Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry.
Immunoglobulin M is an especially important product of the immune system because it plays a critical role in early protection against infections. In this report, the glycosylation pattern of the protective murine monoclonal IgM 12A1 to Cryptococcus neoformans polysaccharide was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Peptide mapping studies covering 88% of the deduced amino acid sequence indicated that of the six potential N-glycosylation sites in this antibody only five were utilized, as the tryptic peptide derived from monoclonal IgM 12A1 containing Asn-260 was recovered without carbohydrates. The oligosaccharide side chains of monoclonal IgM 12A1 were characterized at each of the N-glycosylation sites. Asn-166 possessed 20 monosialylated and nonsialylated, and fucosylated and nonfucosylated complex- and hybrid-type oligosaccharides and one high-mannose-type oligosaccharide. Thirteen oligosaccharides were attached to the site at Asn-401, including six complex-type, four hybrid-type, and three high-mannose-type oligosaccharides. Twelve hybrid-type oligosaccharides were attached to Asn-378, three of which had terminal sialic acids. Eleven hybrid-type oligosaccharides were attached to Asn-331, seven of which had terminal sialic acids. Only two high-mannose type oligosaccharides were attached to Asn-363. These results indicated great complexity in the structure and composition of oligosaccharides attached to individual IgM glycosylation sites.
Wang
Fang
F
Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. fawang@aecom.yu.edu
Nakouzi
Antonio
A
Angeletti
Ruth Hogue
RH
Casadevall
Arturo
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003455:Cryptococcus neoformans / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D007075:Immunoglobulin M / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry*; D010449:Peptide Mapping; D011134:Polysaccharides / Q000276:immunology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013329:Structure-Activity Relationship; D013997:Time Factors
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007075:Immunoglobulin M; D009844:Oligosaccharides; D011134:Polysaccharides