Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry.

Abstract

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.

• Authors: Fang Wang, Antonio Nakouzi, Ruth Hogue Angeletti, Arturo Casadevall
• Journal: Analytical biochemistry (Anal Biochem) Vol. 314 Issue 2 Pg. 266-80 (Mar 15 2003) ISSN: 0003-2697 [Print] United States
• PMID: 12654314 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antibodies, Monoclonal
• Glycopeptides
• Immunoglobulin Heavy Chains
• Immunoglobulin Light Chains
• Immunoglobulin M
• Oligosaccharides
• Polysaccharides

Topics

• Amino Acid Sequence
• Animals
• Antibodies, Monoclonal (analysis, chemistry, genetics)
• Binding Sites
• Chromatography, High Pressure Liquid (methods)
• Cryptococcus neoformans (immunology)
• Enzyme-Linked Immunosorbent Assay
• Glycopeptides (analysis, chemistry, genetics)
• Glycosylation
• Immunoglobulin Heavy Chains (chemistry, genetics)
• Immunoglobulin Light Chains (chemistry, genetics)
• Immunoglobulin M (analysis, chemistry, genetics)
• Mice
• Mice, Inbred BALB C
• Molecular Sequence Data
• Oligosaccharides (analysis, chemistry)
• Peptide Mapping
• Polysaccharides (immunology)
• Spectrometry, Mass, Electrospray Ionization (methods)
• Structure-Activity Relationship
• Time Factors