title: Molecular mechanism of epididymal protease inhibitor modulating the liquefaction of human semen.
abstract: 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.
authors.affiliation: Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. zengjunwang2002@hotmail.com
authors.forename: Zeng-Jun
authors.initials: ZJ
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Ning-Han
authors.initials: NH
authors.lastname: Feng
authors.forename: Ning-Hong
authors.initials: NH
authors.lastname: Song
authors.forename: Hong-Fei
authors.initials: HF
authors.lastname: Wu
authors.forename: Yuan-Geng
authors.initials: YG
authors.lastname: Sui
mesh_terms: 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
chemical_list: 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