Molecular mechanism of epididymal protease inhibitor modulating the liquefaction of human semen.
OBJECTIVE
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 70-723) 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.


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.


CONCLUSIONS
Eppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.
Wang
Zeng-Jun
ZJ
Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. zengjunwang2002@hotmail.com
Zhang
Wei
W
Feng
Ning-Han
NH
Song
Ning-Hong
NH
Wu
Hong-Fei
HF
Sui
Yuan-Geng
YG
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001345:Autoradiography; D006801:Humans; D006868:Hydrolysis; D008297:Male; D017430:Prostate-Specific Antigen / Q000378:metabolism*; D053491:Proteinase Inhibitory Proteins, Secretory / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012661:Semen / Q000166:cytology / Q000378:metabolism*; D029607:Seminal Vesicle Secretory Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism
D000906:Antibodies; C431553:Eppin protein, human; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins; D029607:Seminal Vesicle Secretory Proteins; C424482:seminal vesicle-specific antigen; D017430:Prostate-Specific Antigen