Clustered O-Glycans of IgA1

Abstract

Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1-5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine-and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1) 1 contains both O-and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schö nlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9 -11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques.

The Combined Role of Galactose-Deficient IgA1 and Streptococcal IgA–Binding M Protein in Inducing IL-6 and C3 Secretion from Human Mesangial Cells: Implications for IgA Nephropathy

“…Galactose-deficient polymeric IgA 1 (Ale) was isolated from the plasma of a patient with multiple myeloma using salt precipitation, jacalin precipitation, IgG-depletion on an anti-IgG column and size-exclusion chromatography (37, 38). Heterogeneity of O -glycans of this IgA 1 was fully characterized by high-resolution mass spectrometry (39, 40). …”

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

“…Furthermore, O-glycans still prove to be difficult to remove from the protein by both enzymatic and chemical procedures. 11–13 The most common methodology for characterizing proteins with multiple glycosylation sites is therefore by liquid chromatography–tandem mass spectrometry (LC-MS/MS) of protease-generated glycopeptides. Glycosylated species are then identified from masses that correspond to prior known glycopeptides or by searching fragmentation data (MS/MS spectra) against a database containing known peptide and glycan sequences.…”

Site-Specific Glycosylation of Secretory Immunoglobulin A from Human Colostrum

“…The IgA1 hinge region contains up to nine potential O-glycosylation sites clustering in a 19 amino acid peptide sequence 29 . A variety of MS analytical approaches have been used to characterize the glycan heterogeneity present in this portion of the immunoglobulin with only moderate success 30–33 . Additionally, IgA1 O-glycan site localization has been reported combining different protease digestions and ECD/ETD 29, 32, 33 .…”