Characterization of antibodies to NR2A and NR2B subunits of NMDA receptor

Abstract

Systemic lupus erythematosus (SLE), an autoimmune disease diagnosed principally in women of child-bearing age, is manifested by damage in multiple organs caused by a broad spectrum of autoantibodies. Among the pathogenic autoantibodies, anti-double stranded (ds) DNA antibodies are considered as the hallmark of SLE. High titers of anti-dsDNA antibodies usually correlate with progression of disease, especially nephritis. Involvement of the central nervous system (CNS) has been reported in 14-75% of lupus patients, and is most frequently presented as nonreversible cognitive dysfunction. R4A antibody, a murine monoclonal antibody that binds dsDNA, has been demonstrated to deposit in renal glomeruli of SCID mice. Screening of a phage display library demonstrated that a pentapeptide, Asp/Glu-Trp-Asp/Glu-Tyr-Ser/Gly, is bound by the R4A antibody and, thereby, is regarded capable of serving as a molecular mimic of dsDNA. The consensus sequence is present in the extracellular domains of human and murine NMDA (N-methyl-D-aspartate) receptor subunits NR2A and NR2B. Further studies revealed that a subset of anti-dsDNA antibodies from lupus patients cross-reacts with NR2 and can readily induce neuronal apoptosis both in vivo and in vitro. Accordingly, we set out to ask whether antibodies to other epitopes of the NMDA receptor exist in SLE patients and whether the NMDA receptor induces anti-DNA antibodies. To address the questions, the extracellular amino-terminal domain of human NR2A was expressed and purified to homogeneity. Furthermore, purified NR2A fragments containing partial sequence of the extracellular amino-terminal domain were generated to delineate antigenic epitope(s) on NR2A specific for murine hybridoma monoclonal antibodies. We assayed sera from lupus patients as well as monoclonal Fabs from a combinatorial library derived from spleen cells of a SLE patient. We also immunized BALB/c mice with the purified NR2A. The mice developed a lupus-like serology. Anti-NR2A monoclonal antibodies are generated accordingly by hybridoma fusion, and their cross reactivities against various antigens, such as dsDNA, NR2B, and the Asp/Glu-Trp-Asp/Glu-Tyr-Ser/Gly pentapeptide, were studied. Our study verified that there are multiple epitopes present on the extracellular amino-terminal domain of NR2A, which are capable of binding with antibodies from lupus sera.