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 journalArticle

26 Citations (Scopus)

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

Fingerprint

Glycosylation
Colostrum
Polysaccharides
Immunoglobulins
Secretory Immunoglobulin A
Chemical analysis
Glycopeptides
Immunoglobulin A
Milk
Secretory Component
Hinges
Occupations
Glycoproteins
Mucous Membrane
Proteins
Peptide Hydrolases
Software

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Huang, J., Guerrero, A., Parker, E., Strum, J. S., Smilowitz, J. T., German, J. B., & Lebrilla, C. B. (2015). Site-specific glycosylation of secretory immunoglobulin a from human colostrum. Journal of Proteome Research, 14(3), 1335-1349. https://doi.org/10.1021/pr500826q

Site-specific glycosylation of secretory immunoglobulin a from human colostrum. / Huang, Jincui; Guerrero, Andres; Parker, Evan; Strum, John S.; Smilowitz, Jennifer T.; German, J. Bruce; Lebrilla, Carlito B.

In: Journal of Proteome Research, Vol. 14, No. 3, 06.03.2015, p. 1335-1349.

Research output: Contribution to journalArticle

Huang, J, Guerrero, A, Parker, E, Strum, JS, Smilowitz, JT, German, JB & Lebrilla, CB 2015, 'Site-specific glycosylation of secretory immunoglobulin a from human colostrum', Journal of Proteome Research, vol. 14, no. 3, pp. 1335-1349. https://doi.org/10.1021/pr500826q
Huang J, Guerrero A, Parker E, Strum JS, Smilowitz JT, German JB et al. Site-specific glycosylation of secretory immunoglobulin a from human colostrum. Journal of Proteome Research. 2015 Mar 6;14(3):1335-1349. https://doi.org/10.1021/pr500826q
Huang, Jincui ; Guerrero, Andres ; Parker, Evan ; Strum, John S. ; Smilowitz, Jennifer T. ; German, J. Bruce ; Lebrilla, Carlito B. / Site-specific glycosylation of secretory immunoglobulin a from human colostrum. In: Journal of Proteome Research. 2015 ; Vol. 14, No. 3. pp. 1335-1349.
@article{26508e05fe9c443988298773b05d2d96,
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.",
keywords = "glycan microheterogeneity, glycoproteomics, mass spectrometry, sIgA, site-specific glycosylation",
author = "Jincui Huang and Andres Guerrero and Evan Parker and Strum, {John S.} and Smilowitz, {Jennifer T.} and German, {J. Bruce} and Lebrilla, {Carlito B}",
year = "2015",
month = "3",
day = "6",
doi = "10.1021/pr500826q",
language = "English (US)",
volume = "14",
pages = "1335--1349",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "3",

}

TY - JOUR

T1 - Site-specific glycosylation of secretory immunoglobulin a from human colostrum

AU - Huang, Jincui

AU - Guerrero, Andres

AU - Parker, Evan

AU - Strum, John S.

AU - Smilowitz, Jennifer T.

AU - German, J. Bruce

AU - Lebrilla, Carlito B

PY - 2015/3/6

Y1 - 2015/3/6

N2 - 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.

AB - 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.

KW - glycan microheterogeneity

KW - glycoproteomics

KW - mass spectrometry

KW - sIgA

KW - site-specific glycosylation

UR - http://www.scopus.com/inward/record.url?scp=84924357001&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84924357001&partnerID=8YFLogxK

U2 - 10.1021/pr500826q

DO - 10.1021/pr500826q

M3 - Article

C2 - 25629924

AN - SCOPUS:84924357001

VL - 14

SP - 1335

EP - 1349

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 3

ER -