Abstract
Background: Cervical-vaginal fluid (CVF) hydrates the mucosa of the lower female reproductive tract and is known to contain numerous proteases. The low pH of CVF (4.5 or below in healthy women of reproductive age) is a uniquely human attribute and poses a challenge for the proteolytic functioning of the proteases identified in this complex biological fluid. Despite the abundance of certain proteases in CVF, the proteolytic activity and function of proteases in CVF is not well characterized. Methods: In the present study, we employed fluorogenic substrate screening to investigate the influence of pH and inhibitory compounds on the proteolytic activity in CVF. Activity-based probe (ABP) proteomics has evolved as a powerful tool to investigate active proteases within complex proteomes and a trypsin-specific ABP was used to identify active proteases in CVF. Results: Serine proteases are among the most abundant proteins in the CVF proteome. Labeling human CVF samples with the trypsin-specific ABP revealed serine proteases transmembrane protein serine 11D and kallikrein-related peptidase 13 as active proteases in CVF. Furthermore, we demonstrated that the proteolytic activity in CVF is highly pH-dependent with an almost absolute inhibition of trypsin-like proteolytic activity at physiological pH levels. Conclusions: These findings provide a framework to understand proteolytic activity in CVF. Furthermore, the present results provide clues for a novel regulatory mechanism in which fluctuations in CVF pH have the potential to control the catalytic activity in the lower female reproductive tract.
Highlights
2–4% of the genes in the human genome account for proteases, and in addition, over 150 different protease inhibitors are encoded in the human genome[1]
We changed the following sentences in the discussion section: “This powerful approach resulted in the identification of serine proteases KLK13 and TMPRSS11D as major active proteolytic enzymes in Cervical-vaginal fluid (CVF)
Activity-based probe (ABP) proteomics resulted in the identification of KLK13 and TMPRSS11D as active proteases in this complex biological fluid” as suggested by the reviewer
Summary
2–4% of the genes in the human genome account for proteases, and in addition, over 150 different protease inhibitors are encoded in the human genome[1]. Almost 8% of all proteins identified in CVF are proteases, which highlights the tremendous proteolytic potential of this complex biological fluid[7]. Despite the high abundance of proteases in CVF, their activity and functioning in the lower female reproductive tract remains to be elucidated. Cervical-vaginal fluid (CVF) hydrates the mucosa of the lower female reproductive tract and is known to contain numerous proteases. The low pH of CVF (4.5 or below in healthy women of reproductive age) is a uniquely human attribute and poses a challenge for the proteolytic functioning of the proteases identified in this complex biological fluid. The present results provide clues for a novel regulatory mechanism in which fluctuations in CVF pH have the potential to control the catalytic activity in the lower female reproductive tract
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