Detection and Identification of Active Serine Proteases in the Lower Female Reproductive Tract: A Functional Approach

Proteases irreversibly modify proteins by hydrolyzing the bonds between amino acids and are implicated in virtually every significant biological process. Many proteases have previously been identified in cervical-vaginal fluid (CVF), the fluid covering the tissues of the lower female reproductive tract, and have been linked to a myriad of processes including the susceptibility to sexually transmitted diseases, pain sensitivity during sexual intercourse, extracellular matrix remodeling and vaginal epithelial desquamation. Interestingly, human CVF differs from other mammals in that it is uniquely acidic with levels of 4.5 or less which poses a substantial challenge for proteolytic activity in this environment. An integrated proteomic and peptidomic analysis of CVF confirmed that approximately 8% of the CVF proteome consists of proteases, predominantly serine proteases. Detailed proteomic analysis of CVF samples collected throughout the follicular and luteal phases of the menstrual cycle demonstrated that the CVF proteome remains consistent throughout the menstrual cycle. Functional proteomics using an activity-based probe (ABP) has emerged as a powerful tool to study active proteases within complex proteomes. In the current study, ABP profiling of CVF resulted in the identification of transmembrane protein serine 11D (TMPRSS11D) and kallikrein-related peptidase 13 (KLK13) as active trypsin-like serine proteases. Immunoassays showed that KLK13 is highly abundant in CVF and the expression of a panel of KLKs is not affected by the menstrual cycle phase. The finding that multiple KLKs are present at high abundance levels in CVF strengthens the hypothesis that the reproductive tissues and associated fluids are an important site for proteolytic functioning. We also observed a near complete cessation of trypsin-like proteolytic activity at physiological pH levels in CVF. Therefore, we propose a novel mechanism in which conditions characterized by an elevated pH in CVF, such as male ejaculation, postmenopausal status and bacterial infection, provide a window of opportunity for proteolytic activity in the lower female reproductive tract. Considering the fact that the proteases are involved in virtually every significant biological process, understanding the protease activity and functioning in CVF can provide important insights in their mechanism of action and ultimately lead to novel protease-targeted therapeutics.

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