Molecular characterization of the 32 kDa boar sperm protease.
The purified 32 kDa boar sperm protease, known as the 32 kDa sperminogen, was identified by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis following its partial peptide sequencing. The 32 kDa boar sperm protease was purified from the acid extracts of boar spermatozoa and subjected to CNBr-digestion. The most prominently digested 30 kDa product was purified by HPLC and its peptide sequence was analyzed. NCBI Blast search of the analyzed 21 amino acid sequence revealed that the sequence matched 91% with that of proacrosin. DNA primer was deduced from the analyzed peptide sequence and the 32 kDa protease was further identified by RT-PCR. Upon RT-PCR, 1 Kbp DNA fragment was amplified, which is the expected length if the product was amplified from the proacrosin mRNA, implying that there is no separate mRNA for the 32 kDa sperminogen. To confirm these results, Northern blot analysis was performed. Four DNA probes generated from the exons of proacrosin genomic DNA sequence all detected a single species of mRNA, suggesting that there is no separate mRNA for the 32 kDa sperminogen which might be produced either from the potential separate 32 kDa sperminogen gene or by differential splicing from proacrosin mRNA. These results strongly suggest that the 32 kDa protease is part of the proacrosin/acrosin system, and that the 32 kDa sperminogen might be formed from post-translational processing of proacrosin.
Pyoung
Ilsun
I
Department of Biological Science and the Institute of Basic Sciences, Sungkyunkwan University, Suwon, Korea.
Yi
Lee S H
LS
D000176:Acrosin / Q000235:genetics*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D030562:Databases, Protein; D004792:Enzyme Precursors / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029589:Seminal Plasma Proteins / Q000737:chemistry / Q000235:genetics*; D013094:Spermatozoa / Q000201:enzymology*; D013552:Swine
D004792:Enzyme Precursors; D029589:Seminal Plasma Proteins; D010447:Peptide Hydrolases; C022438:proacrosin; D000176:Acrosin