Site-specific glycosylation of secretory immunoglobulin a from human colostrum

Jincui Huang, Andres Guerrero, Evan Parker, John S. Strum, Jennifer T. Smilowitz, J. Bruce German, Carlito B Lebrilla

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

Secretory immunoglobulin A (sIgA) is a major glycoprotein in milk and plays a key role in mediating immune protection of the gut mucosa. Although it is a highly glycosylated protein, its site-specific glycosylation and associated glycan micro-heterogeneity have still not been fully elucidated. In this study, the site-specific glycosylation of sIgA isolated from human colostrum (n = 3) was analyzed using a combination of LC-MS and LC-MS/MS and in-house software (Glycopeptide Finder). The majority of the glycans found are biantennary structures with one or more acidic Neu5Ac residues; however, a large fraction belonged to truncated complex structures with terminal GlcNAc. Multiple glycosites were identified with nearly 30 glycan compositions located at seven sites on the secretory component, six compositions at a single site on the J chain, and 16 compositions at five sites on the IgA heavy (H) chain. Site-specific heterogeneity and relative quantitation of each composition and the extent of occupation at each site were determined using nonspecific proteases. Additionally, 54 O-linked glycan compositions located at the IgA1 hinge region (HR) were identified by comparison against a theoretical O-glycopeptide library. This represents the most comprehensive report to date detailing the complexity of glycan micro-heterogeneity with relative quantitation of glycoforms for each glycosylation site on milk sIgA. This strategy further provides a general method for determining site-specific glycosylation in large protein complexes.

Original languageEnglish (US)
Pages (from-to)1335-1349
Number of pages15
JournalJournal of Proteome Research
Volume14
Issue number3
DOIs
StatePublished - Mar 6 2015

Keywords

  • glycan microheterogeneity
  • glycoproteomics
  • mass spectrometry
  • sIgA
  • site-specific glycosylation

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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