Nuclear autoantigenic sperm protein (NASP), initially described as a highly autoimmunogenic testis and sperm-specific protein, is a histone-binding protein that is a homologue of the N1/N2 gene expressed in oocytes of Xenopus laevis. Here, we report a somatic form of NASP (sNASP) present in all mitotic cells examined, including mouse embryonic cells and several mouse and human tissue culture cell lines. Affinity chromatography and histone isolation demonstrate that NASP from myeloma cells is complexed only with H1, linker histones. Somatic NASP is a shorter version of testicular NASP (tNASP) with two deletions in the coding region arising from alternative splicing and differs from tNASP in its 5 untranslated regions. We examined the relationship between NASP mRNA expression and the cell cycle and report that in cultures of synchronized mouse 3T3 cells and HeLa cells sNASP mRNA levels increase during Sphase and decline in G 2 , concomitant with histone mRNA levels. NASP protein levels remain stable in these cells but become undetectable in confluent cultures of nondividing CV-1 cells and in nonmitotic cells in various body tissues. Expression of sNASP mRNA is regulated during the cell cycle and, consistent with a role as a histone transport protein, NASP mRNA expression parallels histone mRNA expression. NASP, 1 initially described as a highly autoimmunogenic testis and sperm-specific protein, is present in the nucleus of spermatozoa and spermatogenic cells (1-3), hence the name nuclear autoantigenic sperm protein. Previous studies (4) have demonstrated that human NASP contains three functional histone binding sites: site I (amino acids 116 -127), site II, (amino acids 469 -512), and site III (amino acids 211-244). In vitro, recombinant NASP will bind both linker and core histones (4). 2As a histone-binding protein (4, 5), NASP appears to be a homologue of the N1/N2 gene expressed in oocytes of Xenopus laevis (6 -9) because, in addition to the conserved coding regions, there is an almost 60% identity between the 3Ј untranslated regions of rabbit NASP and Xenopus N1/N2 mRNAs (2). In Xenopus oocytes, the non-chromatin-bound core histones H3 and H4 are associated in a complex with N1/N2 (10, 11), providing a mechanism for the storage of histones for DNA replication in the early embryo. Both mouse (12) and sea urchin oocytes (13) also store histones in a non-chromatin-bound form. Other mammalian histone-binding proteins, for example the nucleosome assembly factor NAP-1 (14 -17), the chromatin assembly factor CAF-1 (18 -21), and the transcription proteins HIRA-HIRIP3 (22), not only bind histones but appear to be important for the assembly of chromatin (19,(23)(24)(25)(26). Consequently NASP may be important not only for both the storage and transport of histones but also for the assembly of chromatin.Here, we report a somatic form of NASP (sNASP) present in almost all tissues examined, including mouse embryonic cells and several mouse and human tissue culture cell lines. Interestingly, an analysis of the histones comp...
In one of our previous studies, the deduced amino acid sequence of the human nuclear autoantigenic sperm protein (hNASP) revealed two conserved histone-binding domains when compared to the Xenopus N1/N2 protein sequence. These histone-binding domains of Xenopus N1/N2 are known to be functional; however, their function in hNASP is unknown. In this study we have determined the number, location, and activity of the histone-binding domains on the primary sequence of hNASP. Purified recombinant polypeptides expressing the full-length hNASP and various deletion constructs covering the entire length of the hNASP sequence were tested by Western blotting and in ELISA for binding to biotin-labeled histones. A positive reaction was detected for the full-length recombinant protein and for the polypeptides spanning the N-terminal region (amino acids [aa] 32-192), and two additional regions: aa 193-352 and aa 353-572. The lack of binding to the expressed C-terminal (aa 573-787), which also contains polyacidic amino acids, suggests that the binding of hNASP to the somatic core histones is a sequence-specific as well as an electrostatic interaction. The removal of flanking sequences from the binding domains did not abrogate their ability to bind histones. We conclude that there are at least three functional histone-binding domains in hNASP, two of them encompassing the predicted histone binding sites homologous to the N1/N2 protein, and a third novel domain. Therefore, hNASP may be defined as a nuclear histone-binding protein found in human testis.
SUMMARYThe human nuclear autoantigenic sperm protein, NASP, is a testicular histone-binding protein of 787 amino acids to which most vasectomized men develop autoantibodies. In this study to define the boundaries of antigenic regions and epitope recognition pattern, recombinant deletion mutants spanning the entire protein coding sequence and a human NASP cDNA sublibrary were screened with vasectomy patients' sera. Employing panel sera from 21 vasectomy patients with anti-sperm antibodies, a heterogeneous pattern of autoantibody binding to the recombinant polypeptides was detected in ELISA and immunoblotting. The majority of sera (20/21) had antibodies to one or more of the NASP fusion proteins. Antigenic sites preferentially recognized by the individual 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 protein in Western blots, cDNA clones positive for the C-terminal domain of the molecule were identified. The number and location of linear epitopes in this region were determined by synthetic peptide mapping and inhibition studies. The epitope-containing segment was delimited to the sequence aa 619±692 and analysis of a series of 74 concurrent overlapping 9mer synthetic peptides encompassing this region revealed four linear epitopes: amino acid residues IREKIEDAK (aa 648±656), KESQRSGNV (aa 656±664), AELALKATL (aa 665±673) and GFTPGGGGS (aa 680±688). All individual patients' sera reacted with epitopes within the sequence IRE¼.GGS (aa 648±688). The strongest reactivity was displayed by peptides corresponding to the sequence AELALKATL (aa 665±673). Thus, multiple continuous autoimmune epitopes in NASP involving sequences in the conserved C-terminal domain as well as in the less conserved testis-specific N-terminal region comprising the histone-binding sites, as predicted for an antigen-driven immune response, may be a target of autoantibodies in vasectomized men and may provide a relevant laboratory variable to describe more accurately the spectrum of autoantibody specificities associated with the clinical manifestation of vasectomy.
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