Kidney Week

Abstract: PO1802

Mass Spectrometric Analysis of IgA₁ O-Glycoforms Reveals the Basis of IgA₁ Galactose Deficiency Detected by Quantitative Lectin ELISA

Session Information

• Glomerular Diseases: IgA, C3G, and FSGS
  October 22, 2020 | Location: On-Demand
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1202 Glomerular Diseases: Immunology and Inflammation

Authors

• Craine, Ellenore P., The University of Alabama at Birmingham, Birmingham, Alabama, United States

Ellenore P. Craine,

• Ueda, Hiroyuki, The University of Alabama at Birmingham, Birmingham, United States

Hiroyuki Ueda,

• Ueda, Yoshimi, The University of Alabama at Birmingham, Birmingham, United States

Yoshimi Ueda,

• Reily, Colin, The University of Alabama at Birmingham, Birmingham, Alabama, United States

Colin Reily,

• Moldoveanu, Zina, The University of Alabama at Birmingham, Birmingham, Alabama, United States

Zina Moldoveanu,

• Hall, Stacy D., The University of Alabama at Birmingham, Birmingham, Alabama, United States

Stacy D. Hall,

• Yokoo, Takashi, The Jikei University School of Medicine, Tokyo, Japan

Takashi Yokoo,

• Rizk, Dana, The University of Alabama at Birmingham, Birmingham, Alabama, United States

Dana Rizk,

• Kiryluk, Krzysztof, Columbia University College of Physicians and Surgeons, New York, New York, United States

Krzysztof Kiryluk,

• Gharavi, Ali G., Columbia University College of Physicians and Surgeons, New York, New York, United States

Ali G. Gharavi,

• Julian, Bruce A., The University of Alabama at Birmingham, Birmingham, Alabama, United States

Bruce A. Julian,

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

Jan Novak,

• Renfrow, Matthew B., The University of Alabama at Birmingham, Birmingham, Alabama, United States

Matthew B. Renfrow,

Background

Patients with IgA nephropathy (IgAN) have elevated serum levels of IgA1 with some hinge-region (HR) O-glycansdeficient in galactose (Gal; Gd-IgA1). Gd-IgA1 is recognized by IgG autoantibodies, resulting in the formation of pathogenic immune complexes. Our group has established a quantitative ELISA for Gd-IgA1 using GalNAc-specific lectins (e.g., from Helix pomatia; HPA). This test enabled determination of genetic basis of IgA1 Gal deficiency and provided a better understanding of IgAN pathogenesis. However, understanding of IgA1 O-glycosylation at a molecular-level is needed.

Methods

We used liquid chromatography with high-resolution mass spectrometry (LC-MS) to analyze and quantify O-glycoforms of monomeric (m) and polymeric (p) IgA1 in sera of IgAN patients (n=31) and healthy controls (HC; n=10). Total serum IgA1 was isolated by lectin affinity chromatography and m and p forms were separated by size-exclusion chromatography. Gd-IgA1 was measured by lectin ELISA. HR glycopeptides, generated by an IgA-specific protease and trypsin, were analyzed by LC-MS using LTQ Orbitrap Velos MS. LC-MS data were analyzed with the Pinnacle software.

Results

Quantitative LC-MS O-glycosylation profiling of IgA1 HR was performed, and results calculated as relative abundance of individual glycoforms and as ratios of Gal-containing vs. Gal-deficient glycoforms. Both LC-MS data and lectin ELISA confirmed that pIgA1 exhibited higher degree of Gal deficiency than mIgA1. LC-MS data provided additional insight into the molecular basis of the variability of Gd-IgA1 serum levels in IgAN patients. For example, IgA1 HR glycoform GalNAc4Gal3 was more abundant for the pIgA1 in the patients with high vs. low serum levels of Gd-IgA1 (p<0.005). LC-MS analysis enabled identification and quantification of individual HR glycoforms and defined the Gd-IgA1 glycoforms detected by lectin ELISA.

Conclusion

High-resolution LC-MS IgA1 glycoprofiling confirmed pIgA1 as the main form of IgA1 detected by Gd-IgA1 lectin ELISA. Furthermore, we identified several different Gal-deficient glycoforms in pIgA1, an observation that enables quantitative molecular-level assessment of Gd-IgA1 glycophenotype.

Funding

• NIDDK Support