Quantification of Seminal Plasma Motility Inhibitor/Semenogelin in Human Seminal Plasma

A 33-kD glycoprotein, known as the "prostate-specific antigen," was purified to homogeneity from human seminal plasma. The prostatic protein was identified as a serine protease, and its NH2-terminal sequence strongly suggests that it belongs to the family of glandular kallikreins. The structural protein of human seminal coagulum, the predominant protein in seminal vesicle secretion, was rapidly cleaved by the prostatic enzyme, which suggests that this seminal vesicle protein may serve as the physiological substrate for the protease. The prostatic enzyme hydrolyzed arginine- and lysine-containing substrates with a distinct preference for the former. All synthetic substrates tested were poor substrates for the enzyme. Synthetic Factor XIa substrate (pyro-glutamyl-prolyl-arginine-p-nitroanilide), and the synthetic kallikrein substrate (H-D-prolyl-phenylalanyl-arginine-p-nitroanilide) were hydrolyzed with maximum specific activities at 23 degrees C of 79 and 34 nmol/min per mg and Km values of 1.0 and 0.45 mM, respectively. Synthetic substrates for plasmin, chymotrypsin, and elastase were either not hydrolyzed by the enzyme at all, or only hydrolyzed very slowly.

The comparison of measurements of fibronectin and lactoferrin in ejaculates from vasectomized men, subjects with functional deficiency or aplasia of the seminal vesicles, and reference subjects provided evidence that both the fibronectin and the lactoferrin in human seminal fluid originate from the seminal vesicles and the ampullae. The fibronectin is incorporated in the framework of the seminal gel formed during the immediate postejaculatory phase, whereas the lactoferrin remains in solution. In the seminal gel fibronectin is linked to its predominant structural protein, a high molecular weight seminal vesicle protein (semenogelin). Both the gel-bound fibronectin and semenogelin are progressively fragmented and solubilized by the abundant prostatic kallikrein-like protease (prostate-specific antigen) during and after seminal gel liquefaction. Lactoferrin remains essentially unaffected by the seminal proteases.

The protease prostate-specific antigen (PSA) is a marker widely used clinically for monitoring prostatic malignancies. Under normal conditions, this enzyme is mainly involved in the post ejaculation degradation of the major human seminal protein, the seminal plasma motility inhibitor precursor/semenogelin I (SPMIP/SgI), which is the predominant protein component of human semen coagulum. PSA primary structure and activity on synthetic substrates predict a chymotrypsin-like activity whose specificity remains to be established. The present study was aimed at characterizing the proteolytic processing of the SPMIP/SgI by PSA. Purified SPMIP/SgI was incubated with PSA in the presence or absence of protease inhibitors. General serine protease inhibitors, heavy metal cations (Zn2+ and Hg2+), and the heavy metal chelator 1,10-phenanthroline partially or totally inhibited the proteolytic activity of PSA toward SPMIP/SgI. Under identical conditions, other proteins, such as bovine serum albumin, ovalbumin, and casein, were very poor substrates for PSA. Hydrolysis products were separated by reverse-phase high-performance liquid chromatography, assayed for sperm motility inhibitory activity, and analyzed by immunoblotting and mass spectrometry. The region responsible for the sperm motility inhibitory activity and containing an SPMI antiserum epitope was localized to the N-terminal portion of the molecule between residues 85 and 136. On the other hand, a monoclonal antibody against a seminal vesicle-specific antigen (MHS-5) recognized fragments derived from the central part of the SPMIP/SgI (residues 198-223). PSA hydrolysis occurred almost exclusively at either leucine or tyrosine residues, demonstrating directly for the first time a restricted chymotrypsin-like activity on a physiological substrate. The results suggest that PSA is the main enzyme responsible for the processing of SPMIP/SgI in human semen and that this protease manifests unusual specificity with respect to hydrolyzable substrates and sites of hydrolysis.