specificity defines the complexity of theglycoproteomeThe view that there is a selective advantage of a ‘general’N-glycosylation system is further supported by a phylogeneticanalysis of the central enzyme of the pathway.The Stt3 protein accounts alone for OST activity in someorganisms. In fact, bacterial and archaeal Stt3 homologs(called PglB or AglB) glycosylate proteins and peptides[32,33,34 ,35]. Similarly, some protists express Stt3proteins that are self-sufficient for oligosaccharide transfer[36,37]. By contrast, in complex eukaryotes such asfungi, animals and plants, the OST works as a multiproteincomplex made of up to eight polypeptides, builtas an extension of the Stt3 core [38].In multimeric OSTs, some of the additional subunits finetunethe glycosylation process. Ost1p acts as a chaperon topromote glycosylation of selected protein clients [39–41].Ost3/6p exhibit oxidoreductase activity and bind specificpolypeptides, both non-covalently and via transient disulfidebonds [42]. These subunits are thought to slow downthe early stages of protein folding, thereby maintaining thepolypeptide chain in a glycosylation-competent conformation.Interactions among OST subunits and the transloconmight also increase the versatility of the system byexecuting glycosylation on the nascent polypeptides,before folding of the protein [43]. In this view, some ofthe auxiliary OST subunits enlarge the substrate range ofthe enzyme (Figure 2). Alternatively, other eukaryotesextend their glycosylation ability by duplication of theSTT3 gene and diversification of Stt3 specificity. Trypanosomabrucei Stt3 paralogs exhibit distinct acceptor specificity:TbStt3A is highly selective for acidic peptideCurrent Opinion in Structural Biology 2011, 21:576–582 www.sciencedirect.com

N-linked protein glycosylation Schwarz and Aebi 579Figure 2EukaryotesOST complexMultiple single-subunitsSaccharomyces cerevisiaeOst1pWbp1pSwp1p Stt3pOst3/6pOst5p Ost2p Ost4pTrypanosoma bruceiStt3A Stt3B Stt3CBacteriaSingle-subunitCampylobacterPglBX-X-X-X-N-X-S/T-X sequence modifiedCurrent Opinion in Structural BiologyThe composition of OST correlates to the complexity of the glycoproteome. The x axis describes the range of peptides that can be modified by distinctOSTs. Stt3 acceptor specificity is indicated in green. Campylobacter possesses a single-subunit OST and generally modifies peptides of sequence D/E-X 1 -N-X +1 -S/T. The light green bar represents the relaxed specificity of C. lari OST. T. brucei Stt3 proteins feature distinct, yet overlappingspecificities for the acceptor sequon. In S. cerevisiae, the range of peptides is extended by the distinct activity of OST subunits (indicated in red), whichcan bind specific polypeptides and expose them to the Stt3 catalytic subunit.acceptor sites, TbStt3B has a broad specificity for theacceptor sites, whereas TbStt3C preferentially glycosylatesacidic peptide acceptors [36]. This alternative strategyresults in a proficient system, in which the complexity ofthe glycoproteome is the result of the cumulative activity ofmultiple OSTs (Figure 2). Multiple copies of STT3 genesare also found within bacterial and archaeal genomes[44 ,45]. Interestingly, whereas some STT3 homologs areclosely grouped and probably derive from gene duplicationevents as in protists, others are distantly related. By contrast,for bacterial systems the consensus sequence N-X-S/T is extended at the amino-terminus to present aspartic orglutamic acid in the -2 position relative to the asparagine[10,12,46]. Therefore, bacterial OSTs exhibit a more stringentspecificity for the acceptor site than the eukaryoticcounterpart. This restricts glycosylation to a narrow set ofpolypeptides (Figure 2).In light of the above, it is tempting to speculate thatN-glycosylation is a process that descends from anarchaeal ancestor. N-glycosylation is ubiquitous amongarchaea, where there is the highest diversity of glycanstructures, and multiple N-glycosylation pathways arefound within the same genome. Deep-sea vents mighthave been the environment where the geneticinformation for N-glycosylation was transferred fromthermophilic archaea (usually possessing the glycosylationgenes organized in a cluster) to some e-proteobacteria[44 ,47]. Evident similarities between the lipidcycles that assist peptidoglycan and O-antigen biosynthesisin bacteria and N-glycosylation in archaea madethe functional transfer of this protein modification tobacteria possible [48]. Along the same line of argumentation,the eukaryotic progenitor probably contained aglycosylation machinery of the prokaryotic type, featuringa single subunit OST and the cytosolic segment ofthe LLO biosynthetic pathway. It is this pathway thatmight be the most primitive eukaryotic N-glycosylationsystem. Lower eukaryotes producing smaller versionsof the Glc 3 Man 9 GlcNAc 2 glycan therefore representevolutionary intermediates lacking some of the ERglycosyltransferases. However, alternative views startingfrom a complete ER-glycosylation machinery havebeen proposed [49].A functional view of eukaryotic N-glycanstructureThe biosynthetic route of the eukaryotic LLO is dividedinto two branches (Figure 3). The first part resemblesthat of archaea, as it occurs at the cytosolic side andwww.sciencedirect.com Current Opinion in Structural Biology 2011, 21:576–582

580 Carbohydrates and glucoconjugatesFigure 3Alg3Alg9Alg12Alg6Alg8Alg10NPPPPNPPPAlg7Alg1Alg2Alg11cytosolic GTs ER-resident GTs Golgi-resident GTsancientrecentprotein stabilityquality controlERADcell-cell communicationmodulation of activityCurrent Opinion in Structural BiologyRelating oligosaccharide structures of eukaryotes to biological function. Glycosyltransferases involved in the process of N-glycosylation are divided inthree groups reflecting their location, their phylogenetic origin, and their function in N-glycan biosynthesis. The resulting structures are associated tothe different functions of N-linked glycans.glycosyltransferases build the Man 5 -GlcNAc 2 glycan byusing nucleotide activated monosaccharides. After translocationof LLO to the ER lumen, glycosyltransferases ofa different class complete the glycan structure by utilizingdolichyl phosphate-activated monosaccharides.Notably, these glycosyltransferases seem to share a commonancestor [50], and cluster in the same family ofenzymes that build glycosylphosphatidylinositol (GPI)membrane anchors.Hydrophilic N-glycans are major contributors to thermodynamicstability and solubility of proteins [51,52 ,53].This general effect is not directly related to the glycanstructure, and it is also detectable in organisms where N-glycosylation is not essential for viability, such as archaeaand bacteria [54,55 ].By contrast, the oligosaccharide portions formed in theER are a distant appendix that do not interact with theprotein backbone. Terminal glucoses and mannoses incombination with lectin receptors create a system ableto assist folding of newly synthesized proteins and tosort out and direct misfolded polypeptide to degradation[56]. The ability to control folding of proteinsmight have been the driving force for the evolution ofthe Glc 3 -Man 9 -GlcNAc 2 oligosaccharide and its conservationamong eukaryotes. The selective advantagegiven by this system probably resides in the capacityof producing proteins of increased complexity and ofnovel function.The topological division of the N-glycosylation pathwayinto ER and Golgi marks both a biosynthetic and afunctional difference of oligosaccharides. Golgi glycosyltransferasesbelong to the family of type II membraneproteins and use nucleotide-activated monosaccharidesas donors to elaborate the core glycans into complexstructures. Whereas the transfer of the Glc 3 -Man 9 -GlcNAc 2 occurs en bloc and thus results in homogeneousglycoconjugates, these later additions of monosaccharidesoccur sequentially and allow for a superior flexibilityin the generation of terminal elaborations. This‘explosion of diversity’ [30] occurred in plants andanimals and resulted in a tremendous diversity of carbohydratestructures displayed at the cell surface. Novelfunctions such as cell–cell interaction required in processeslike development or host-pathogen interactionsare built on N-glycan structure [57].Current Opinion in Structural Biology 2011, 21:576–582 www.sciencedirect.com

N-linked protein glycosylation Schwarz and Aebi 581Conclusions and perspectivesThe model that we propose allows for a phylogeneticinterpretation of the N-glycosylation process. This view isbased on a structural framework characterized in differentmodel systems. The better we understand the differentmechanisms of N-linked glycosylation at a molecularlevel, the clearer we can formulate the underlying principles.For instance, determining the catalytic mechanismof OST and NGT will be important to recognize howthese two enzymes activate the poorly reactive amidegroup. These data will also shed light on the drivingforces that selected the N-X-S/T consensus sequence as ageneral recognition element. The definition of the occurrenceof NGT-based N-glycosylation in a proteome willdelineate the molecular constraints that lead on the oneside to a seemingly specific protein modification system(NGT), and on the other side to the unique system wherea single enzyme (OST) can modify thousands of siteswithin a given proteome.AcknowledgementsResearch in the Aebi laboratory is supported by the Swiss National ScienceFoundation and the ETH Zurich.References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:1. of special interest of outstanding interestChoi KJ, Grass S, Paek S, St Geme JW 3rd, Yeo HJ: TheActinobacillus pleuropneumoniae HMW1C-likeglycosyltransferase mediates N-linked glycosylation of theHaemophilus influenzae HMW1 adhesin. PLoS ONE 2010,5:e15888.See reference [3 ].2. 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