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.

Figures

Figure 1

Extracted Compound chromatogram (ECC) and associated structural assignments of glycopeptides from pronase digested sIgA isolated from an individual donor. (A) ECC of major glycopeptides eluted between 6 and 38 min; (B) ECC of all glycopeptides eluted between 7 and 21 min; (C) ECC of all glycopeptides eluted between 21 to 29 min. Glycosites corresponding to the four polypeptides was denoted in different colors. Green Circles, yellow circles, blue squares, red triangles, and purple diamonds represent mannose, galactose, GlcNAc, fucose and Neu5Ac, respectively.

Figure 2

Deconvoluted MS/MS spectra of three N-linked glycopeptides from sIgA. (A) MS/MS data for a N-linked peptide from J chain; (B) MS/MS data for a neutral N-linked peptide from SC; (C) MS/MS data for an acidic N-linked peptide from SC. Green Circles, yellow circles, blue squares, red triangles, and purple diamonds represent mannose, galactose, GlcNAc, fucose and Neu5Ac, respectively.

Figure 3

Extracted compound chromatogram (ECC) of glycopeptides from pronase digested commercial pooled human colostrum IgA with corresponding structural assignments. Different colors represent various sites. Green Circles, yellow circles, blue squares, red triangles, and purple diamonds represent mannose, galactose, GlcNAc, fucose and Neu5Ac, respectively.

Figure 4

(A–C) Glycosite occupancy and heterogeneity in pronase digested sIgA isolated from an individual donor, SC, J chain, and IgA1/IgA2, respectively. (D–F) Glycosite occupancy and heterogeneity in pooled commercial sIgA, SC, J chain, and IgA1/IgA2, respectively. Circle shading under each site is representative of site occupancy ratio to the most occupied site in 92Asn from IgA2. Circle shading under each glycan is representative of the relative glycan abundance at each site.

Figure 4

(A–C) Glycosite occupancy and heterogeneity in pronase digested sIgA isolated from an individual donor, SC, J chain, and IgA1/IgA2, respectively. (D–F) Glycosite occupancy and heterogeneity in pooled commercial sIgA, SC, J chain, and IgA1/IgA2, respectively. Circle shading under each site is representative of site occupancy ratio to the most occupied site in 92Asn from IgA2. Circle shading under each glycan is representative of the relative glycan abundance at each site.

Figure 5

MS spectra of three tryptic O-glycopeptides from sIgA and the corresponding isotopic patterns and tandem spectra. (A) O-glycopeptide with glycan composition HexNAc5Hex4Neu5Ac3; (B) O-glycopeptide with glycan composition HexNAc5Hex4Neu5Ac2; (C) O-glycopeptide with glycan composition HexNAc5Hex4. Red boxes are representative of the predicted isotopic pattern.

Figure 6

Extracted compound chromatograms (ECCs) of four sIgA HR O-glycopeptides containing same number of N-acetylgalactosamine and galactose residues and 0–3 sialic acid monosaccharides.