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Abstract: PO1450

Structural Characterization of Autoreactive IgG Antibodies in the Context of IgA Nephropathy

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Lingo, Jordan, The University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Knoppova, Barbora, The University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Hall, Stacy D., The University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Novak, Jan, The University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Green, Todd J., The University of Alabama at Birmingham, Birmingham, Alabama, United States
Background

IgA nephropathy (IgAN) is the most prevalent glomerulonephritis in the world. The pathology of IgAN is characterized by deposition of immune complexes in the kidneys. These complexes can trigger inflammation and mesangial cell proliferation in patients, where up to 40% of cases progress to renal failure. The pathogenic immunodeposits in kidneys of IgAN patients have been shown to contain IgA1, which has O-linked glycans in the hinge region of the heavy chain that are deficient in galactose (galactose-deficient IgA1; Gd-IgA1) and IgG autoantibodies to Gd-IgA1. Sequencing of IgG heavy chain variable regions from IgAN patients has identified a correlation between a serine residue, introduced by somatic hypermutation into the framework leading into the third hypervariable loop of the heavy chain, with an increased association of IgG autoantibodies with Gd-IgA1. We set out to understand how this single amino acid mutation affects the IgG Fab structure and antigen recognition with the aid of protein crystallography, reverse engineering, and binding studies.

Methods

Recombinant IgG constructs (rIgG) based on IgAN patient sample sequences and site-specific mutants were expressed in 293F cells and purified. Fabs were cleaved from IgG, purified, crystallized, and their protein structures determined. Binding differences between Gd-IgA1 and intact rIgGs were determined with ELISA-based experiments.

Results

Comparisons of rIgG Fabs structures derived from a healthy control patient and an IgAN patient showed conformational differences that possibly influence the recognition of Gd-IgA1. Using this structural data and bioinformatics to guide antibody design, we reverse engineered the IgG from a healthy patient control to modulate binding to Gd-IgA1. Site-directed mutagenesis and binding studies indicated that residues in our selected regions of the variable domains alter Gd-IgA1-IgG complex formation.

Conclusion

We have determined the structures of Fabs from IgGs that form complexes with Gd-IgA1, and we have further identified residues in the Fabs that are important for Gd-IgA1-binding. This knowledge could inform future strategies to inhibit pathogenic immune complex formation.

Funding

  • NIDDK Support