Characterization of the glycosylation of a human IgM produced by a human-mouse hybridoma.
We analysed the oligosaccharides of a human IgM produced by a human-human-mouse hybridoma at each of its five conserved heavy chain glycosylation sites. Consistent with previous reports, this IgM possesses sialylated oligosaccharides at Asn171, Asn332 and Asn395, and high-mannose-type oligosaccharides at Asn402. In contrast to previous reports for human IgMs, we find that Asn563 is not occupied by oligosaccharide on perhaps 25% of IgM heavy chains, while occupied Asn563 sites contain both high-mannose-type and sialylated oligosaccharides. These latter results are consistent with the glycosylation at Asn563 previously reported for the mouse MOPC 104E IgM. We demonstrate that both the human hybridoma IgM and the mouse MOPC 104E IgM are mixtures of pentamers and hexamers, raising the possibility that the unique findings concerning the glycosylation at Asn563 in this study and the previous study of the MOPC 104E IgM could be related, at least in part, to the different packing requirements of the hexameric geometry and the accessibility of oligosaccharides in the hexameric geometry for processing to complex type. In addition, we used high-pH anion-exchange (HPAE) chromatography, neutral anion-exchange chromatography, fluorophore-assisted carbohydrate electrophoresis and Western blots to compare the oligosaccharide compositions of the human hybridoma IgM, pooled human serum IgM and two mouse monoclonal IgMs (MOPC 104E and TEPC 183). Of note is the presence of N-glycolylneuraminic acid (NeuGc) and N-acetylneuraminic acid (NeuAc) at a 2:1 ratio in the oligosaccharides of the human hybridoma IgM. The presence of both NeuGc and NeuAc complicates the interpretation of HPAE chromatographs.
Monica
T J
TJ
Department of Chemical Engineering, Stanford University, CA 94305-5025, USA.
Williams
S B
SB
Goochee
C F
CF
Maiorella
B L
BL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007075:Immunoglobulin M / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D009439:Neuraminidase; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D007075:Immunoglobulin M; D009844:Oligosaccharides; D009439:Neuraminidase