Article thumbnail
Location of Repository

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

By 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 (k(cat)/K-M = 7.93 x 10(2) s(-1)M(-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

Topics: Corneum Chymotryptic Enzyme, Ovarian-Cancer Cells, Stratum-Corneum, Gene Family, Active-Site, Hormonal-Regulation, Serine Proteases, Catalytic Triad, Specificity, Expression, 1000 General
Publisher: Nature Publishing Group
Year: 2017
DOI identifier: 10.1038/s41598-017-06680-4
OAI identifier: oai:espace.library.uq.edu.au:UQ:6694896
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • https://espace.library.uq.edu.... (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.