Asian Journal of Andrology 2008;10(5):770-775

doi:10.1111/j.1745-7262.2008.00393.x

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

Zeng-Jun WANG 1 ; Wei ZHANG ; Ning-Han FENG ; Ning-Hong SONG ; Hong-Fei WU ; Yuan-Geng SUI

Affiliations

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Country

China

Language

English

MeSH

Abstract

AIMTo study the molecular mechanism of epididymal protease inhibitor (Eppin) modulating the process of prostate specific antigen (PSA) digesting semenogelin (Sg).

METHODSHuman Sg cDNA (nucleotides 82-849) and Eppin cDNA (nucleotides 70-723) were generated by polymerase chain reaction (PCR) and cloned into pET-100D/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.

RESULTSEppin 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.

CONCLUSIONEppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.