RESUMO
Serum antibodies that recognize carbohydrate antigens play a fundamental role in immune defense, homeostasis, and autoimmunity. In addition, they serve as potential biomarkers for a variety of medical applications. For most anti-glycan antibodies found in human serum, however, the origins, regulation, and biological significance are not well understood. Antibody subpopulations that are relevant to a particular biological process or disease are often difficult to identify from the myriad of anti-glycan antibodies present in human serum. While prior studies have examined anti-glycan IgG and/or IgM repertoires, little is known about IgA repertoires or how IgA, IgG, and IgM are related. In this study, we describe the development of a multiplex assay to simultaneously detect IgA, IgG, and IgM on a glycan microarray and its application to studying anti-glycan repertoires in healthy subjects. The multiplex glycan microarray assay revealed unique insights and systems-level relationships that would be difficult to uncover using traditional approaches. In particular, we found that anti-glycan IgA, IgG, and IgM expression levels appear to be tightly regulated, coordinated within individuals, and stable over time. Additionally, our results help define natural fluctuations over time, which is critical for identifying changes that are beyond normal biological variation.
RESUMO
Lectins have been used at length for basic research and clinical applications. New insights into the molecular recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell-based assays, glycan microarrays, and X-ray crystallography to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI-60 panel; in particular, it inhibited growth of melanoma cancer cells (LOX IMVI) by over 95%. BfL is dimeric in solution and highly specific for binding of oligosaccharides and glycopeptides with terminal N-acetylgalactosamine (GalNAc). BfL was found to have especially strong binding (apparent Kd = 0.5-1.0 nm) to the tumor-associated Tn antigen. High-resolution crystal structures were determined for the ligand-free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive analysis of the eight crystal structures and comparison to structures of related lectins revealed several unique features of GalNAc recognition. Of special note, the carboxylate group of Glu126, lining the glycan-binding pocket, forms H-bonds with both the N-acetyl of GalNAc and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any GalNAc-specific lectin. Taken together, the results provide new insights into the molecular recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications. DATABASE: Structural data are available in the PDB under the accession numbers 5T50, 5T52, 5T55, 5T54, 5T5L, 5T5J, 5T5P, and 5T5O.
