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 AF311312), encoding a human sperm component, was determined to consist of 3818 bp with a reading frame of 2778 bp encoding a deduced polypeptide composed of 926 amino acids. A 0.7 kb fragment containing three immunological epitopes of HSD-3.8 cDNA was prepared and used to construct recombinant expression vectors. The constructs were transformed into E.coli BL-21, and the fusion proteins were expressed, isolated and purified. Using the polyclonal antibodies raised against the purified expressed fusion proteins, positive immunostaining occurred over the surface of the postacrosomal zone of human spermatozoa and of germ cells within the seminiferous epithelium of human testis. 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 exhibited marked reduction in their fertility. Analysis of the deduced HSD-3.8 polypeptide revealed the presence of a tetratricopeptide repeat (TPR) motif, a P-loop sequence that acts as a binding site for ATP/GTP and phosphorylation sites for PKC, CK2 and cAMP/cGMP-dependent protein kinases. A blot overlay assay with [alpha-(32)P]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 conclusion, HSD-3.8 is a GTP binding protein and its activity may be regulated by phosphorylation.
Lin
W
W
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 5 Dong Dan 3-Tiao, Beijing 100005, People's Republic of China.
Zhou
X
X
Zhang
M
M
Li
Y
Y
Miao
S
S
Wang
L
L
Zong
S
S
Koide
S S
SS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface; D001667:Binding, Competitive; D015153:Blotting, Western; D018076:DNA, Complementary / Q000302:isolation & purification; D005260:Female; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013094:Spermatozoa / Q000737:chemistry / Q000502:physiology; D016708:Synaptophysin; D013737:Testis / Q000737:chemistry*
D000954:Antigens, Surface; D018076:DNA, Complementary; D011994:Recombinant Proteins; D016708:Synaptophysin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C483942:SPAG1 protein, human