Analysis of O-glycoforms of the IgA1 hinge region by sequential deglycosylation

Yukako Ohyama,Matthew B Renfrow,Naotake Tsuboi,Jan Novak,Yukio Yuzawa,Hisateru Yamaguchi,Kazuki Nakajima,Kazuo Takahashi,Daijo Inaguma,Yasuto Yokoi,Yukihiro Fukamachi,Midori Hasegawa,Tomohiro Mizuno

doi:10.1038/s41598-020-57510-z

Yukako Ohyama, Matthew B Renfrow + Show 11 more

Open Access

https://doi.org/10.1038/s41598-020-57510-z

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Abstract

A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes. IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine with β1,3-linked galactose and variable sialylation. IgA1 glycoforms with some galactose-deficient (Gd) HR O-glycans play a key role in IgAN pathogenesis. The clustered and variable O-glycans make the IgA1 glycomic analysis challenging and better approaches are needed. Here, we report a comprehensive analytical workflow for IgA1 HR O-glycoform analysis. We combined an automated quantitative analysis of the HR O-glycopeptide profiles with sequential deglycosylation to remove all but Gd O-glycans from the HR. The workflow was tested using serum IgA1 from healthy subjects. Twelve variants of glycopeptides corresponding to the HR with three to six O-glycans were detected; nine glycopeptides carried up to three Gd O-glycans. Sites with Gd O-glycans were unambiguously identified by electron-transfer/higher-energy collision dissociation tandem mass spectrometry. Extracted ion chromatograms of isomeric glycoforms enabled quantitative assignment of Gd sites. The most frequent Gd site was T236, followed by S230, T233, T228, and S232. The new workflow for quantitative profiling of IgA1 HR O-glycoforms with site-specific resolution will enable identification of pathogenic IgA1 HR O-glycoforms in IgAN.

Highlights

A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes
After the removal of all GalNAc-Gal disaccharides from IgA1, HRpeptides were produced by digestion with trypsin and analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) with electron-transfer dissociation (ETD) combined with supplemental higher energy collision dissociation (HCD) activation (EThcD) to fragment hinge region (HR) Gd-glycopeptides to identify glycosylation sites (Fig. 2)
As determined by anti–galactose-deficient IgA1 (Gd-IgA1) monoclonal antibodies (mAbs) and GalNAc-specific lectins, the serum levels of Gd-IgA1 are elevated in individuals with IgA nephropathy (IgAN) and glomerular immunodeposits in these individuals are enriched for Gd-IgA1

Summary

Introduction

A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes. The serum levels of IgA1 with Gal-deficient (Gd) O-glycans, with terminal or sialylated GalNAc residues, are elevated in IgAN25–27. These observations stem from a lectin-based enzyme-linked immunosorbent assay (ELISA) using Helix aspersa agglutinin (HAA), a lectin recognizing terminal GalNAc. IgA1 in glomerular immunodeposits is enriched for Gd-IgA1 glycoforms, as revealed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and lectin ELISA28,29. O-glycoforms of IgA1 in renal immunodeposits have not been characterized at the molecular level This type of analysis to elucidate the O-glycan structure of IgA1 involved in the pathogenesis of IgAN will require quantitative O-glycopeptide profiling and determination of Gd O-glycan attachment sites

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