A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein.
We have previously indicated that at least in mouse, sperm serine protease(s) other than acrosin probably act on the limited proteolysis of egg zona pellucida to create a penetration pathway for motile sperm, although the participation of acrosin cannot be ruled out completely. A 42-kDa gelatin-hydrolyzing serine protease present in mouse sperm is a candidate enzyme involved in the sperm penetration of the zona pellucida. In this study, we have PCR-amplified an EST clone encoding a testicular serine protease, termed TESP5, and then screened a mouse genomic DNA library using the DNA fragment as a probe. The DNA sequence of the isolated genomic clones indicated that the TESP5 gene is identical to the genes coding for testicular testisin and eosinophilic esp-1. Immunochemical analysis using affinity-purified anti-TESP5 antibody revealed that 42- and 41-kDa forms of TESP5 with the isoelectric points of 5.0 to 5.5 are localized in the head, cytoplasmic droplet, and midpiece of cauda epididymal sperm probably as a membranous protein. Moreover, these two forms of TESP5 were selectively included into Triton X-100-insoluble microdomains, lipid rafts, of the sperm membranes. These results show the identity between TESP5/testisin/esp-1 and the 42-kDa sperm serine protease. When HEK293 cells were transformed by an expression plasmid carrying the entire protein-coding region of TESP5, the recombinant protein produced was released from the cell membrane by treatment with Bacillus cereus phosphatidylinositol-specific phospholipase C, indicating that TESP5 is glycosylphosphatidylinositol-anchored on the cell surface. Enzymatic properties of recombinant TESP5 was similar to but distinguished from those of rat acrosin and pancreatic trypsin by the substrate specificity and inhibitory effects of serine protease inhibitors.
Honda
Arata
A
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan.
Yamagata
Kazuo
K
Sugiura
Shin
S
Watanabe
Katsuto
K
Baba
Tadashi
T
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004804:Eosinophils / Q000378:metabolism; D030501:Epididymal Secretory Proteins / Q000378:metabolism; D005091:Exons; D020224:Expressed Sequence Tags; D058851:GPI-Linked Proteins; D015723:Gene Library; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007438:Introns; D007525:Isoelectric Focusing; D008297:Male; D021962:Membrane Microdomains / Q000201:enzymology*; D008565:Membrane Proteins; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000201:enzymology*; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D013737:Testis / Q000378:metabolism; D014018:Tissue Distribution; D010738:Type C Phospholipases / Q000378:metabolism
D003902:Detergents; D030501:Epididymal Secretory Proteins; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; C426183:Lcn5 protein, rat; D008565:Membrane Proteins; D011994:Recombinant Proteins; D017830:Octoxynol; D004247:DNA; D010738:Type C Phospholipases; C120203:PRSS21 protein, human; C456340:Prss21 protein, mouse; D012697:Serine Endopeptidases