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

ObjectiveTo study the molecular mechanism of epididymal protease inhibitor (Eppin) modulating the liquafication of semen.MethodsHuman semenogelin cDNA (nucleotides 82-849) and Eppin cDNA (nucleotides 70-423) were generated by PCR and cloned into pET-100D/TOPO. Recombinant Eppin and Sg were produced by BL21 (DE3). The association of Eppin with Sg was studied by far-western and radioautography. In vitro the digestion of Sg by PSA in the presence or absence of recombinant Eppin 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 C-terminal Eppin (aa75-133). Recombinant Sg was digested with PSA, many low molecular weight fragments were produced, when Eppin is bound to Sg, then digested by PSA, producing incomplete digestion and a 14.5-14.8 kDa fragmen. Antibody binding to the N-terminal of Eppin did not affect Sg digestion. Addition of antibodies to the C-terminal of Eppin inhibited the modulating effects of Eppin.ConclusionEppin modulates the digestion activity of PSA through binding Sg. The active site locates at C-terminal.