Serine Protease HTRA1 as a Novel Target Antigen in Primary Membranous Nephropathy

Abstract

BackgroundIdentification of target antigens PLA2R, THSD7A, NELL1, or Semaphorin-3B can explain the majority of cases of primary membranous nephropathy (MN). However, target antigens remain unidentified in 15%–20% of patients.MethodsA multipronged approach, using traditional and modern technologies, converged on a novel target antigen, and capitalized on the temporal variation in autoantibody titer for biomarker discovery. Immunoblotting of human glomerular proteins followed by differential immunoprecipitation and mass spectrometric analysis was complemented by laser-capture microdissection followed by mass spectrometry, elution of immune complexes from renal biopsy specimen tissue, and autoimmune profiling on a protein fragment microarray.ResultsThese approaches identified serine protease HTRA1 as a novel podocyte antigen in a subset of patients with primary MN. Sera from two patients reacted by immunoblotting with a 51-kD protein within glomerular extract and with recombinant human HTRA1, under reducing and nonreducing conditions. Longitudinal serum samples from these patients seemed to correlate with clinical disease activity. As in PLA2R- and THSD7A- associated MN, anti-HTRA1 antibodies were predominantly IgG4, suggesting a primary etiology. Analysis of sera collected during active disease versus remission on protein fragment microarrays detected significantly higher titers of anti-HTRA1 antibody in active disease. HTRA1 was specifically detected within immune deposits of HTRA1-associated MN in 14 patients identified among three cohorts. Screening of 118 “quadruple-negative” (PLA2R-, THSD7A-, NELL1-, EXT2-negative) patients in a large repository of MN biopsy specimens revealed a prevalence of 4.2%.ConclusionsConventional and more modern techniques converged to identify serine protease HTRA1 as a target antigen in MN.

How Times Have Changed! A Cornucopia of Antigens for Membranous Nephropathy

“…This technique was subsequently followed by protein G immunoprecipitation (IP) from biopsy tissue, specifically focusing on immune complexes eluted from kidney tissue, followed by MS for identification of antigenic targets (Figure 1C). This tissue IP was the main method of identification of NCAM1 and TGFBR3, and additionally supported the discovery of HTRA1 (23). Functional validation of targeted antigens was performed by colocalization of each target antigen with IgG within the glomerular immune deposits.…”

“…Western blotting for the detection of glomerular antigens remains an effective method by which to identify additional candidate antigens and to further validate tissue-based discovery. Moreover, analysis of bands differentially recognized by serial serum samples under different clinical states (nephrosis vs. remission vs. relapse) (19,22,23), followed by immunoprecipitation and analysis by MS remains a viable technique for antigen identification (Figure 1A).…”

“…In the field of MN, the use of sequential methods of LCM followed by MS has allowed the discovery of multiple new autoantigens including the exostosin 1/2 complex (EXT1/2) (21), neural epidermal growth factor-like 1 (NELL1) (22), semaphorin 3B (SEMA3B) (30), and protocadherin 7 (PCDH7) (31). It was also utilized as an ancillary technique in confirmation of the new autoantigens serine protease HTRA1 (HTRA1) (23), neural cell adhesion molecule 1 (NCAM1) (32), and type III transforming growth factor-beta receptor (TGFBR3) (33). Additionally, the LCM-MS methodology confirmed the appropriate target antigen in cases of PLA2R-and THSD7Aassociated MN cases, validating its utility for all known subtypes of MN.…”

“…Autoimmune profiling, a new technology based on peptide or protein fragment microarrays for the analysis of the antibody repertoire in various autoimmune conditions, has only been implemented in a few studies (34)(35)(36). This technique has been applied to MN as an ancillary technique for the discovery of autoantibodies against a novel MN antigen serine protease HTRA1 (23), demonstrating proof-of-concept for its use in this disease.…”

“…HTRA1 was identified by an inter-institutional collaborative group including these three authors, utilizing four independent but adjunctive methodologies (23). Western blotting of human glomerular proteins with serum from the index patient identified a 50 kDa candidate antigen and mass spectrometry of immunoprecipitates from serum at the time of nephrosis but not remission revealed the identity of this protein as the serine protease HTRA1.…”