Molecular Biology - The Scripps Research Institute

236 MOLECULAR BIOLOGY 2005
teins in cell-cell adhesion and cell signaling. Projects
fall into 2 main areas: (1) functions of glycan-binding
proteins expressed on leukocytes and (2) regulation of
the synthesis of the carbohydrate ligands of the proteins
during leukocyte activation and differentiation. Our multidisciplinary
approach is complemented by a diverse
group of chemists, biochemists, cell biologists, and
molecular biologists.
SIGLEC FAMILY OF CELL ADHESION PROTEINS
A total of 11 human and 8 mouse siglecs have been
identified so far, and most siglecs are expressed on
leukocytes. The siglecs are a subfamily of the immunoglobulin
superfamily. They have variable numbers of
extracellular Ig domains, including a unique, homologous
N-terminal Ig domain that confers the ability to bind
to sialic acid–containing carbohydrate groups (sialosides)
of glycoproteins and glycolipids. The cytoplasmic
domains of the siglecs typically contain one or more
immunoreceptor tyrosine-based inhibitory motifs characteristic
of accessory proteins that regulate transmembrane
signaling of cell-surface receptor proteins.
To dissect the biology of the siglecs, we use novel
carbohydrate probes that modulate the function of the
proteins. We use chemoenzymatic approaches to synthesize
sialoside analogs recognized by siglecs. The
analogs range from potent inhibitors to multivalent
probes of siglec binding to monovalent sialic acid analogs
that can be fed to cells and incorporated into cell-surface
glycoproteins to add chemical functionality or alter the
affinity of sialoside ligands for cell-surface siglecs. Projects
on several members of the siglec family are ongoing.
CD22 (siglec-2) is an accessory molecule of the
B-cell receptor complex; it has both positive and negative
effects on receptor signaling. The carbohydrate
ligand recognized by CD22 is the sequence sialic acid
α-2-6-galactose, which commonly terminates N-linked
carbohydrate groups of glycoproteins. Significantly, ablation
of the gene that encodes β-galactoside α-2,6-sialyltransferase
I, the enzyme responsible for synthesis of
this carbohydrate in mice, causes a marked deficiency
in antibody production in response to vaccination with
T cell–dependent or T cell–independent antigens, establishing
the importance of the ligand in CD22 function.
We developed a novel method for in situ photoaffinity
cross-linking of CD22 to its ligands on the same cell
(cis) or on an adjacent cell (trans); we use a 9-arylazide-sialic
acid that is taken up by cells and incorporated
into cell-surface glycoproteins, allowing the
glycoproteins to be cross-linked to CD22 upon expo-
Published by TSRI Press ®. ©Copyright 2005,
The Scripps Research Institute. All rights reserved.
sure to ultraviolet light (Fig. 1). The striking finding is
that microdomain localization of CD22, not glycan
structure alone, strongly influences the glycoprotein
ligands CD22 interacts with, providing insights into
how glycan ligands influence the function of this molecule.
This basic observation on siglec-ligand interactions
most likely is recapitulated by other members of
the siglec family.
Fig. 1. Bioengineering of cell-surface glycoproteins to carry 9-arylazide-sialic
acids for in situ photoaffinity cross-linking of CD22 to
its ligands.
Other members of the siglec family differ from CD22
both in cellular distribution and in specificity for recognition
of sialic acid–containing oligosaccharides. We are
evaluating the roles of siglec-7 and siglec-9 in regulation
of human T-cell receptor signaling, the role of siglec-F
in the biology of eosinophils in mice, and the role of
myelin-associated glycoprotein (siglec-4) in regulation
of neurite formation. We recently developed a novel
approach in which a robotically printed glycan array is
used for combinatorial assessment of the effects of sialoside
analogs on the affinity of siglecs. This method
should be a rapid one for developing high-affinity sialoside
probes for each of the siglecs to facilitate investigations
into siglec biology.
REGULATION OF LEUKOCYTE GLYCOSYLATION
Activation of lymphocytes and other leukocytes
induces programmed changes in glycosylation. Such
changes regulate leukocyte trafficking and can modulate
the functions of carbohydrate-binding proteins. We
are systematically investigating the changes in glycosylation
that occur in B and T lymphocytes after activation
in order to elucidate the underlying molecular
mechanisms for these changes and their biological relevance.
To this end, in collaboration with S. Head and
the Consortium for Functional Glycomics (http://www
.functionalglycomics.org), we participated in the development
and use of a custom microarray of glycosyl-