The human kallikrein protein 5 (hK5) is enzymatically active, glycosylated and forms complexes with two protease inhibitors in ovarian cancer fluids

Abstract

The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Binding of kallikreins to protease inhibitors is an important mechanism for regulating their enzymatic activity and may have potential clinical applications. Human kallikrein gene 5 ( KLK5) is a member of this family and encodes for a secreted serine protease (hK5). This kallikrein was shown to be differentially expressed at the mRNA and protein levels in diverse malignancies. Our objective was to study the enzymatic activity and the interaction of recombinant hK5 protein with protease inhibitors. Recombinant hK5 protein was produced in yeast and mammalian expression systems and purified by chromatography. HPLC fractionation, followed by ELISA-type assays, immunoblotting and radiolabeling experiments were performed to detect the possible interactions between hK5 and proteinase inhibitors in serum. Enzymatic deglycosylation was performed to examine the glycosylation pattern of the protein. The enzymatic activity of hK5 was tested using trypsin and chymotrypsin-specific synthetic fluorogenic substrates. In serum and ascites fluid, in addition to the free (∼40 kDa) form, hK5 forms complexes with α 1-antitrypsin and α 2-macroglobulin. These complexes were detected by hybrid ELISA-type assays using hK5-specific coating antibodies and inhibitor detection antibodies. The ability of hK5 to bind to these inhibitors was further verified in vitro. Spiking of serum samples with 125I-labeled hK5 results in the distribution of the protein in two higher molecular mass (bound) forms, in addition to the unbound form. The hK5 mature enzyme is active and shows trypsin, but not chymotrypsin-like, activity. The pro-form of hK5 is not active. Recombinant hK5 shows a higher than predicted molecular mass due to glycosylation. hK5 is partially complexed with α 1-antitrypsin and α 2-macroglobulin in serum and ascites fluid of ovarian cancer patients. The recombinant protein is glycosylated and its mature form shows trypsin-like activity.