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Research Chemical glycobiology Our group investigates the roles of glycan binding proteins that mediate cellular processes central to immune regulation 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 multi-disciplinary 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 thirteen sialic acid binding proteins that function as cell signaling co-receptors and are primarily expressed on various leukocytes that mediate acquired and innate immune functions including B cells, eosinophils, macrophages, dendritic cells and NK cells. Siglecs are a subfamily of the immunoglobulin superfamily that have a unique N-terminal Ig domain that binds sialic acid containing carbohydrate groups (sialosides) of glycoproteins and glycolipids as ligands. The cytoplasmic domains of most siglecs contain tyrosine based 'ITIM' motifs characteristic of accessory proteins that regulate trans-membrane signaling and endocytosis of cell surface receptor proteins. The diverse specificity for their sialoside ligands and 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 is CD22 (Siglec-2), an accessory molecule of the B-cell receptor complex (BCR) that exhibits both positive and negative effects on receptor signaling.  .
The carbohydrate ligand recognized by CD22 is the sequence Siaa2-6Galb1-4GlcNAc found on both neighboring glycoproteins of the same B cell (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 have shown that CD22 is predominately associated with clathrin-coated pits in resting B cells while the BCR is minimally associated with clathrin domains. In mice deficient in the ligand for CD22 there is greater co-localization of CD22 and the BCR in fused raft-clathrin domains, accounting for the immuno-suppression in these mice. Following antigen activation in wild type mice, 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 we have shown can result in internalization of high affinity ligands of CD22 (Figure 1).
A major barrier to studying the ligand binding properties of siglecs and their role in siglec biology is the creation of synthetic probes that compete with endogenous (cis) ) ligands. Only highly multivalent polymers containing high affinity ligands will compete with the abundant natural cis ligands. A major focus is investigation of multivalent protein scaffolds that provide favorable geometry for presentation of bi-functional ligands to cell surface siglecs. In this regard, we collaborate with the M.G. Finn laboratory to use of viral capsids that can be functionalized to carry variable numbers of synthetic siglec ligands with controled geometry. IIn a recent development we have found that bi-functional molecules comprising a high affinity ligand of CD22 (BPCNeuAc) coupled to an antigen (NP) will dock an anti-NP antibody (IgM, IgA or IgG) to CD22 on the surface of B cells. In effect, the antibody serves as a multivalent protein scaffold that promotes spontaneous assembly of an immune complex on the surface of B cells driven by the bi-functional ligand of CD22 (Figure 2).
A major focus is investigation of multivalent protein scaffolds that provide favorable geometry for presentation of bi-functional ligands to cell surface siglecs. In this regard, we are collaborating with the M.G. Finn laboratory on the use of viral capsids that can be functionalized to carry variable numbers of synthetic ligands. We are pursing this finding as an approach to active targeting of B cells for therapy of B cell leukemia and B cell depletion therapy.
Sialoside analog glycan arrays. With the understanding gained from development of ligand based probes of CD22 we have embarked on a major effort to identify high affinity ligand analogs of each siglec to produce ligand-based tools to investigate their biological roles in innate and adaptive immunity. To this end we have developed a robotically printed glycan array displaying sialoside analogs to assess the affinity of siglecs for unnatural substituents at the C-9 and C-5 positions of sialic acids (see also Consortium for Functional Glycomics below). Even in the initial experiments with 65 acyl substituents at the C-9 position of sialic acid, it is proving to be a powerful method for identifying substituents that increase the affinity of the natural ligand for siglecs by 100 fold or more (see example in Figure 3).  Results from the array can be rapidly assimilated into the synthesis of high affinity ligands and ligand based probes of the corresponding siglec using flexible chemo-enzymatic synthesis strategies.
Consortium for Functional Glycomics. Our group also comprises two scientific cores for the Consortium for Functional Glycomics (CFG), organized to elucidate the mechanisms by which glycan binding proteins mediate cell communication (http://www.functionalglycomics.org/). The Mouse Transgenics Core led by Bo Ma has created eight novel knock-out mouse strains from C57Bl/6 ES cells deficient in key glycan binding proteins affecting immune function. The Glycan Array Synthesis Core led by Nahid Razi produces a library of synthetic glycans by chemo-enzymatic synthesis for use in numerous applications. In addition, the TSRI DNA Microarray Core led by Steve Head designed and conducts investigator-initiated analysis on a custom Affimetrix based microarray with genes of relevance for the CFG. A major achievement by the Glycan Array Synthesis Core includes the development of the world largest glycan microarray, which currently has over 400 unique structures, most of which are synthetic glycans produced by chemo-enzymatic synthesis. Now produced in collaboration with the DNA microarray Core at TSRI, it is widely used by investigators around the world to assess the specificity of glycan binding proteins that mediate a broad scope of biological interactions. An exemplary TSRI collaboration with lab of Ian Wilson and has used this array to investigate the specificity of the 1918 pandemic influenza and the more recent avian influenza (H5N1) to identify mutations required to switch specificity from avian receptors (NeuAca2-3Gal) to human type receptors (NeuAca2-6Gal). KEY WORDS: Carbohydrate; Siglec; CD22; lymphocyte; signaling; sialic acid; glycan; macrophage; dendritic cell |
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