Abstract: TH-OR114
Characterization of Recombinant IgG Autoantibody That Binds Galactose-Deficient IgA1 and Forms Immune Complexes Mimicking Those in IgA Nephropathy
Session Information
- Mostly IgA Nephropathy
November 07, 2019 | Location: Ballroom C, Walter E. Washington Convention Center
Abstract Time: 05:42 PM - 05:54 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Authors
- Knoppova, Barbora, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Moldoveanu, Zina, Univ 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
- Novak, Jan, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Green, Todd J., University of Alabama at Birmingham, Birmingham, Alabama, United States
Background
Immune complexes (IC) containing galactose-deficient IgA1 (Gd-IgA1) and Gd-IgA1-specific IgG autoantibodies (autoAbs) play a key role in the pathogenesis of IgA nephropathy (IgAN). However, the molecular interactions between autoAb and Gd-IgA1 in IgAN are not well understood. To gain a better insight, we used a recombinant IgG (rIgG) autoAb derived from an IgAN patient and assessed its structural and functional features.
Methods
rIgG autoAb was produced in Expi293F cells. Gd-IgA1 was isolated from plasma of a patient with IgA myeloma. Surface plasmon resonance was used for kinetic analysis of autoAb binding to Gd-IgA1. Fab of rIgG was used for crystallographic studies and the structure was solved by molecular replacement method. We formed in vitro engineered IC (EIC) that mimic those in IgAN patients by incubating Gd-IgA1 and rIgG in the presence of human serum. The formed EIC were isolated by size-exclusion chromatography and their biological activity was evaluated using human primary mesangial cells (MC). Furthermore, IC formed from purified Gd-IgA1 and rIgG were injected to immunodeficient mice and then the kidney histopathology was assessed.
Results
The rIgG formed complexes with Gd-IgA1. Kinetic analysis showed intermediate affinity of rIgG to Gd-IgA1 (KD=3.16 E-07 M). The structure of the Fab was solved at the resolution of 1.69 Å. The structure revealed a loop in the heavy chain that adopts a unique conformation, unveiling a surface-accessible pocket located in close proximity to the CDR3. Binding modes from an in silico docking study of a glycopeptide mimicking the hinge region of Gd-IgA1 showed potential binding to this region. The EIC, but not Gd-IgA1 alone, stimulated proliferation of cultured MC (2.88±0.69-fold increase over control). IC injected into immunodeficient mice increased glomerular cellularity (48.4±13.3 nuclei per glomerulus vs. 40.4±10.5 in control; p<0.0001; n=4 each).
Conclusion
This study provides the first structure of an autoAb that binds Gd-IgA1 and forms biologically active EIC. We envision that better understanding of the interactions of Gd-IgA1 and autoantibodies will enable design of inhibitors to block the formation of pathogenic IC in IgAN.
Funding
- NIDDK Support