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Type: Journal article
Title: Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency
Author: Silva, L.
Stoll, T.
Kryza, T.
Stephens, C.
Hastie, M.
Irving-Rodgers, H.
Dong, Y.
Gorman, J.
Clements, J.
Citation: Scientific Reports, 2017; 7(1):6789-1-6789-12
Publisher: Nature Publishing Group
Issue Date: 2017
ISSN: 2045-2322
Statement of
Lakmali Munasinghage Silva, Thomas Stoll, Thomas Kryza, Carson Ryan Stephens, Marcus Lachlan Hastie, Helen Frances Irving-Rodgers, Ying Dong, Jeffrey John Gorman and Judith Ann Clements
Abstract: The cleavage preferences of Kallikrein-related peptidase 7 (KLK7) have previously been delineated using synthetic peptide libraries of fixed length, or single protein chains and have suggested that KLK7 exerts a chymotryptic-like cleavage preference. Due to the short length of the peptides utilised, only a limited number of subsites have however been assessed. To determine the subsite preferences of KLK7 in a global setting, we used a mass spectrometry (MS)-based in-depth proteomics approach that utilises human proteome-derived peptide libraries of varying length, termed Proteomic Identification of protease Cleavage Sites (PICS). Consistent with previous findings, KLK7 was found to exert chymotryptic-like cleavage preferences. KLK7 subsite preferences were also characterised in the P2-P2' region, demonstrating a preference for hydrophobic residues in the non-prime and hydrophilic residues in the prime subsites. Interestingly, single catalytic triad mutant KLK7 (mKLK7; S195A) also showed residual catalytic activity (kcat/KM = 7.93 × 10²s⁻¹M⁻¹). Catalytic inactivity of KLK7 was however achieved by additional mutation in this region (D102N). In addition to characterising the cleavage preferences of KLK7, our data thereby also suggests that the use of double catalytic triad mutants should be employed as more appropriate negative controls in future investigations of KLK7, especially when highly sensitive MS-based approaches are employed.
Keywords: Humans; Pichia; Kallikreins; Proteome; Amino Acid Substitution; Catalytic Domain; Substrate Specificity; Mass Spectrometry; HEK293 Cells; Proteolysis
Description: Published online: 28 July 2017
Rights: © The Author(s) 2017. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit
RMID: 0030090071
DOI: 10.1038/s41598-017-06680-4
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Appears in Collections:Obstetrics and Gynaecology publications

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