1 research outputs found
Site-Specific Glycosylation of Secretory Immunoglobulin A from Human Colostrum
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 (<i>n</i> = 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