Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency

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...

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Autores principales:	Lakmali Munasinghage Silva, Thomas Stoll, Thomas Kryza, Carson Ryan Stephens, Marcus Lachlan Hastie, Helen Frances Irving-Rodgers, Ying Dong, Jeffrey John Gorman, Judith Ann Clements
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0ce0071cd33d4881b0d18b43301cfac5
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spelling	oai:doaj.org-article:0ce0071cd33d4881b0d18b43301cfac52021-12-02T12:32:55ZMass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency10.1038/s41598-017-06680-42045-2322https://doaj.org/article/0ce0071cd33d4881b0d18b43301cfac52017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-06680-4https://doaj.org/toc/2045-2322Abstract 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 × 102 s−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.Lakmali Munasinghage SilvaThomas StollThomas KryzaCarson Ryan StephensMarcus Lachlan HastieHelen Frances Irving-RodgersYing DongJeffrey John GormanJudith Ann ClementsNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-12 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Lakmali Munasinghage Silva Thomas Stoll Thomas Kryza Carson Ryan Stephens Marcus Lachlan Hastie Helen Frances Irving-Rodgers Ying Dong Jeffrey John Gorman Judith Ann Clements Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency
description	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 × 102 s−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.
format	article
author	Lakmali Munasinghage Silva Thomas Stoll Thomas Kryza Carson Ryan Stephens Marcus Lachlan Hastie Helen Frances Irving-Rodgers Ying Dong Jeffrey John Gorman Judith Ann Clements
author_facet	Lakmali Munasinghage Silva Thomas Stoll Thomas Kryza Carson Ryan Stephens Marcus Lachlan Hastie Helen Frances Irving-Rodgers Ying Dong Jeffrey John Gorman Judith Ann Clements
author_sort	Lakmali Munasinghage Silva
title	Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency
title_short	Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency
title_full	Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency
title_fullStr	Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency
title_full_unstemmed	Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency
title_sort	mass spectrometry-based determination of kallikrein-related peptidase 7 (klk7) cleavage preferences and subsite dependency
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/0ce0071cd33d4881b0d18b43301cfac5
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Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency

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