Analysis of the autoimmune epitopes on human testicular NASP using recombinant and synthetic peptides

The human nuclear autoantigenic sperm protein, NASP, is a testicular histon e-binding protein of 787 amino acids to which most vasectomized men develop autoantibodies. In this study to define the boundaries of antigenic region s and epitope recognition pattern, recombinant deletion mutants spanning th e entire protein coding sequence and a human NASP cDNA sublibrary were scre ened with vasectomy patients' sera. Employing panel sera from 21 vasectomy patients with anti-sperm antibodies, a heterogeneous pattern of autoantibod y binding to the recombinant polypeptides was detected in ELISA and immunob lotting. The majority of sera (20/21) had antibodies to one or more of the NASP fusion proteins. Antigenic sites preferentially recognized by the indi vidual patients' sera were located within aa 32-352 and aa 572-787. Using a patient's serum selected for its reactivity to the whole recombinant prote in in Western blots, cDNA clones positive for the C-terminal domain of the molecule were identified. The number and location of linear epitopes in thi s region were determined by synthetic peptide mapping and inhibition studie s. The epitope-containing segment was delimited to the sequence aa 619-692 and analysis of a series of 74 concurrent overlapping 9mer synthetic peptid es encompassing this region revealed four linear epitopes: amino acid resid ues IREKIEDAK (aa 648-656), KESQRSGNV (aa 656-664), AELALKATL (aa 665-673) and GFTPGGGGS (aa 680-688). All individual patients' sera reacted with epit opes within the sequence IRE....GGS (aa 648-688). The strongest reactivity was displayed by peptides corresponding to the sequence AELALKATL (aa 665-6 73). Thus, multiple continuous autoimmune epitopes in NASP involving sequen ces in the conserved C-terminal domain as well as in the less conserved tes tis-specific N-terminal region comprising the histone-binding sites, as pre dicted for an antigen-driven immune response, may be a target of autoantibo dies in vasectomized men and may provide a relevant laboratory variable to describe more accurately the spectrum of autoantibody specificities associa ted with the clinical manifestation of vasectomy.