Identification of Proteins Associated with IgA<sub>1</sub>-Containing Circulating Immune Complexes in Patients with IgA Nephropathy
October 22, 2020 | 10:00 AM - 12:00 PM
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Identification of Proteins Associated with IgA1-Containing Circulating Immune Complexes in Patients with IgA Nephropathy
- 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
- Bunten, Mary A., University of Alabama at Birmingham, Birmingham, Alabama, United States
- Holloway, Amanda, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Hargett, Audra A., University of Alabama at Birmingham, Birmingham, Alabama, United States
- Hall, Stacy D., University of Alabama at Birmingham, Birmingham, Alabama, United States
- Huang, Zhi qiang, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Maillard, Nicolas, Hospital and University Jean Monnet of Saint-Etienne, Saint-Etienne, France
- Julian, Bruce A., University of Alabama at Birmingham, Birmingham, Alabama, United States
- Novak, Jan, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Renfrow, Matthew B., University of Alabama at Birmingham, Birmingham, Alabama, United States
Mary A. Bunten,
Audra A. Hargett,
Stacy D. Hall,
Zhi qiang Huang,
Bruce A. Julian,
Matthew B. Renfrow,
IgA1-containing immune complexes (IgA1-ICs), consisting of galactose-deficient IgA1 (Gd-IgA1) bound by IgG specific for Gd-IgA1, are central to the pathogenesis of IgA nephropathy (IgAN). We have shown that Gd-IgA1 alone is not sufficient to induce mesangial-cell proliferation and that additional serum proteins are required for IgA1-ICs to become nephritogenic. To elucidate the composition of IgA1-ICs, we have developed a novel proteomic-bioinformatic workflow to identify proteins in IgA1-ICs in patients with IgAN.
IgA1-ICs from sera of 20 patients and 20 healthy controls were isolated by lectin affinity chromatography followed by size-exclusion chromatography (SEC). Quality-control test confirmed that most IgA1-ICs and free IgA1 were captured by affinity chromatography. IgA1-ICs were separated by SEC from monomeric and polymeric IgA1. After IgA-specific protease and LC-MS sequence-grade trypsin digestion, each IgA1-IC sample was analyzed by liquid chromatography coupled on line with mass spectrometry (LC-MS). After standard proteomic database searches, LC-MS results were extensively curated by use of Scaffold perSPECtives to identify proteins enriched in IgA1-ICs of IgAN patients vs. healthy controls. Additional comparisons included polymeric and monomeric IgA1.
Seventy-nine proteins were identified in IgA1-ICs samples from IgAN patients, with a false discovery rate of 1%. After proteomic-bioinformatic curation, we generated a list of 38 proteins with high-confidence identification that were uniquely enriched in the IgA1-ICs from patients with IgAN. Using Principle Component Analysis, we confirmed that protein content differentiated the three molecular forms of IgA1, monomeric, polymeric, and IgA1-IC. Pathway analysis indicated that proteins in IgA1-ICs were part of the complement cascade, with seemingly more enrichment in the regulation of complement, and the plasma lipoprotein pathway.
Our new workflow enabled targeted identification and evaluation of proteins associated with IgA1-ICs in IgAN. These proteins represent new targets to be evaluated for their roles in the formation and activity of the nephritogenic IgA1-ICs in IgAN.
- NIDDK Support