Scientific Report 2005

Molecular Biology

Chemical Glycobiology in the Immune System

J.C. Paulson, P. Bengtson, O. Blixt, B.E. Collins, S. Han, T. Islam, H. Tateno, Q. Yan

We investigate the roles of glycan-binding proteins that mediate cellular processes central to immunoregulation and human disease. We work at the interface of biology and chemistry to understand how the interaction of glycan-binding proteins with their ligands modulates the functions of the proteins 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-aryl-azide-sialic acid that is taken up by cells and incorporated into cell-surface glycoproteins, allowing the glycoproteins to be cross-linked to CD22 upon exposure to ultraviolet light (Fig. 1).

Fig. 1. Bioengineering of cell-surface glycoproteins to carry 9-aryl-azide-sialic acids for in situ photoaffinity cross-linking of CD22 to its ligands.

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.

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 transferase genes, and in collaboration with A. Dell, Imperial College London, London, England, we correlated dramatic changes in gene expression with changes in the glycan profiles of the resting and activated B and T cells.

Publications

Amado, M., Yan, Q., Comelli, E.M., Collins, B.D. Paulson, J.C. Peanut agglutinin high phenotype of activated CD8⁺ T cells results from de novo synthesis of CD45 glycans. J. Biol. Chem. 279:36689, 2004.

Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan, M.C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D.J., Skehel, J.J., van Die, I., Burton, D.R., Wilson, I.A., Cummings, R., Bovin, N., Wong, C-H., Paulson, J.C. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U. S. A. 101:17033, 2004.

Blixt, O., Vasiliu, D., Allin, K., Jacobsen, N., Warnock, D., Razi, N., Paulson, J.C., Bernatchez, S., Gilbert, M., Wakarchuk, W. Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a. Carbohydr. Res. 340:1963, 2005.

Bryan, M.C., Fazio, F., Lee, H.-K., Huang, C.-Y., Chang, A., Best, M.D., Calarese, D.A., Blixt, O., Paulson, J.C., Burton, D., Wilson, I.A., Wong, C.-H. Covalent display of oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 126:8640, 2004.

Collins, B.E., Paulson, J.C. Cell surface biology mediated by low affinity multivalent protein-glycan interactions. Curr. Opin. Chem. Biol. 8:617, 2004.

Goldberg, D., Sutton-Smith, M., Paulson, J.C., Dell, A. Automatic annotation of matrix-assisted laser desorption/ionization N-glycan spectra. Proteomics 5:865, 2005.

Han, S., Collins, B.E., Bengtson, P., Paulson, J.C. Homomultimeric complexes of CD22 in B cells revealed by protein-glycan cross-linking. Nat. Chem. Biol. 1:93, 2005.

Ikehara, Y., Ikehara, S.K., Paulson, J.C. Negative regulation of T cell receptor signaling by Siglec-7 (p70/AIRM) and Siglec-9. J. Biol. Chem. 279:43117, 2004.

Kitazume, S., Nakagawa, K., Oka, R., Tachida, Y., Ogawa, K., Luo, Y., Citron, M., Shitara, H., Taya, C., Yonekawa, H., Paulson, J.C., Miyoshi, E., Taniguchi, N., Hashimoto, Y. In vivo cleavage of α2,6-sialyltransferase by Alzheimer’s β-secretase. J. Biol. Chem. 280:8589, 2005.

Vyas, A.A., Blixt, O., Paulson, J.C., Schnaar, R.L. Potent glycan inhibitors of myelin-associated glycoprotein enhance axon outgrowth in vitro. J. Biol. Chem. 280:16305, 2005.