Kidney Week

Abstract: SA-PO596

Quantitative Assessment of Sites with Galactose-Deficient O-Glycans in the Hinge Region of IgA1

Session Information

• Glomerular Diseases: Immunology, Inflammation - II
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1202 Glomerular Diseases: Immunology and Inflammation

Authors

• Ohyama, Yukako, Fujita Health University, Toyoake, Japan

Yukako Ohyama,

• Yamaguchi, Hisateru, Fujita Health University, Toyoake, Japan

Hisateru Yamaguchi,

• Nakajima, Kazuki, Fujita Health University, Toyoake, Japan

Kazuki Nakajima,

• Hayashi, Hiroki, Fujita Health University, Toyoake, Japan

Hiroki Hayashi,

• Koide, Shigehisa, Fujita Health University, Toyoake, Japan

Shigehisa Koide,

• Inaguma, Daijo, Fujita Health University, Toyoake, Japan

Daijo Inaguma,

• Hasegawa, Midori, Fujita Health University, Toyoake, Japan

Midori Hasegawa,

• Novak, Jan, University of Alabama at Birmingham, Birmingham, Alabama, United States

Jan Novak,

• Hiki, Yoshiyuki, Fujita Health University, Toyoake, Japan

Yoshiyuki Hiki,

• Tsuboi, Naotake, Fujita Health University, Toyoake, Japan

Naotake Tsuboi,

• Yuzawa, Yukio, Fujita Health University, Toyoake, Japan

Yukio Yuzawa,

• Takahashi, Kazuo, Fujita Health University, Toyoake, Japan

Kazuo Takahashi,

Background

Serum level of IgA1 with galactose (Gal)-deficient hinge-region (HR) O-glycans (Gd-IgA1) is elevated in patient with IgA nephropathy (IgAN) and the glomerular immunodeposits of IgAN patients are enriched for Gd-IgA1. Lectins and a monoclonal antibody (mAb) specific for Gd-IgA1 have been used in ELISA to measure Gd-IgA1 serum levels. mAb specific for Gd-IgA1 stained glomerular immunodeposits in IgAN, further supporting the role of Gd-IgA1 in pathogenesis of IgAN. As mAb for Gd-IgA1 recognizes Gal-deficient O-glycans at a specific site(s), quantitative assessment of Gal-deficient sites is needed to identify disease-specific IgA1 HR O-glycoforms. Here, we describe a new method for quantitative assessment of sites with Gal- deficient O-glycans in IgA1 HR.

Methods

IgA1 from sera of 5 healthy controls was purified by affinity chromatography. After neuraminidase treatment and trypsin digestion, IgA1 HR glycosylation heterogeneity was analyzed by liquid chromatography-high-resolution mass spectrometry (LC-MS). Area under the peaks of extracted ion chromatogram (XIC) of identified IgA1 HR O-glycopeptides was calculated and expressed as relative abundance (RA) for each glycopeptide. The sites with Gal-deficient O-glycans were identified after selective quantitative removal of galactosylated O-glycans with O-glycanase by electron-transfer dissociation (ETD) tandem MS.

Results

Approximately 60% of IgA1 HR O-glycoforms contained one to three Gal-deficient O-glycans. ETD tandem MS unambiguously identify sites with Gal-deficient O-glycans and XIC based on isomeric glycoforms enabled quantitative assessment of Gal-deficient sites. The most common Gal-deficient sites included T²³⁶ followed by S²³⁰, S²³²/T²³³ and T²²⁸. HR O-glycoforms with 2 or 3 Gal-deficient O-glycans predominantly included combinations of Gal-deficient sites at S²³⁰, T²³³, and/or T²³⁶.

Conclusion

The quantitative assessment of sites with Gal-deficient O-glycans in IgA1 HR of healthy controls will enable future differentiation of IgAN vs. controls and identification of disease-specific IgA1 HR O-glycoforms associated with IgA1 glomerular deposition.

Funding

• Government Support - Non-U.S.