Identification of the Immunodominant Epitope Region in Phospholipase A2 Receptor-Mediating Autoantibody Binding in Idiopathic Membranous Nephropathy

Abstract

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Recent clinical studies established that >70% of patients with idiopathic (also called primary) MN (IMN) possess circulating autoantibodies targeting the M-type phospholipase A₂ receptor-1 (PLA₂R) on the surface of glomerular visceral epithelial cells (podocytes). In situ, these autoantibodies trigger the formation of immune complexes, which are hypothesized to cause enhanced glomerular permeability to plasma proteins. Indeed, the level of autoantibody in circulation correlates with the severity of proteinuria in patients. The autoantibody only recognizes the nonreduced form of PLA₂R, suggesting that disulfide bonds determine the antigenic epitope conformation. Here, we identified the immunodominant epitope region in PLA₂R by probing isolated truncated PLA₂R extracellular domains with sera from patients with IMN that contain anti-PLA₂R autoantibodies. Patient sera specifically recognized a protein complex consisting of the cysteine-rich (CysR), fibronectin-like type II (FnII), and C-type lectin-like domain 1 (CTLD1) domains of PLA₂R only under nonreducing conditions. Moreover, absence of either the CysR or CTLD1 domain prevented autoantibody recognition of the remaining domains. Additional analysis suggested that this three-domain complex contains at least one disulfide bond required for conformational configuration and autoantibody binding. Notably, the three-domain complex completely blocked the reactivity of autoantibodies from patient sera with the full-length PLA₂R, and the reactivity of patient sera with the three-domain complex on immunoblots equaled the reactivity with full-length PLA₂R. These results indicate that the immunodominant epitope in PLA₂R is exclusively located in the CysR-FnII-CTLD1 region.