Expression of a hepatocyte growth-factor activator protein in turkey (Meleagris gallopavo) deferent duct epithelial cells.
In previous research, we discovered that turkey deferent duct epithelial cells express a serine protease. Our experimental objective was to identify the gene that encodes this protein. A lambda phage cDNA library from duct cell mRNA was constructed. The library was screened using monoclonal antibodies previously produced against the turkey deferent-duct serine protease. Phage containing the protease cDNA was excised and re-circularized into plasmids. E. coli were transformed with plasmids containing protease cDNA, which was then isolated for sequencing. NCBI BLAST searches within the GenBank database returned 63.5 and 61.7% identity with murine and human hepatocyte growth-factor activator (HGFA) precursor, respectively. The turkey protease cDNA was then cloned into the pQE-32 expression vector and transformed into M15 cells for HIS-tagged expression of the recombinant protein, which was then purified using nickel-chelated Sepharose spin columns. Afterwards, Western blot analysis of the purified recombinant turkey protein revealed recognition by a monoclonal antibody specific to the proteolytic subunit of the turkey deferent duct protease. Therefore, these findings indicate that the recombinant HGFA precursor isolated from the deferent duct is the turkey seminal plasma protease that is secreted from the deferent duct. HGFA, a member of the Kringle-serine proteinase superfamily, can initiate diverse mitogenic, morphogenic and motogenic effects through its substrate hepatocyte growth factor. Although the presence of hepatocyte growth factor and its c-MET receptor have been reported in male mammalian reproductive tracts, our novel findings on the secretion of HGFA precursor from turkeys may help to elucidate the regulation of activated hepatocyte growth factor.
Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 29634, USA.
D R
DR
Holsberger
A E
AE
Becker
R J
RJ
Thurston
C D
CD
Rice
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012661:Semen / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014422:Turkeys / Q000033:anatomy & histology / Q000378:metabolism*; D014649:Vas Deferens / Q000166:cytology* / Q000378:metabolism
D011994:Recombinant Proteins; C081092:HGF activator; D012697:Serine Endopeptidases