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Functional proteomic profiling reveals KLK13 and TMPRSS11D as active proteases in the lower female reproductive tract.

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.

SUBMITTER: Muytjens CMJ

PROVIDER: S-EPMC6329257 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Genes involved in post-mating processes of multiple mating organisms are known to evolve rapidly due to coevolution driven by sexual conflict among male-female interacting proteins. In the malaria mosquito Anopheles gambiae - a monandrous species in which sexual conflict is expected to be absent or minimal - recent data strongly suggest that proteolytic enzymes specifically expressed in the female lower reproductive tissues are involved in the processing of male products transferred to females during mating. In order to better understand the role of selective forces underlying the evolution of proteins involved in post-mating responses, we analysed a cluster of genes encoding for three serine proteases that are down-regulated after mating, two of which specifically expressed in the atrium and one in the spermatheca of A. gambiae females.The analysis of polymorphisms and divergence of these female-expressed proteases in closely related species of the A. gambiae complex revealed a high level of replacement polymorphisms consistent with relaxed evolutionary constraints of duplicated genes, allowing to rapidly fix novel replacements to perform new or more specific functions. Adaptive evolution was detected in several codons of the 3 genes and hints of episodic selection were also found. In addition, the structural modelling of these proteases highlighted some important differences in their substrate specificity, and provided evidence that a number of sites evolving under selective pressures lie relatively close to the catalytic triad and/or on the edge of the specificity pocket, known to be involved in substrate recognition or binding. The observed patterns suggest that these proteases may interact with factors transferred by males during mating (e.g. substrates, inhibitors or pathogens) and that they may have differently evolved in independent A. gambiae lineages.Our results - also examined in light of constraints in the application of selection-inference methods to the closely related species of the A. gambiae complex - reveal an unexpectedly intricate evolutionary scenario. Further experimental analyses are needed to investigate the biological functions of these genes in order to better interpret their molecular evolution and to assess whether they represent possible targets for limiting the fertility of Anopheles mosquitoes in malaria vector control strategies.

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Functional proteomic profiling reveals KLK13 and TMPRSS11D as active proteases in the lower female reproductive tract.

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