Site‐specific analysis of the N‐glycans on murine polymeric immunoglobulin A using liquid chromatography/electrospray mass spectrometry

Abstract

The site-specific distribution of oligosaccharides on murine polymeric immunoglobulin A (pIgA) consisting of two or more immunoglobulin A (IgA) antibodies connected through J-chain was analysed by liquid chromatographylelectrospray mass spectrometry. Glycopeptides from pIgA were localized in a reversed-phase tryptic peptide chromatogram by collision excitation scanning and their amino acid sequences determined by electrospray tandem mass spectrometry and Edman degradation. Two glycosylation sites on IgA and a single glycosylation site on J-chain were identified. Using biosynthetic constraints on carbohydrate structures, molecular mass information on the glycan moieties of the glycopeptides was translated into specific carbohydrate structure proposals. The glycopeptide incorporating the single glycosylation site at Am49 in J-chain carried fucosylated and non-fucosylated di-and triantennary N-acetyllactosamine type carbohydrate chains terminated by N-acetyl-and/or Nglycolylneuraminic acid residues. The glycosylation site in the IgA heavy chain at Asn446 contained two oligomannose-type carbohydrate chains, one carrying five and the other six mannose residues. To the other glycosylation site in the IgA heavy chain at A d 5 5 one oligomannose structure, hybrid structures with the lactosamine branch terminated by either an additional galactose residue, N-glycolylneuraminic acid or N-acetylneuraminic acid, and non-fucosylated N-acetyllactosamine-type structures carrying the same terminating residues were attached. This glycosylation site was present in two separate glycopeptides differing only in their degree of carboxymethylation and yielding identical oligosaccharide distributions, thus providing additional confidence in the assignment method.

Expression, purification and biochemical characterization of recombinant murine secretory component: a novel tool in mucosal immunology

“…Murine cell lines and tissues are known to add terminal α1-3galactosyl residues to N-linked carbohydrates, resulting in the structure Galα1-3Galβ1-4GlcNAc-R [25,33]. Further, IgA antibodies secreted by murine hybridomas are known to carry αgalactosylated oligosaccharides [34][35][36]. The presence of α1-3-…”

Interaction of Human, Rat, and Mouse Immunoglobulin A (IgA) with Staphylococcal Superantigen-like 7 (SSL7) Decoy Protein and Leukocyte IgA Receptor

“…Glycan analysis by mass spectroscopy has previously identified N-linked glycosylation of mouse IgA at two sites, one in the CH1 domain and one in the tail piece (30). This is to our knowledge the first report of glycosylation at Asn-442 in the FG loop of CH3.…”

Expression, purification and biochemical characterization of recombinant murine secretory component: a novel tool in mucosal immunology

“…Murine cell lines and tissues are known to add terminal α1-3galactosyl residues to N-linked carbohydrates, resulting in the structure Galα1-3Galβ1-4GlcNAc-R [25,33]. Further, IgA antibodies secreted by murine hybridomas are known to carry αgalactosylated oligosaccharides [34][35][36]. The presence of α1-3-…”