Oligosaccharides of mouse immunoglobulin-M: Structural variations in hybridoma and myeloma cells.
AdvisorGrimes, William J.
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PublisherThe University of Arizona.
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AbstractMany protein-linked oligosaccharides are believed to impart biological specificities to the molecules. The knowledge of detailed structural characteristics of oligosaccharides is essential for understanding their functions. In order to develop methodology for characterization of oligosaccharides of glycoproteins, and to compare glycosylation patterns of different immunoglobulins, oligosaccharides of IgM from two cell lines, MOPC 104E and PC 700, were analyzed. Homogeneous preparations of glycopeptides carrying individual glycosylation sites of the heavy chain were obtained from the two IgM's. The oligosaccharides of these glycopeptides were prepared by hydrazinolysis, and fractionated by HPLC under conditions that resolve oligosaccharides by charge and size, and by affinity chromatography on Concavalin A-Sepharose. Structures of some of these oligosaccharides were determined by 400 MHz NMR spectroscopy. HPLC fractionation by charge resolved oligosaccharides with zero, one, two, and three sialic acids. As indicated by HPLC analyses, oligosaccharides at all the glycosylation sites of both the IgM's were highly heterogeneous. A comparative study on oligosaccharides prepared by peptide-N-glycosidase F digestion of glycopeptides showed a similar degree of heterogeneity. Therefore, it was concluded that the observed heterogeneity of oligosaccharides was not an artefact caused by hydrazinolysis. Major differences between the glycosylation patterns of the two IgM's were evident from analyses of the oligosaccharides by both chromatographic techniques and NMR spectroscopy. MOPC IgM contained a high proportion of sialylated oligosaccharides when compared to PC IgM. Furthermore, the major oligosaccharide structures of MOPC IgM were of triantennary type whereas PC IgM contained biantennary oligosaccharides as its major species. In both the IgM's, a decreased trend of oligosaccharide processing was observed from the N-terminus to the C-terminus.