Site-specific N-glycosylation of recombinant pentameric and hexameric human IgM

Edward S X Moh, Chi Hung Lin, Morten Thaysen-Andersen, Nicolle H. Packer*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Citations (Scopus)

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. [Figure not available: see fulltext.]

Original languageEnglish
Pages (from-to)1143-1155
Number of pages13
JournalJournal of the American Society for Mass Spectrometry
Volume27
Issue number7
DOIs
Publication statusPublished - 1 Jul 2016

Keywords

  • IgM
  • Site-specific glycosylation
  • Glycopeptide
  • Pentamer
  • Hexamer

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