Objective: To study the molecular mechanism of epididymal protease inhibitor(Eppin) modulating the liquafication of semen. Methods: Human semenogelin cDNA(nucleotides 82-849) and Eppin cDNA(nucleotides 70-423) were ge...Objective: To study the molecular mechanism of epididymal protease inhibitor(Eppin) modulating the liquafication of semen. Methods: Human 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. Results: Eppin 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. Conclusion: Eppin modulates the digestion activity of PSA through binding Sg.The active site locates at C-terminal.展开更多
文摘Objective: To study the molecular mechanism of epididymal protease inhibitor(Eppin) modulating the liquafication of semen. Methods: Human 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. Results: Eppin 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. Conclusion: Eppin modulates the digestion activity of PSA through binding Sg.The active site locates at C-terminal.
