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Molecular mechanism of epididymal protease inhibitor modulating the liquefaction of human semen

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Abstract

Aim: To study the molecular mechanism of epididymal protease inhibitor (Eppin) modulating the process of prostate specific antigen (PSA) digesting semenogelin (Sg). Methods: Human Sg cDNA (nucleotides 82–849) and Eppin cDNA (nucleotides 70–423) were generated by polymerase chain reaction (PCR) and cloned into pET-lOOD/TOPO. Recombinant Eppin and Sg (rEppin and rSg) were produced by BL21 (DE3). The association of Eppin with Sg was studied by far-western immunoblot and radioautography In vitro the digestion of rSg by PSA in the presence or absence of rEppin was studied. The effect of anti-Q20E (N-terminal) and C-terminal of Eppin on Eppin-Sg binding was monitored. Results: Eppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin (amino acids 75–133). rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results. Antibody binding to the N-terminal of rEppin did not affect rSg digestion. Addition of antibodies to the C-terminal of rEppin inhibited the modulating effect of rEppin. Conclusion: Eppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.
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Keywords: epididymal protease inhibitor; prostate specific antigen; semenogelin

Document Type: Original Article

Affiliations: Laboratory of Reproductive Medicine, Department of Urology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China

Publication date: 2008-09-01

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