ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on X

Kidney Week

Abstract: SA-PO846

The C-Terminal Region of HTRA1 Is the Predominant Target for Autoimmunity in HTRA1-Associated Membranous Nephropathy (MN)

Session Information

Category: Glomerular Diseases

  • 1401 Glomerular Diseases: From Inflammation to Fibrosis

Authors

  • Al-Rabadi, Laith, University of Utah Health, Salt Lake City, Utah, United States
  • Reinhard, Linda, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Ehrmann, Michael, Universitat Duisburg-Essen, Duisburg, Nordrhein-Westfalen, Germany
  • Hoxha, Elion, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Beck, Laurence H., Boston Medical Center, Boston, Massachusetts, United States
Background

We previously identified HTRA1 as a novel antigen in 4.2% of uncharacterized MN patients. This secreted serine protease is expressed in various tissues with high homology across human and rodent species (~92%). The trypsin-like serine protease domain in HTRA1 is responsible for its proteolytic activity whereas its PDZ domain facilitates protein-protein interactions by tethering HTRA1 to specific sites in the extracellular matrix.

Methods

Immunoblotting/cyclic constrained epitope mapping of patient serum from two independent cohorts with various truncated recombinant HTRA1 proteins. WT and HTRA1 KO mice were immunized with full-length human HTRA1.

Results

Sera from patients with HTRA1-associated MN is known to recognize native and recombinant human HTRA1 even under reducing conditions, which are expected to disrupt the conformation of the highly disulfide bonded N-terminal region of HTRA1. Accordingly, both circulating and glomerulus-eluted antibodies from patients with HTRA1-associated MN recognize the HTRA1 C-terminus, which encompasses its PDZ and protease domains. Immunization of WT or HTRA1 KO mice with human HTRA1 generates mouse Ab against HTRA1. Importantly, these mouse autoantibodies recognized the HTRA1 C-terminus (similar to what is seen with patient-derived autoantibodies). To further approximate the location of the targeted epitopes of HTRA1 autoantibodies, immunoblotting of reactive sera with various truncated recombinant HTRA1 proteins showed that HTRA1 autoantibody-containing human sera recognized full length HTRA1 and HTRA lacking AA 1-158 (referred to as DeltaMac25). To identify any outliers or conformation-specific epitopes, the commercially available PEPperMAP Cyclic assay was performed. This analysis revealed the top three antibody responses were against non-conformational peptides with the consensus motifs AIINYGNSGGPL (AA 312-332, which encompasses the AA 328 site known to be required for HTRA1 protease function), and GGPLVNLDGEV (AA 329-339) in the protease domain, and IEVIPD (AA 415-420, required for HTRA binding activity) in the PDZ domain.

Conclusion

Together, these findings suggest that the targeted epitope in HTRA1 is non-conformational and located within the C-terminus which raises the possibility that anti-HTRA1 antibodies may interfere with biological function and/or binding.

Funding

  • Other NIH Support