title: Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM.
abstract: Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling. Graphical Abstract ᅟ.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.
authors.forename: Edward S X
authors.initials: ES
authors.lastname: Moh
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.|ARC Centre of Excellence in Nanoscale BioPhotonics, Macquarie University, Sydney, NSW, 2109, Australia.
authors.forename: Chi-Hung
authors.initials: CH
authors.lastname: Lin
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia. nicki.packer@mq.edu.au.|ARC Centre of Excellence in Nanoscale BioPhotonics, Macquarie University, Sydney, NSW, 2109, Australia. nicki.packer@mq.edu.au.
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D006020:Glycopeptides; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D007075:Immunoglobulin M / Q000737:chemistry*; D011134:Polysaccharides; D053719:Tandem Mass Spectrometry
chemical_list: D006020:Glycopeptides; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011134:Polysaccharides
keywords: Glycopeptide; Hexamer; IgM; Pentamer; Site-specific glycosylation