Studies on a serine peptidase inhibitor locus on human chromosome 20 Characterization of novel genes encoding proteins with WFDC and kunitz domains

Abstract: An unlimited number of proteolytic reactions are carried out by enzymes in biological systems. These enzymes are a necessity of life, but they can also be dangerous if they are not controlled. Protease inhibitors regulate proteolytic enzymes by interacting and forming complexes with them, thereby preventing the substrate from gaining access to the active site of the enzyme. We have recently identified a locus on human chromosome 20 that encompasses several genes that encode proteins containing one or more of the WFDC and kunitz serine protease domains. Among these genes are the previously described PI3 and SLPI. These new genes were first detected by their sequence similarity to the genes that encode the predominant gel-forming proteins in semen, semenogelin I and semenogelin II. To evaluate the function of these novel proteins, we used RT-PCR to study their expression pattern in a panel of tissues and cell lines. Most genes are ubiquitously expressed, although higher levels of expression have been detected in the male reproductive organs, including the seminal vesicles, epididymis, testes, and prostate. Expression has also been observed in the LNCaP and PC3 prostate cancer cell lines, the former of which is androgen sensitive and the latter androgen independent. The semenogelin genes have previously been described to be rapidly evolving. We identified and compared the locus of four mammalian species, and the results demonstrate that the genes are under evolutionary pressure, which is consistent with those involved in reproduction and/or immune defence. Three recombinant proteins were constructed in a prokaryote system to study their anti-proteolytic capacity. We did not detect any inhibitory effect on a panel of enzymes, possibly due to misfolding of the recombinant proteins. To circumvent this problem, we plan to construct new recombinant proteins in a eukaryotic system to ensure correct folding and disulphide paring.