Scientific Report 2006

Molecular Biology

Chemical Glycobiology

J.C. Paulson, O. Blixt, L.K. Allin, H. Andersson-Sand, O.V. Bohorov, B.E. Collins, S. Han, J. Hoffman, D. Lebus, L. Liao, X. Liu, B. Ma, M. O’Reilly, N. Razi, P. Sobieszczuk, L. Stewart, H. Tateno, H. Tian, D. Vasiliu, Y. Zeng

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 mediates cell-cell interactions, endocytosis, and cell signaling. Our multidisciplinary approach is complemented by a diverse group of chemists, biochemists, cell biologists, and molecular biologists.

Biological Roles of Siglecs

The siglecs are a family of 11 sialic acid–binding proteins that function as cell-signaling coreceptors. They are expressed on glial cells and on a variety of leukocytes that mediate acquired and innate immune functions, including B cells, eosinophils, macrophages, dendritic cells, and natural killer cells. Siglecs are a subfamily of the immunoglobulin superfamily that have in common a unique 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 most siglecs contain tyrosine-based inhibitory motifs characteristic of accessory proteins that regulate transmembrane signaling and endocytosis of cell-surface receptor proteins. The diverse specificity for their sialoside ligands and their variable cytoplasmic regulatory elements provide siglecs with attributes for unique roles in the cell-surface biology of each cell that expresses them.

The best understood siglec is CD22 (siglec-2), an accessory molecule of the B-cell receptor (BCR) complex that has both positive and negative effects on receptor signaling. The carbohydrate ligand recognized by CD22 is the sequence Siaα2-6Galbβ1-4GlcNAc found on glycoproteins of both B cells (cis ligands) and on cells that interact with B cells (e.g., T cells, trans ligands). Interactions of CD22 with cis or trans ligands regulate aspects of B-cell activation, proliferation, and development.

We found that CD22 is predominately associated with clathrin-coated pits in resting B cells, whereas BCRs are minimally associated with clathrin domains. Mice deficient in the ligand for CD22 have greater colocalization of CD22 and the BCR in fused raft-clathrin domains than do mice that have the ligand, accounting for the immunosuppression in deficient mice. In wild-type mice, after antigen activation, the BCR is endocytosed via raft-clathrin domains, a logical site for the dampening of B-cell signaling by CD22. In resting cells, CD22 undergoes constitutive endocytosis, which can result in internalization of high-affinity ligands of CD22 (Fig. 1).

Fig. 1. Relationship between microdomain localization of the BCR and CD22, a regulator of BCR signaling that binds glycan ligands.

We also study siglec-F (murine) and siglec-8 (human), which are predominately expressed on eosinophils and recognize the sialoside Siaα2-3(6-SO₄⁼)Galβ1-4GlcNAc and are targets for modulating eosinophil activation. Another siglec being actively investigated is myelin-associated glycoprotein (siglec-4). This siglec is expressed on glial cells and recognizes the sialoside Siaα2-3Galβ1-3(Siaα2-6)GalNAc-R found on O-linked glycans of glycoproteins and glycolipids. Functionally, myelin-associated glycoprotein stabilizes interactions between glial cells and axons essential for normal organization of myelin and inhibits axonal regeneration, which is currently a target for pharmaceutical intervention to promote nerve regeneration.

A major barrier to studying the ligand-binding properties of siglecs and their role in siglec biology is the difficulty in creating synthetic probes that compete with endogenous (cis) ligands. Even highly multivalent polymers containing the natural glycan sequence recognized by a siglec will not bind to cells unless cis ligands are first destroyed. However, we found that high-affinity analogs of the natural sialoside ligand of CD22 bind to native B cells and are carried into the cell by receptor-mediated endocytosis. Similar constructs with the ligand of siglec-F are also bound and endocytosed by eosinophils, but by a different endocytic mechanism. We have also developed potent inhibitors of myelin-associated glycoprotein that reverse its ability to block axon growth, and in collaborative studies with R. Schnaar, Johns Hopkins University, Baltimore, Maryland, we are investigating the potential of the inhibitors to promote nerve growth in vivo.

With these successes, we have embarked on a major effort to identify high-affinity analogs of each siglec to produce ligand-based tools to investigate the biological roles of the siglecs in innate and adaptive immunity.

Sialoside Analog Glycan Arrays

We have developed a robotically printed glycan array that displays sialoside analogs to assess the affinity of siglecs for unnatural substituents at the C-9 and C-5 positions of sialic acids. Even in the initial experiments with 65 acyl substituents at the C-9 position of sialic acid, the method was a powerful one for identifying substituents that increase the affinity of siglecs by 100-fold or more (Fig. 2). In collaboration with K.B. Sharpless, Department of Chemistry, we have created another 80 analogs by using by click chemistry to couple members of a library of alkynes to sialosides containing 9-azido-N-acetyl-neuraminic acid. Results from the array can be rapidly assimilated into the synthesis of high-affinity ligands and ligand-based probes of the corresponding siglec by using our flexible chemoenzymatic synthesis strategies.

Fig. 2. Sialoside analog glycan microarray reveals high-affinity ligands for CD22. A, Sialoside ligands of CD22 with amino-terminated linkers are printed on N-hydroxyl succinimide (NHS)–activated glass slides, resulting in a covalent amide bond. B, The natural ligand (3) with various substituents (1, 2, 4, 6) and a nonligand control (5) are printed in 10 replicates at 10 two-fold diluted printing concentrations. Overlay with a fluorescence-labeled CD22-Ig chimera reveals the increased binding to various substituents compared with the natural ligand.

Bioengineering of Cell-Surface Sialosides

Sialic acids with substituents at the C-9 and C-5 positions are readily taken up by cells and incorporated into cell-surface glycans of glycoproteins and glycolipids by the natural glycosylation pathways. Taking advantage of this concept, we developed a novel method for in situ photoaffinity cross-linking of CD22 to its ligands on the same cell (cis) or adjacent cell (trans) by using a 9-aryl-azide-sialic acid. When exposed to ultraviolet light, CD22 is rapidly cross-linked to its cis ligands through protein-glycan covalent bonds (Fig. 3). The striking finding is that in addition to glycan structure, microdomain localization of CD22 strongly influences the glycoprotein ligands that CD22 interacts with. In fact, the predominant cis ligands of CD22 were glycans of neighboring CD22 molecules, showing homomultimeric complexes of CD22 mediated by CD22’s ligand-binding domain.

Another application is to incorporate sialic acid analogs that increase or decrease the affinity of a siglec for its natural ligand to perturb the dynamics of interactions of the siglec with its cis ligand. For example, a 9-biphenylcarboxyl substituent (Fig. 3) increases the affinity for CD22 by 100-fold, resulting in the strong CD22-mediated aggregation of B cells. These basic approaches will be of general value in elucidating the biology of other members of the siglec family.

Fig. 3. Bioengineering of cell-surface glycoproteins to carry substituents at the 9-position of N-acetyl–neuraminic acid that increase affinity (biphenylcarboxyl) or allow in situ photoaffinity cross-linking (9-aryl-azide) of CD22 to its ligands.

Consortium for Functional Glycomics

Members of our laboratory also staff 2 scientific cores for the Consortium for Functional Glycomics, organized to elucidate the mechanisms by which glycan-binding proteins mediate cell communication (http://www.functionalglycomics.org/). In the past year, scientists in the Mouse Transgenics Core, led by Peter Sobieszczuk, created 6 novel mouse strains from C57Bl/6 embryonic stem cells that are deficient in genes for key glycan-binding proteins that affect immune function. Scientists in the Glycan Array Synthesis Core, led by Ola Blixt, have produced a library of synthetic glycans by chemoenzymatic synthesis for use in numerous applications. In addition, scientists in the Scripps DNA Microarray Core, led by Steve Head, designed and conduced investigator-initiated analysis with a custom-based microarray with genes of relevance for the consortium.

A major achievement by staff in the Glycan Array Synthesis Core is the development of the world largest glycan microarray, which currently has more than 300 unique structures, mostly synthetic glycans produced by chemoenzymatic synthesis. Now produced in collaboration with the DNA Microarray Core, the microarray is widely used by investigators around the world to assess the specificity of glycan-binding proteins that mediate a broad scope of biological interactions. In an exemplary collaboration with I.A. Wilson and J. Stevens, Department of Molecular Biology, this array was used to investigate the specificity of the 1918 pandemic influenza and related H1 avian influenza viruses and the more recent avian influenza virus (H5N1) to identify mutations required to switch specificity from avian receptors to human-type receptors.

Publications

Bochner, B.S., Alvarez, R.A., Mehta, P., Bovin, N.V., Blixt, O., White, J.R., Schnaar, R.L. Glycan array screening reveals a candidate ligand for siglec-8. J. Biol. Chem. 280:4307, 2005.

Collins, B.E., Blixt, O., Han, S., Duong, B., Li, H., Nathan, J.K., Bovin, N., Paulson, J.C. High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells. J. Immunol. 177:2994, 2006.

Collins, B.E., Smith, B.A., Bengtson, P., Paulson, J.C. Ablation of CD22 in ligand-deficient mice restores B cell receptor signaling. Nat. Immunol. 7:199, 2006.

Comelli, E.M., Head, S.R., Gilmartin, T., Whisenant, T., Haslam, S.M., North, S.J., Wong, N.K., Kudo, T., Narimatsu, H., Esko, J.D., Drickamer, K., Dell, A., Paulson, J.C. A focused microarray approach to functional glycomics: transcriptional regulation of the glycome. Glycobiology 16:117, 2006.

Comelli, E.M., Sutton-Smtih, M., Yan, Q., Amado, M., Panico, M., Gilmartin, T., Whisenant, T., Lanigan, C.M., Head, S.R., Goldberg, D., Morris, H., Dell, A., Paulson, J.C. Activation of murine CD4⁺ and CD8⁺ T lymphocytes leads to dramatic remodeling of N-linked glycans. J. Immunol. 177:2431, 2006.

Han, S., Collins, B.E., Paulson, J.C. Synthesis of 9-substituted sialic acids as probes for CD22-ligand interactions on B. Oxford University Press, New York, in press. ACS Symposium Series.

Leppanen, A., Stowell, S., Blixt, O., Cummings, R.D. Dimeric galectin-1 binds with high affinity to α2,3-sialylated and non-sialylated terminal N-acetyllactosamine units on surface-bound extended glycans. J. Biol. Chem. 280:5549, 2005.

Paulson, J.C., Blixt, O., Collins, B.E. Sweet spots in functional glycomics. Nat. Chem. Biol. 2:238, 2006.

Raman, R., Raguram, S., Venkataraman, G., Paulson, J.C., Sasisekharan, R.
Glycomics: an integrated systems approach to structure-function relationships of glycans. Nat. Methods 2:817, 2005.

Singh, T., Wu, J.H., Peumans, W.J., Rouge, P., Van Damme, E.J., Alvarez, R.A., Blixt, O., Wu, A.M. Carbohydrate specificity of an insecticidal lectin isolated from the leaves of Glechoma hederacea (ground ivy) towards mammalian glycoconjugates. Biochem. J. 393:331, 2005.

Stevens, J., Blixt, O., Glaser, L., Taubenberger, J.K., Palese, P., Paulson, J.C., Wilson, I.A. Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J. Mol. Biol. 355:1143, 2006.

Stevens, J., Blixt, O., Paulson, J.C., Wilson, I.A. Glycan microarray technologies: tools to survey host specificity of influenza viruses. Nat. Rev. Microbiol. 4:857, 2006.

Stevens, J., Blixt, O., Tumpey, T.M., Taubenberger, J.K., Paulson, J.C., Wilson, I.A. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312:404, 2006.

Taniguchi, N., Nakamura, K., Narimatsu, H., von der Lieth, C.W., Paulson, J.C. Human Disease Glycomics/Proteome Initiative workshop and the 4th HUPO Annual Congress. Proteomics 6:12, 2006.

Tateno, H., Crocker, P.R., Paulson, J.C. Mouse siglec-F and human siglec-8 are functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6′-sulfo-sialyl Lewis X as a preferred glycan ligand. Glycobiology 15:1125, 2005.

van Vliet, S.J., van Liempt, E., Saeland, E., Aarnoudse, C.A., Appelmelk, B., Irimura, T., Geijtenbeek, T.B., Blixt, O., Alvarez, R., van Die, I., van Kooyk, Y. Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells. Int. Immunol. 17:661, 2005.

Vasiliu, D., Razi, N., Zhang, Y., Jacobsen, N., Allin, K., Liu, X., Hoffmann, J., Bohorov, O., Blixt, O. Large-scale chemoenzymatic synthesis of blood group and tumor-associated poly-N-acetyllactosamine antigens. Carbohydr. Res. 3451:1447, 2006.

Westerlind, U., Hagback, P., Tidback, B., Wiik, L., Blixt, O., Razi, N., Norberg, T. Synthesis of deoxy and acylamino derivatives of lactose and use of these for probing the active site of Neisseria meningitidis N-acetylglucosaminyltransferase. Carbohydr. Res. 340:221, 2005.