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.
Highlights
Human Kallikrein-related peptidase 7 (KLK7) is a member of a family composed of 15 serine peptidases clustered on chromosome 19q13.41–3 and reported to be involved in many pathological conditions, including skin inflammation, ovarian and many other cancers[4,5,6,7,8], its mechanism of action is still not known
To further characterise the substrate specificity of KLK7, we employed a peptide-centric proteomics approach, which utilises human proteome-derived peptide libraries and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), termed Proteomic Identification of protease Cleavage Sites (PICS)[13] to determine KLK7 substrate preferences on both the prime and non-prime sides simultaneously
We produced S195A mKLK4, which showed residual catalytic activity. These observations of residual catalytic activity of single mKLK4 and mutant KLK7 (mKLK7) when mutated at the Ser residue led us to make an additional point mutation in both the mKLK7 and mKLK4 catalytic triad, D102N, which showed no catalytic activity in subsequent assays
Summary
Human Kallikrein-related peptidase 7 (KLK7) is a member of a family composed of 15 serine peptidases clustered on chromosome 19q13.41–3 and reported to be involved in many pathological conditions, including skin inflammation, ovarian and many other cancers[4,5,6,7,8], its mechanism of action is still not known. Thereby, we determined that KLK7 shows chymotryptic-like cleavage specificity by cleaving C-terminal to hydrophobic amino acids, tyrosine www.nature.com/scientificreports/. This study is the first study to determine KLK7 cleavage specificities in a more global manner, analyzing subsite preferences of KLK7 on both prime and non-prime sites simultaneously. Mutant KLK7 (mKLK7) carrying the single mutation (S195A) in the catalytic triad has been widely used in in vitro cell-based protease research[14], the activity has not been measured using more highly sensitive in-depth proteomics approaches. We observed that the single mKLK7 has residual chymotryptic-like activity by cleaving C-terminal to Tyrosine (Y), Leucine (L) and Phenylalanine (F) at the P1 position in line with active KLK7 (kcat/KM = 7.93 × 102 s−1M−1). We produced S195A mKLK4, which showed residual catalytic activity (kcat/KM = 6.13 × 103 s−1M−1). These observations of residual catalytic activity of single mKLK4 and mKLK7 when mutated at the Ser residue led us to make an additional point mutation in both the mKLK7 and mKLK4 catalytic triad, D102N (asparagine), which showed no catalytic activity in subsequent assays
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