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Immunoglobulin A Carries Sulfated and O-acetylated N-glycans Primarily at the Tailpiece Site

ABSTRACT: A site-specific description of a recently discovered HexNAc-sulfated N-glycans of human immunoglobulin A (IgA) was still pending. Here we provide an in-depth N-glycoproteomic analysis of human serum IgA. This in-depth N-glycoproteomic analysis, developed by us, integrates a new strategy for identifying sulfated and other rare N-glycans in IgA. In our study, we implemented two strategies for identifying the new N-glycan compositions: screening for rare glycopeptides using oxonium marker ions and wildcard search to identify glycans holding a rare modification attached to peptides. The dataset provided here contains primarily IgA N-glycopeptides identified from technical quadruplicates of two commercial human serum IgA samples. A comparison of the N-glycosylation profiles of two commercial human serum IgA samples demonstrated that sulfated N-glycans are mainly present in the tailpiece site. Also, complex-type N-glycan compositions with O-acetylated sialic acid were identified in the tailpiece. Surprisingly, N-glycans bearing glucuronic acid were identified in the commercial IgA samples, but from peptides of contaminant glycoproteins.These N-glycans have not been included in previously published IgA micro-heterogeneity analyses. We expect that a broader micro-heterogeneity description of clinically relevant glycoproteins, such as IgA, can expand the screening for biomarkers or treatment options.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Marcus Hoffmann Udo Reichl Greta Lemke Erdmann Rapp Frania Jaqueline Zuniga-Banuelos

PROVIDER: MSV000096980 | MassIVE | Tue Jan 28 06:43:00 GMT 2025

SECONDARY ACCESSION(S): PXD060281

REPOSITORIES: MassIVE

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Project description:Glycoproteomics, TNF-alpha, membrane proteomics, insulin resistance, adipocyte, SILAC Insulin resistance (IR) is a complex process arising from both environmental and genetic perturbations and leads to a variety of diseases including type-2 diabetes (T2D). The expansion of adipose tissue during obesity results in secretion of proinflammatory cytokines which significantly impairs insulin sensitivity throughout the entire body. Inflammation modulates glycosylation in a variety of cell types however, an investigation of changes in glycosylation following inflammation-induced IR in adipocytes has not been performed. In the present study, we performed a quantitative N-glycomic analysis of TNF-alpha induced IR in adipocytes and identified the regulation of specific N-glycans including an increase in terminal di-galactose- and decrease in di-sialic acid-containing glycans with alpha-2,3 linkages. Quantitative analysis of the membrane-associated proteome in TNF-alpha treated adipocytes identified the regulation of specific glycosyltransferases and glycosidases which correlated with the regulated N-glycans. This included the up- and down-regulation of B4GalT5 and ST3Gal6, respectively at both the protein and mRNA level. To identify the protein and glycosylation sites modified with these regulated N-glycans, we performed a site-specific quantitative glycoproteomic analysis of enriched N-glycopeptides from TNF-alpha treated adipocytes with and without deglycosylation. The combined workflow provided a relative quantification of changes in protein abundance verses N-glycosylation occupancy verses site-specific N-glycans on a proteome-wide level. This revealed the modulation of a subset of N-glycan compositions on specific proteins including those previously associated with insulin-stimulated GLUT4 trafficking to the plasma membrane. Finally, knockdown of B4GalT5 with siRNA and analysis of released N-glycans resulted in the down-regulation of di-galactose containing glycans, confirming the involvement of this enzyme in the TNF-alpha regulated N-glycome.

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Immunoglobulin A Carries Sulfated and O-acetylated N-glycans Primarily at the Tailpiece Site

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