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Abstract: PO1805

Functional Studies of IgG Autoantibodies in IgA Nephropathy

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Knoppova, Barbora, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Hansen, Alyssa L., University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Moldoveanu, Zina, 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
  • Novak, Lea, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Julian, Bruce A., University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Renfrow, Matthew B., University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Novak, Jan, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Green, Todd J., University of Alabama at Birmingham, Birmingham, Alabama, United States
Background

IgA nephropathy (IgAN) is an autoimmune disease wherein galactose-deficient IgA1 (Gd-IgA1) is recognized by IgG autoantibodies (autoAbs), resulting in the formation of immune complexes (ICs), some of which deposit in the kidneys and induce glomerular injury. We have shown that a recombinant IgG (rIgG) autoAb derived from an IgAN patient can bind to Gd-IgA1, form ICs, and induce pathologic mesangioproliferative changes in a passive mouse model of IgAN. However, the interaction between the key elements, Gd-IgA1 and IgG autoAbs, has not been fully clarified. After solving the Fab 3-D structure, we focused on functional characterization of this rIgG autoAb.

Methods

Based on solved 3-D structure of the Fab of rIgG autoAb, we used site-directed mutagenesis to replace specific amino-acid (aa) residues in the rIgG autoAb. rIgGs were expressed in Expi293F cells and purified by affinity chromatography. ELISA was used to assess the binding of rIgGs to Gd-IgA1. Fab fragments of two selected mutants of rIgG were purified and used for hanging-drop crystallization. Liquid chromatography coupled with mass spectrometry (LC-MS) analysis was used to identify Gd-IgA1 O-glycoforms recognized by rIgG autoAb.

Results

We generated rIgG variants with aa replacements in several heavy-chain segments: junction of framework 1 (FR1) and complementarity-determining region 1 (CDR1) and in the CDR3.The FR1-CDR1 mutations reduced or disabled rIgGs binding to Gd-IgA1, depending on the specific aa residue used for replacement. Mutations in CDR3 completely impaired the binding of rIgG to Gd-IgA1. LC-MS analyses indicated that rIgG autoAb preferentially binds to a subset of IgA1 molecules, resulting in enrichment of hinge-region (HR) glycoforms with 4 O-glycans, including HR glycoform GalNAc4Gal3.

Conclusion

Our study identified specific aa residues in the FR1-CDR1 and CDR3 regions of the rIgG autoAb that are critical for Gd-IgA1 binding. The ongoing structural studies of different variants of this rIgG will elucidate the nature of autoantigen recognition by IgG autoAbs. This knowledge, together with the understanding what are the main targeted HR glycoforms, will enable the design of future therapeutic approaches based on blockade of these pathogenic IgG autoAbs in IgAN.

Funding

  • NIDDK Support