Stable oligosaccharide microheterogeneity at individual glycosylation sites of a murine major histocompatibility antigen derived from a B-cell lymphoma.
The H-2Kk glycoprotein has been isolated by monoclonal antibody affinity chromatography, and an analysis of the asparagine-linked oligosaccharides present at the two major glycosylation sites has been performed. Antigen obtained from the AKTB-1b B-cell lymphoma that had been labeled with [2,6-3H]mannose for 5 or 21 h or for 5 h followed by a 5-h chase was digested exhaustively with trypsin. Each glycosylation site was then isolated by reverse phase high performance liquid chromatography using a C18 column. After removal from the peptide backbone by the almond emulsin peptide: N-glycosidase, the oligosaccharides from each isolated site were analyzed by gel filtration, ion exchange chromatography, concanavalin A affinity chromatography, and glycosidase treatment to assess the contribution of sialic acid and branching patterns of the oligosaccharide backbones to the overall microheterogeneity. The glycosylation of the H-2Kk antigen derived from several different AKTB-1b tumor preparations was examined during a period covering 1 year, during which time the tumor was passaged continuously in vivo in 2-week cycles. Our results conclusively demonstrate that the pattern of oligosaccharide microheterogeneity at the two glycosylation sites of the H-2Kk antigen derived from AKTB-1b cells is stable and that each site differs as to the specific array of oligosaccharide types found on the fully processed glycoprotein. In addition, this report describes an analytical scheme employing reverse phase high performance liquid chromatography to follow oligosaccharide processing and hydrolysis of the N-glycosidic bond by the peptide: N-glycosidase.
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D006020:Glycopeptides / Q000032:analysis; D006183:H-2 Antigens / Q000302:isolation & purification*; D008223:Lymphoma / Q000276:immunology*; D008285:Major Histocompatibility Complex; D051379:Mice; D008806:Mice, Inbred AKR; D009844:Oligosaccharides / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D006020:Glycopeptides; D006183:H-2 Antigens; C024755:H-2K(K) antigen; D009844:Oligosaccharides; D010446:Peptide Fragments; D014357:Trypsin