title: Site-specific glycosylation of secretory immunoglobulin A from human colostrum.
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.
authors: Department of Chemistry, ‡Foods for Health Institute, §Department of Food Science and Technology, and ∥Department of Biochemistry and Molecular Medicine, University of California , Davis, California 95616, United States.
authors: Jincui
authors: J
authors: Huang
authors: Andres
authors: A
authors: Guerrero
authors: Evan
authors: E
authors: Parker
authors: John S
authors: JS
authors: Strum
authors: Jennifer T
authors: JT
authors: Smilowitz
authors: J Bruce
authors: JB
authors: German
authors: Carlito B
authors: CB
authors: Lebrilla
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003126:Colostrum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007071:Immunoglobulin A, Secretory / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D007071:Immunoglobulin A, Secretory; D011134:Polysaccharides
keywords: glycan microheterogeneity; glycoproteomics; mass spectrometry; sIgA; site-specific glycosylation
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