Microarray based strategy for functional analyses of complex cellular glycomes.
- Identifies functionally relevant glycans.
- Allows focused structural analyses of biologically important glycans.
- New approach to label glycosphingolipids that retains their protein binding.
Glycans mediate crucial cell-cell, cell-protein and cell-lipid interactions. Recognition and binding of glycans by glycan binding proteins (GBPs) is central to a variety of biological processes, such as movement and trafficking of white blood cells in inflammation and host pathogen recognition. In addition, many glycans are bound by antibodies and this is important in host defense as well as autoimmune disorders. Identifying functional glycans as ligands for GBPs, as well as antigenic glycans, and characterizing the highly complex cellular glycomes remains extremely challenging. The technology developed at Emory can be used to analyze glycans released from glycoproteins and cellular glycosphingolipids (GSLs) in a shotgun format. These isolated and purified glycans can then be used for identification of GBPs, and thereby define functionally relevant glycans. This strategy allows focused structural analyses on biologically important glycans, and will be especially useful in developing glycan-based therapeutics and diagnostics.
Researchers at Emory University have developed a novel shotgun glycomics strategy that allows identification of functional glycans derivatized from GSLs as ligands for GBPs. Derivatization of glycans from GSLs for functional glycomics is complicated since enzymatic release of glycans from GSLs might compromise their GBP binding. This new approach fluorescently labels GSLs that permits easy derivatization, quantification, and separation by HPLC and immobilization to glass slides to generate GSL shotgun microarrays. Probing of these microarrays by GBPs and antibodies identifies functional or antigens GSLs, which can then be characterized by mass spectrometry. Glycoprotein derived glycans could also potentially be analyzed using this technique.
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Anti-glycolipid antibodies have been identified in sera from patients with Lyme disease, and GSL microarrays and tagged glycan libraries (TGLs) have been prepared from human erythrocytes and PC3 cells.