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TMPRSS11D As Active Proteases in the Lower Female Reproductive Tract [Version 2; Peer Review: 2 Approved]

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TMPRSS11D As Active Proteases in the Lower Female Reproductive Tract [Version 2; Peer Review: 2 Approved] F1000Research 2018, 7:1666 Last updated: 26 JUL 2021 RESEARCH ARTICLE Functional proteomic profiling reveals KLK13 and TMPRSS11D as active proteases in the lower female reproductive tract [version 2; peer review: 2 approved] Carla M.J. Muytjens1,2, Yijing Yu1,2, Eleftherios P. Diamandis 1-3 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada 2Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Toronto, Canada 3Department of Clinical Biochemistry, University Health Network, Canada, Toronto, Canada v2 First published: 19 Oct 2018, 7:1666 Open Peer Review https://doi.org/10.12688/f1000research.16255.1 Latest published: 31 Dec 2018, 7:1666 https://doi.org/10.12688/f1000research.16255.2 Reviewer Status Invited Reviewers Abstract Background: Cervical-vaginal fluid (CVF) hydrates the mucosa of the 1 2 lower female reproductive tract and is known to contain numerous proteases. The low pH of CVF (4.5 or below in healthy women of version 2 reproductive age) is a uniquely human attribute and poses a challenge (revision) report for the proteolytic functioning of the proteases identified in this 31 Dec 2018 complex biological fluid. Despite the abundance of certain proteases in CVF, the proteolytic activity and function of proteases in CVF is not version 1 well characterized. 19 Oct 2018 report report 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) 1. Georgios Pampalakis , University of proteomics has evolved as a powerful tool to investigate active Patras, Patras, Greece proteases within complex proteomes and a trypsin-specific ABP was used to identify active proteases in CVF. 2. Céline Deraison , INSERM, Toulouse, Results: Serine proteases are among the most abundant proteins in France the CVF proteome. Labeling human CVF samples with the trypsin- specific ABP revealed serine proteases transmembrane protein serine Any reports and responses or comments on the 11D and kallikrein-related peptidase 13 as active proteases in CVF. article can be found at the end of the article. 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. Keywords cervical-vaginal fluid, serine proteases, activtiy-based probe proteomics Page 1 of 19 F1000Research 2018, 7:1666 Last updated: 26 JUL 2021 Corresponding author: Eleftherios P. Diamandis ([email protected]) Author roles: Muytjens CMJ: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Yu Y: Data Curation, Formal Analysis, Investigation, Validation, Visualization, Writing – Review & Editing; Diamandis EP: Conceptualization, Project Administration, Supervision, Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2018 Muytjens CMJ et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Muytjens CMJ, Yu Y and Diamandis EP. Functional proteomic profiling reveals KLK13 and TMPRSS11D as active proteases in the lower female reproductive tract [version 2; peer review: 2 approved] F1000Research 2018, 7:1666 https://doi.org/10.12688/f1000research.16255.2 First published: 19 Oct 2018, 7:1666 https://doi.org/10.12688/f1000research.16255.1 Page 2 of 19 F1000Research 2018, 7:1666 Last updated: 26 JUL 2021 to study active proteases within complex proteomes13,14. ABPs, REVISE D Amendments from Version 1 designed to target certain classes of active proteases, have been The following changes were made in the manuscript: successfully applied to investigate ABP-mediated profiling Methods: of complex proteomes, inhibitor screening and enzymatic 15,16 Validation of KLK13 in CVF section target identification . We provided a reference for the production of the monoclonal KLK13 antibody used in this study. In the current study, ABP profiling of CVF resulted in the iden- Results: tification of transmembrane protein serine 11D (TMPRSS11D) Investigation of proteolytic activity in CVF section and kallikrein-related peptidase 13 (KLK13) as active trypsin- Figure 2: we have adjusted the wording in the legend to include like proteases. In addition, we observed a near complete the explanation of the relative trypsin-like activity. cessation of trypsin-like proteolytic activity at physiological Identification of active proteases TMPRSS11D and KLK13 pH levels in CVF. Therefore, we propose a novel mechanism in section which fluctuations in CVF pH have the potential to regulate Revised Figure 4 to highlight KLK13 and TMPRSS11D. We have the catalytic activity in the lower female reproductive tract. adjusted the legend of the figure to reflect this change. Understanding the protease activity and functioning in CVF Validation of TMPRSS11D and KLK13 in CVF section can provide important insights in their mechanism of action and We have changed the wording in the manuscript from 225NRT to ultimately lead to novel protease targeted therapeutics. 225N. Discussion: Methods We changed the following sentences in the discussion section: Sample collection “This powerful approach resulted in the identification of serine CVF samples were collected from 10 healthy, non-pregnant proteases KLK13 and TMPRSS11D as major active proteolytic enzymes in CVF. ABP proteomics resulted in the identification female volunteers using a softcup collection device (Instead Inc. 7,17 of KLK13 and TMPRSS11D as active proteases in this complex San Diego, CA) as described previously . Informed consent biological fluid” as suggested by the reviewer. was obtained from all participants prior to sample collection. We have included the appropriate reference to support the The collection and use of human samples was approved by the biological concentrations of KLKs in the CVF and the male Research Ethic Board at Mount Sinai Hospital in Toronto (MSH reproductive system. REB 16-0137-E). All women abstained from sexual activity and See referee reports douching at least 3 days prior to sample collection and had no self-reported vaginal infections. The sample was collected after 90 minutes via centrifugation (Eppendorf centrifuge, model Introduction 5415R) of the softcup inside a 50mL conical tube at 200xg for Approximately 2–4% of the genes in the human genome account 2 minutes. CVF was transferred to a 1.5mL tube and centri- for proteases, and in addition, over 150 different protease inhibi- fuged at 15,000xg for 20 minutes to remove cellular debris. Total tors are encoded in the human genome1. Proteases irreversibly protein of each CVF sample was determined using a Bradford modify proteins via peptide bond hydrolysis and thereby impact protein assay (Thermo Fisher Scientific, #1856209)). The samples on both protein structure and function. As a result, proteases are were stored at -80°C until further use. implicated in nearly every significant biological process. Estimation of protease abundance in CVF proteome Cervical-vaginal fluid (CVF) hydrates the mucosa of the vagina The estimation of protease abundance in the CVF proteome and lower part of the cervix. Situated directly at the interface was based on the CVF proteome dataset from healthy non- between the external environment and the internal milieu, pregnant women7. Proteases were identified by comparison with CVF plays key roles in sexual intercourse, conception and the Merops database and grouped according to their catalytic provides a barrier against microbial invasion2–6. Almost 8% of type1. The log transformed MS1 area data were used as a proxy all proteins identified in CVF are proteases, which highlights for the relative abundance of each protein within the dataset. the tremendous proteolytic potential of this complex biological CVF proteins and proteases were subsequently ranked accord- fluid7. Interestingly, human CVF differs from other mammals ing to the relative abundance and plots were produced using R in that it is uniquely acidic with levels of 4.5 or less in healthy (R version 3.5.0). women of reproductive age8,9. The low pH is estrogen dependent and results from the conversion of glycogen to lactic acid by Investigation of proteolytic activity in CVF the vaginal microbiome or, alternatively, the vaginal lumen The trypsin-like proteolytic activity in CVF was measured by is acidified via active proton secretion across the vaginal incubating 0.1µg total protein CVF sample with 0.5mM of the epithelial membrane10–12. The acidity levels in CVF pose a fluorogenic t-butoxycarbonyl-tri-peptide-7amino-4-methylcou- substantial challenge for protease activity in this environment. marin (AMC) synthetic peptide (Bachem, #4003460) with a Despite the high abundance of proteases in CVF, their activity valine
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