title: Identification of a major epitope recognized by PLA2R autoantibodies in primary membranous nephropathy.
abstract: Phospholipase A2 receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD). However, in nondenaturing conditions the epitope was protected against reduction in larger fragments, including the full-length extracellular region of PLA2R. To determine the composition of the epitope, we isolated immunoreactive tryptic fragments by Western blotting and analyzed them by mass spectrometry. The identified peptides were tested as inhibitors of autoantibody binding to PLA2R by surface plasmon resonance. Two peptides from the ricin domain showed strong inhibition, with a longer sequence covering both peptides (31-mer) producing 85% inhibition of autoantibody binding to PLA2R. Anti-PLA2R antibody directly bound this 31-mer peptide under nondenaturing conditions and binding was sensitive to reduction. Analysis of PLA2R and the PLA2R-anti-PLA2R complex using electron microscopy and homology-based representations allowed us to generate a structural model of this major epitope and its antibody binding site, which is independent of pH-induced conformational change in PLA2R. Identification of this major PLA2R epitope will enable further therapeutic advances for patients with idiopathic membranous nephropathy, including antibody inhibition therapy and immunoadsorption of circulating autoantibodies.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Manchester, United Kingdom;
authors.forename: Maryline
authors.initials: M
authors.lastname: Fresquet
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Manchester, United Kingdom;
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Jowitt
authors.affiliation: Manchester Institute of Biotechnology, Manchester, United Kingdom;
authors.forename: Jennet
authors.initials: J
authors.lastname: Gummadova
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Manchester, United Kingdom;
authors.forename: Richard
authors.initials: R
authors.lastname: Collins
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Manchester, United Kingdom;
authors.forename: Ronan
authors.initials: R
authors.lastname: O'Cualain
authors.affiliation: Manchester Institute of Biotechnology, Manchester, United Kingdom;
authors.forename: Edward A
authors.initials: EA
authors.lastname: McKenzie
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Manchester, United Kingdom; Institute of Human Development, Manchester, United Kingdom; Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom; and paul.brenchley@manchester.ac.uk Rachel.Lennon@manchester.ac.uk.
authors.forename: Rachel
authors.initials: R
authors.lastname: Lennon
authors.affiliation: Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom; and Institute of Cardiovascular Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Brenchley
mesh_terms: D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005353:Fibronectins / Q000276:immunology; D015433:Glomerulonephritis, Membranous / Q000097:blood / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037181:Lectins, C-Type / Q000276:immunology; D008969:Molecular Sequence Data; D054507:Receptors, Phospholipase A2 / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D000939:Epitopes; D005353:Fibronectins; D037181:Lectins, C-Type; D054507:Receptors, Phospholipase A2
keywords: Anti-PLA2R; PLA2R; autoantibody; epitope; membranous nephropathy