Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)-A CleavEx Based Analysis

Abstract

Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we developed a peptide, fusion protein-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences. Initial assessment identified ten MMP activation domain sequences which were validated by Edman degradation. The analysis revealed that membrane-type MMPs (MT-MMPs) are targeted by KLK14 for activation. Correspondingly, proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT-MMPs was confirmed by Edman degradation. The effectiveness of proMMP activation was analyzed by gelatin zymography, confirming the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation. As both, KLKs and MT-MMPs, are implicated in cancer, their cross-activation may constitute an important factor in tumor progression and metastasis.

Keywords: CleavEx; fusion protein; kallikrein 14; membrane-type MMP; zymogen activation.

Figure 1

Effect of KLK14 on the CleavEx_proMMP fusion proteins. (A) Western blot analysis of each 25 ng CleavEx_proMMP protein incubated with 50 and 250 nM KLK14 after 1 h at 37 °C. Each fusion protein with its respective activation sequence is listed with the native site of hydrolysis indicated by an asterisk. (B) Schematic representation of the CleavEx_proMMP fusion proteins from the Western blots in panel A. Scoring was performed by densitometry analysis using ImageJ. The shading is based on the quartile of change: 100–75% of control sample intensity is presented as white (no degradation); 75–50% as light grey; 50–25% as dark grey; and 25% and lower as black. KLK = kallikrein-related peptidase; CleavEx = Cleavage of exposed amino acid sequences; MMP = matrix metalloproteinase.

Figure 2

Concentration-dependent processing of proMMPs by KLK14. Each respective proMMP was incubated with increasing concentrations of KLK14 (A,C,E–G) or furin (B,D) for 1 h at 37 °C in the presence of 5 µM batimastat, and the reaction products were analyzed by SDS-PAGE and visualized with Coomassie staining. Bands denoted with arrows were identified by N-terminal sequencing using Edman degradation (Table 2). KLK = kallikrein-related peptidase; MMP = matrix metalloproteinase.

Figure 3

Recombinant human proMMP14,15, 16, and 17 are processed by KLK14 in a time dependent manner. (A–D) Respective proMMPs were incubated with KLK14 for 5, 15, 30, 60, 120, and 180 min, at 37 °C in the presence of 5 µM batimastat. The reaction products at indicated timepoints were analyzed by SDS-PAGE and visualized via Coomassie staining. KLK = kallikrein-related peptidase; MMP = matrix metalloproteinase.

Figure 4

Gelatin zymography of proMMPs by KLK14-mediated processing. Activation of proMMPs by KLK14 results in a fully functional mature enzyme. Each proMMP was incubated with the indicated concentrations of KLK14 for 1 h at 37 °C. The reaction was stopped by the addition of KLK14-specific inhibitors, and the reaction mixture was analyzed by SDS-PAGE, followed by a zymogram with gelatin as a substrate. The proMMP2 (A) negative control was not activated. ProMMP14 (B), proMMP15 (C), and proMMP16 (D) were activated, whereas proMMP17 (E) did not show hydrolysis of gelatin; yet a shift corresponding to the loss of the profragment was observed (note that an amino acid substitution was introduced in proMMP17 by the manufacturer (R&D Systems, Abingdon, United Kingdom)). KLK = kallikrein-related peptidase; MMP = matrix metalloproteinase.

Figure 5

Comparison of recombinant human proMMP14 activation by KLK14 and furin as a functional peptidase. ProMMP14 (10 nM) was incubated with KLK14 (3 nM) and furin (3 nM) in the presence of a fluorogenic substrate Mca-KPLGL-Dpa-AR-NH2. The final concentration of the substrate was 10 μM in the MMP reaction buffer (50 mM Tris, 3 mM CaCl2, 1 µM ZnCl2, pH 8.5). Hydrolysis was recorded for 180 min at 37 °C. The velocities were plotted in triplicates as mean ± SD using GraphPad Prism. Please note that the error bars may be occluded by the point markers and are not visible for some of the data points. KLK = kallikrein-related peptidase; MMP = matrix metalloproteinase.

Figure 6

Processing of cell surface proMMP14 by KLK14. Murine fibroblasts stably expressing human MMP14 (MT1-MMP) were treated with KLK14 and furin. Selective inhibitors serine protease inhibitor Kazal-type 6 (SPINK6) (KLK14) and dec-RVKR-CMK (furin) were used to inhibit KLK and furin in the control samples. Cell surface proteins were then biotinylated, and streptavidin bead immunoprecipitates were subjected to immunoblotting using an anti-MMP14 antibody. Each sample contained the 63 kDa proMMP14 form, whereas an increase in the active 58 kDa MMP14 form was observed after KLK14 incubation. Additionally, a lower molecular weight MMP14 form at 56 kDa was detected only in the KLK14 treated sample. KLK = kallikrein-related peptidase; MMP = matrix metalloproteinase.

Figure 7

KLK14-activated MMPs cluster together in phylogenetic analysis. The full sequences of all human MMPs were obtained from the uniport database and analyzed in BioEdit using built-in multiple alignment ClustalW algorithm. The resulting alignment was visualized using an online tool (www.phylogeny.fr). The full black branches represent the MMPs in which the profragment activation sequence was hydrolyzed by KLK14. An asterisk denotes MMPs processed by KLK14 as verified in vitro. MMP = matrix metalloproteinase; MT-MMP = membrane-type matrix metalloproteinase.