The C-Terminal Region of HTRA1 Is the Predominant Target for Autoimmunity in HTRA1-Associated Membranous Nephropathy (MN)
- Glomerular Diseases: From Inflammation to Fibrosis - III
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: From Inflammation to Fibrosis
- 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
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.
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.
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.
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.
- Other NIH Support