Expression and function of the HSD-3.8 gene encoding a testis-specific protein

The nucleotide sequence of the full length HSD-3.8 cDNA (accession number A F311312), encoding a human sperm component, was determined to consist of 38 18 bp with a reading frame of 2778 bp encoding a deduced polypeptide compos ed of 926 amino acids. A 0.7 kb fragment containing three immunological epi topes of HSD-3.8 cDNA was prepared and used to construct recombinant expres sion vectors. The constructs were transformed into E.coli BL21, and the fus ion proteins were expressed, isolated and purified. Using the polyclonal an tibodies raised against the purified expressed fusion proteins, positive im munostaining occurred over the surface of the postacrosomal. zone of human spermatozoa and of germ cells within the seminiferous epithelium of human t estis. Intense staining of large pachytene primary spermatocytes occurred. The capacity of the recombinant protein to reduce fertility as an immunogen in adult female rats was assessed. Immunized animals were infertile or exh ibited marked reduction in their fertility. Analysis of the deduced HSD-3.8 polypeptide revealed the presence of a tetratricopeptide repeat (TPR) moti f, a P-loop sequence that acts as a binding site for ATP/GTP and phosphoryl ation sites for PKC, CK2 and cAMP/cGMP-dependent protein kinases. A blot ov erlay assay with [alpha-P-32]GTP showed that the polypeptide encoded by the 0.7 kb fragment of HSD-3.8 is a GTP binding protein. It was also shown to possess GTPase activity and to be phosphorylated by PKC in vitro. In conclu sion, HSD-3.8 is a GTP binding protein and its activity may be regulated by phosphorylation.