1. Introduction
The extracellular matrix (ECM) consists of a complex array of locally secreted macromolecules interacting as a meshwork of proteins, glycoproteins, glycosaminoglycans, and proteoglycans [
1]. Proteases present within the ECM are essential for turnover of structural proteins or core protein components. In addition, proteolytic enzymes are required for the activation of growth factors and other ligands of cell surface receptors. Thereby, proteolytic processing of ECM components and the molecules stored therein provides both the structural organization and essential initial steps in cell-cell communication. By initiation of the signal transduction, proteases effectively regulate gene transcription eventually impacting on cell differentiation, proliferation, growth, and apoptotic programs [
2]. Proteolytic enzymes essential for ECM homeostasis predominantly belong to the families of a disintegrin and metalloproteinase (ADAMs) [
3], matrix metalloproteinases (MMPs) [
1], and tissue kallikrein-related peptidases (KLKs) [
4]. The MMP family is considered especially important for ECM remodeling [
5,
6]. In particular, the MMP proteases of the membrane-type subfamily (MT-MMPs) are essential in regulating cell migration during physiological wound healing [
7] but are also implicated in cancer progression as they often involve in the initial steps of cancer cell metastasis [
8,
9,
10]. Specifically, MMP14 (MT1-MMP) associates with integrins in endothelial cells at distinct cellular domains, which may initiate cell junction processing during invasion [
11]. Furthermore, MMP14 is attributed as one of the main MT-MMPs implemented in activating cancer associated proMMP2 [
12].
It is corroborated that the activation of MT-MMPs happens intracellularly by means of furin-mediated processing such that the active forms of MT-MMPs decorate the cell surface right upon secretory release of cellular products by exocytosis. This pathway is not exclusive, as extracellular processing of proMT1-MMP (proMMP14) has been reported [
13,
14]. About one-third of all proMMPs activation domains contain an Arg/Lys-rich motif, compatible with furin specificity, but also targeted by serine proteases, particularly plasmin [
15,
16] and transmembrane serine proteinases hepsin and TMPRSS2 [
17].
The family of human kallikrein-related peptidases consists of a total of 15 different serine proteases with trypsin-like, chymotrypsin-like, or mixed substrate specificity. Physiological roles of KLKs include regulation of cell growth and tissue remodeling [
18]. Typically, KLKs and MMPs are co-expressed in many cell types and tissues [
19,
20,
21,
22]; therefore, understanding their interactions may provide a novel perspective for deciphering regulation of MMP activities in health and disease. A recent report has identified a pericellular network of KLK4, KLK14, MMP3, and MMP9 that are involved in initiation and promotion of prostate cancer cell metastasis [
17]; however, the function of KLK14 in this complex was not identified.
Herein, we employed a new unbiased technique CleavEx (Cleavage of exposed amino acid sequences), which allowed us to test for the activation of MMP zymogen forms by KLK14. Our approach was based on the recombinant attachment of an MMP activation site sequence to the proteolytically stable heme-binding (HmuY) protein. Hybrid proteins displaying all 23 activation sequences of human proMMPs was expressed and purified. These proteins were then incubated with KLK14 and the generated products were subsequently analyzed to identify the potential processing. CleavEx analysis was followed by investigation of KLK14-mediated processing of native proMMP14-17. Lastly, we identified the cell surface processing of proMMP14 by KLK14. To our knowledge, this is the first study examining the potential of KLK family member to activate the proMMP family.
3. Discussion
Matrix metalloproteinases are essential enzymes in the maintenance of tissue homeostasis, namely by regulation of its self-reorganization, through growth factor activation and processing, as well as involvement in the regulation of immune cell functions [
29]. Herein, we present biochemical evidence of selective activation of membrane-type MMPs by the kallikrein-related protease family member KLK14.
Our assessment is based on an unbiased approach, where all proMMP-specific activation sequences were displayed utilizing a hybrid protein system, allowing for easy production, purification and fragmentation analysis. The described herein CleavEx (
Cleavage of
exposed amino acid sequences) system facilitated a rapid selection of KLK14-specific candidate proMMPs for further analysis with recombinant proteins. The carrier protein, HmuY, is produced by the bacterium responsible for periodontitis,
Porphyromonas gingivalis, which uses an arsenal of proteolytic enzymes as virulence factors [
30]. Stability studies indicate an unsurpassed resistance of the HmuY protein to proteolytic digestion by trypsin and bacterial proteinases alike [
23] and suggest enhanced stability in situ (i.e., in inflamed tissue). Furthermore, structural studies revealed that the N- and C-termini of HmuY are exposed to the solvent and are thus easily accessible, while the overall globular structure of HmuY ensures good solubility and ease of folding when produced recombinantly [
23]. The N-terminal HisTag serves as an affinity label for purification and additionally as a degradation-specific tag for follow-up of peptide bond hydrolysis, even in complex protein mixtures. Previously, a similar approach, but using a modified fibroblast growth factor-1 (FGF-1), was implemented by another group in the analysis of a proKLK cascade activation [
31,
32] and MMP-dependent activation of KLKs [
33]. Yet, we believe the choice of HmuY used herein to be superior. The approximate 25 kDa molecular masses of the CleavEx fusion proteins and the ~2.5 kDa N-terminal fragment removal allowed for an easy follow-up of the reaction products by SDS-PAGE and Western blot in comparison to the FGF-1-based system, characterized by a ~1kDa shift only.
Out of all 23 sequences, a total of 10 CleavEx
proMMP fusion proteins were hydrolyzed by KLK14, specifically the ones corresponding to membrane-type MMPs (MMP14-17 and MMP24-25), stromelysin-3 (MMP11), MMP21, femalysin (MMP23), and epilysin (MMP28) activation sequences (
Figure 1). Importantly, CleavEx
proMMP hydrolysis by KLK14 was specific and limited to sequences derived from a subset of metalloprotease zymogens. Strikingly, the majority of all proMMPs recognized by KLK14 in the CleavEx analysis, cluster together constituting the group of membrane-type (MT) MMPs. This subgroup of the MMP proteinases is bound to the plasma membrane, either by type I transmembrane domains (MMP14-16, MMP24) or by means of phosphatidylinositol-anchors attached to the protein chains (MMP17 and MMP25) [
20]. Therefore, recognition by KLK14 reflects not only the similarities in the activation domain of MT-MMPs but also the common evolutionary ancestry and functional homology of the processed proteinases. Together, these results indicate that the group of cell-surface proMMPs can be nearly exclusively targeted by KLK14-mediated activation (
Figure 7).
Commercially available recombinant proMMPs were investigated to confirm the initial CleavEx
proMMP analysis. KLK14 was an effective convertase for proMMP14 and proMMP16 but less-efficiently, for proMMP15 and proMMP17 in a time dependent manner (
Figure 3). N-terminal sequencing of the concentration-dependent KLK14-released products identified the expected hydrolysis sites for proMMP14, proMMP15, and proMMP16, respectively, indicating native-like processing of these enzymes by KLK14 [
13,
24] (
Table 2). ProMMP14 processing by KLK14 and furin revealed a single band corresponding to the active form (
Figure 2C,D). No ~12 kDa prodomain was visible upon KLK14 treatment, while furin released an intact profragment observed on SDS-PAGE. This KLK14-mediated prodomain processing may be vital in the disruption of the non-covalent association between the MMP14 prodomain and catalytic domain. As observed in the activity assay using a synthetic substrate, KLK14-mediated MMP14 activation was more efficient than furin, which may be due to the prodomain still being non-covalently attached serving as inhibitor of MMP14 [
34] (
Figure 5). In addition, the identified G
298–I
299 internal cleavage of MMP15 was located in the hinge region connecting the catalytic domain and hemopexin domain [
35] (
Table 2). It is difficult to predict the impact of this cleavage on the protease activity of the resulting MMP15 and its specificity towards protein substrates. However, the appearance of a corresponding band in the zymogram analysis indicated ability to cleave gelatin and suggested that this specific auto-processing by initial KLK14-mediation may be responsible for shedding of the active catalytic domain of MMP15 from the cell surface (
Figure 4C). Interestingly, the cell surface localization of MMP15 was essential for its ability to cleave collagen in cell-based assays, and it was required for exhibition of invasive cancer cell phenotypes [
36,
37], while soluble MMP15 was found to display a nearly 13-fold higher activity on triple-helical substrates compared to the cell-surface located form [
38]. This suggests, that release of the mature MMP15 from the cell surface may be an important regulatory step, resulting in enhanced triple helical collagen I degradation. Furthermore, lower concentrations of KLK14 induced step-wise MMMP16 profragment processing, as revealed by two truncated proforms, one form corresponding to KLK14 specificity (KKPR
100↓CG) and the other to the specificity of MMP16 [
25] (
Figure 2F and
Table 2). The processing of KKPR
100↓CG, illustrates that KLK14 recognizes the site adjacent to cysteine-sulfhydryl group within the prodomain that associates with the catalytic zinc, which may disrupt the noncovalent interaction vital in releasing an active MMP16. Lastly, two distinct bands were present in the proMMP17 untreated samples both with identical N-termini, suggesting the difference between these products is in the C-terminal region (
Figure 2G). KLK14 processed both proMMP17 forms and generated two bands, both again with the same N-termini, three amino acids upstream at the motif TQAR
122↓RRPQ (
Table 2). However, as described in the manufacturer documentation, the proMMP17 activation site R
125 was exchanged to P
125 therefore the native activation site is not accessible for proteolysis, which in turn targets a secondary activation site.
The verification of the hydrolysis sites at the expected activation location (except for proMMP17) indicates the proper processing of the proMMP by KLK14; however, it does not confirm the production of the active metalloproteinase from its zymogen form. To validate that KLK14-mediated processing does in fact release mature and functional MMP proteinases, gelatin zymography was employed (
Figure 4). It confirmed MMP14, 15, and 16 as functional proteinases since clear bands appeared at the expected molecular mass shifts of processed MMPs. Despite the correct mass shift, proMMP17 displayed no gelatinolytic activity, either due to the abovementioned modification of the native activation site or the natural low activity of MMP17 on gelatin as a substrate [
26]. It is worth noting, however, that all commercially available proMMP14-17 preparations do not contain the membrane-interacting domain, located in the C-terminal region of the mature forms, which may impact activation kinetics in vivo. Therefore, demonstrating whether KLK14 activates MT-MMP in a cellular system provides increasing evidence of an inter-activating KLK and MT-MMP network.
Recently, a KLK4/KLK14 activity system on the cell surface of COS-7 cells was described [
17]. The authors propose a cell surface-located organization of a proteinase complex, containing hepsin and transmembrane serine protease 2 (TMPRSS2), two membrane-bound serine proteinases, which recruit KLK4 and KLK14 and activate proMMP3 and proMMP9. In this complex, KLK14 undergoes specific activation and accumulates in the active form, bound to the plasma membrane by a so far unknown mechanism. The role of cell surface-bound KLK14 in that system was not identified. However, based on the data presented in our study, we believe and propose that the proximity of MT-MMPs to membrane-attached, proteolytically active KLK14 may provide a yet undescribed platform for the activation of MT-MMPs.
As exemplified by the archetypical member of the subfamily, MT1-MMP (MMP14), this subclass of MMPs was reported to undergo furin-dependent activation in the compartments of the late secretory pathway; however, a body of research challenges this earlier assumption [
13]. An alternative activation pathway was identified in furin-deficient RPE.40 cells which were able to display active MMP14 with the same N-terminus as the native form found in furin-expressing COS-7 cells [
13]. As furin expressing CHO-K1 cells processed the membrane-bound form to the same extent as RPE.40 cells, while also converting the soluble form of MMP14, the authors concluded, that furin is required only for the maturation of the soluble form. Other, yet unidentified enzymes are responsible for the processing of membrane bound MMP14 [
13]. Similarly, the non-constitutive, furin-independent processing of proMMP14 was also reported to occur in fibrosarcoma HT-1080 and CCL-137 normal fibroblast cells, where the 63 kDa proform of MMP14 was detected in both, the tumor-derived cell line and in normal cells upon PMA stimulation [
39,
40]. Thus, a furin-independent activation pathway needs to be further elucidated since active MMP14 was still present in furin-deficient cells [
41,
42,
43].
MMP14 is expressed quite low in cells normally between 100,000–200,000 sites/cell, which makes it difficult to perform biochemical analysis and investigate functional parameters [
44]. To delve into its processing, the
MT1-MMP (
MMP14) gene is transfected either transiently or stably, which results in MMP14 overexpression, leading to a plethora of cell surface forms; 63 kDa proenzyme, 54 kDa enzyme; and a 39 – 45 kDa degradation products [
27]. The work presented here shows MMP14 to be processed on the cell surface by KLK14 using murine fibroblasts stably overexpressing MMP14 (
Figure 6). The ~63 and 58 kDa forms detected by immunoprecipitation are consistent with previous reports and were identified as proMMP14 and active MMP14, respectively [
27,
28]. Indeed, the intensity of the 58 kDa band increased upon KLK14 treatment. In addition, a lower-molecular weight MMP14 form of around 56 kDa appeared. Intriguingly, this may present an alternative location of processing the membrane anchored proMMP14 by KLK14, as potential locations corresponding with KLK14 specificity are present ~1 and 4 kDa downstream in the MMP14 sequence (TPK
134 and IRK
146, respectively). Furthermore, in our experiments, furin did not process cell surface proMMP14, also consistent with previous reports, indicating that furin acts exclusively in the Golgi apparatus and/or on soluble, not-membrane-bound forms of MMP14 [
41].
Researchers confirmed that there is a 2-step activation process for MT1-MMP. The linkage (RRPRC
93GVPD) in the prodomain maintains the latent proenzyme by chelating the cysteine-sulfhydryl group to the active site zinc, which furin hydrolysis alone is not enough to disrupt the interaction [
34]. Most importantly, mutant forms of the MMP dependent cleavage site (PGD↓L
50) and furin activation site (RRKR
111↓Y
112) sequences in HT 1080 cells showed both proenzyme and enzyme forms of MMP14 on their cell surface, thus again confirming a furin-independent pathway of MMP14 processing [
34]. This cell-surface processing sheds more light on the potential of various secreted MMP14 forms to be differentially regulated in normal and cancer cells by extracellular KLK14. In addition to KLK14 hydrolyzing the furin recognition site (RRKR
111↓Y
112), KLK14 may also recognize the trypsin-like sequence, RRPR
92CGVPD, which is specifically adjacent to the site of the cysteine-sulfhydryl group within the prodomain that associates with the catalytic zinc in the active site. In the work presented here (
Figure 2C,D), when recombinant proMMP14 was incubated with furin, the released prodomain was intact and visible, which confirms furin hydrolysis is limited to only the furin recognition site [
45]. On the other hand, KLK14 further degraded the released prodomain since it was no longer visible at 12 kDa as compared to furin. Thus, this indicates the additional KLK14 hydrolysis in the prodomain, which may be vital in the release non-covalent association of the prodomain and catalytic domain once KLK14 or furin cleave at the furin recognition site. Intriguingly, KLK14 recognized this highly conserved cysteine linkage sequence, KKPR
100↓CGVPD, within the prodomain of recombinant MMP16 (
Table 2). This illustrates the possibility of KLK14 to disrupt the prodomain association with the catalytic zinc in other proMMPs in vivo, providing a universal activation mechanism for the entire proMMP family by KLK14. Nonetheless, recombinant proMMP2 also contains the corresponding
97MRKPR
101C sequence; yet no KLK14-mediated activation was observed, indicating that accessibility to this sequence may be selective and depend on protein conformation (
Figure 2A).
The proteolytic network interactions between the KLKs and MMPs are starting to emerge. In vitro analysis studies illustrate that MMPs can modulate KLK activity, such as MMP20 activating proKLK1, 4, and 6, as well as MMP3 activating proKLK4 [
33,
46]. Furthermore, major MMPs involved in tumor progression (proMMP1, 2, and 9) have been shown to be activated by KLK1, which may lead to enhanced tissue homeostasis or dysregulation [
47]. Cell surface-located, membrane type MMPs have been identified as important players in bone remodeling, as well as in wound healing, growth factor signaling, and in immune functions [
48,
49,
50], while KLKs are implicated in immune functions and TGFβ activation [
51]. Interestingly, the presence of both MT-MMPs and KLKs was identified in many types of cancer [
50,
52]. A body of research indicates MMP14 as the main membrane type MMP essential for cancer cell escape and hence, tumor progression [
53,
54], which is partially attributed to proMMP2 activation, but also for the ability of MMP14 to directly degrade type I collagen and to promote cellular invasion in 3D collagen matrices [
55]. Similarly, MMP15 and MMP16 were shown to promote invasiveness of tumor cells in 3D fibrin matrices [
10,
56], while, in particular, MMP16 is highly expressed in aggressive melanoma [
10]. MMP17 was implicated as a protease critical for breast cancer metastasis in animal models [
57]. KLK14 was similarly implicated in many human tumors. Most notably, KLK14 levels were found to predict poor outcomes in prostate cancer patients [
58], and elevated levels of both transcript and protein were found in malignant breast cancer tissues [
59,
60]. In correlation, increased levels of MMP14 were detected in prostate cancer cells [
61,
62], and active MMP2 was found to be inversely correlated with the disease-free survival time in prostate cancer patients [
61]. Furthermore, elevated levels of MMP14 were associated with poor prognosis and invasiveness in breast cancer [
63,
64]. Recently, the involvement of MMP14 was also indicated in epithelial-to-mesenchymal transition in squamous cell carcinoma [
65,
66] and prostate cancer alike [
67].
It is therefore exciting to speculate, that the herein described potential of KLK14 to activate membrane-type MMPs may provide a novel mechanism facilitating prostate and breast tumorigenesis, initial cancer cell invasion, and subsequent tumor progression at the sites of metastases formation. In addition, future studies include profiling more KLKs utilizing the CleavEx analysis to better understand the proteolytic network interaction between KLKs and proMMPs.
4. Materials and Methods
4.1. Cloning of HmuY-Based CleavEx Fusion Proteins
The CleavEx fusion proteins were constructed based on a proteolysis-resistant
Porphyromonas gingivalis HmuY protein (accession number ABL74281.1) as a carrier via PCR cloning. Firstly, the HmuY gene was amplified using primers forward: 5′–atatgcggccgcagacgagccgaaccaaccctcca–3′ and reverse: 5′–atactcgagttatttaacggggtatgtataagcgaaagtga–3′ from whole-genomic DNA isolated from
P. gingivalis strain W83. PCR was conducted for 35 cycles with initial denaturation at 98 °C, followed by 40s annealing at 68 °C and 30 s extension at 72 °C, using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The HmuY PCR product was further amplified in three consecutive PCR reactions with primers specific to the 5′ HmuY fragment and a 3′ specific primer introducing additional nucleotides dependent on the designed sequence (
Table 3) at the same conditions. All sequences were designed based of the accession number from the Uniprot database (
www.uniprot.org): MMP1 (P03956), MMP2 (P08253), MMP3 (P08254), MMP7 (P09237), MMP8 (P22894), MMP9 (P14780), MMP10 (P09238), MMP11 (P24347), MMP12 (P39900), MMP13 (P45452), MMP14 (P50281), MMP15 (P51511), MMP16 (P51512), MMP17 (Q9ULZ9), MMP19 (Q99542), MMP20 (O60882), MMP21 (Q8N119), MMP23 (O75900), MMP24 (Q9Y5R2), MMP25 (Q9NPA2), MMP26 (Q9NRE1), MMP27 (Q9H306), and MMP28 (Q9H239). Lastly, the final PCR reaction was ligated into a modified pETDuet plasmid, according to the manufacturers protocol, with potential tryptic cleavage sites removed from the MCS using QuickChange (Agilent Technologies, Santa Clara, CA, USA). An alternative method was also used for the fusion protein-encoding sequences by using Phusion Site-Directed Mutagenesis (Thermo Fisher Scientific, Waltham, MA, USA) via sequence exchange from a previously prepared CleavEx construct (
Table 4). The final product was transformed into competent
E. coli T10 cells and then purified and sequenced. All CleavEx
proMMP DNA sequences were identified to be as intended.
4.2. Expression and Purification of CleavEx Fusion Proteins
The designed fusion proteins covering proMMP activation sequences were expressed using an E. coli BL21 expression system. Following the 0.5 mM IPTG induction at OD600nm = 0.5–0.6, the bacterial culture protein production was facilitated for 3 h at 37 °C, with shaking. Then, the bacteria were spun down, and the pellet was suspended in buffer A (10 mM sodium phosphate, 500 mM NaCl, and 5 mM imidazole, pH 7.4) and sonicated (15 min at 16 °C, pulse 6s, amplitude 70%). Supernatant of the soluble proteins was then loaded onto the HisTrap™ Excel (GE Healthcare, Chicago, IL, USA) column in buffer A and eluted with a linear gradient of 0–100% of 1 M imidazole in buffer A in 20 column volumes (CV). Protein containing fractions were pooled together and exchanged into 50 mM Tris pH 7.5 and then purified by ion exchange chromatography using a MonoQ 4.6/100 PE column (GE Healthcare, Chicago, IL, USA) with a linear gradient of 0–100% 50 mM Tris pH 7.5, 1 M NaCl in 15 CV. Purity of all the products was verified by SDS-PAGE.
4.3. Expression and Production of KLK14
The gene encoding human proKLK14 was custom-synthesized by Life Technologies (Carlsbad, CA, USA) with a codon usage optimized for
Leishmania tarentolae and cloned into the pLEXSY_I-blecherry3 plasmid (Cat. No. EGE-243, JenaBioscience, Jena, Germany) using NotI and XbaI restriction sites. All preparations for transfection, selection, and expression in host strain T7-TR of
L. tarentolae were performed according to the JenaBioscience protocol for inducible expression of recombinant proteins secreted to the medium. Expression of proKLK14 was induced with 15 µg/mL of tetracycline (BioShop, Burlington, Canada) and was carried out for 3 days. Next, the media was spun down (20 min at 3000 rcf), and the supernatant was precipitated with 80% ammonium sulfate for 1 h at 4 °C and then centrifuged (30 min at 15,000 rcf). The pellet was suspended in 10 mM sodium phosphate, 500 mM NaCl, and 5 mM imidazole, pH 7.4 in the presence of 5 mM benzamidine and dialyzed overnight at 4°C in the same buffer. The following day, the solution was loaded onto HisTrap™ Excel (GE Healthcare) as described above (
Section 4.2). Obtained fractions were analyzed by SDS PAGE and fractions containing proKLK14 were concentrated with Vivaspin® 2 (Sartorius, Göttingen, Germany) and further purified on Superdex s75 pg (GE Healthcare, Chicago, IL, USA) in 20 mM Tris pH 7.5, 0.5 M NaCl. After purification and self-activation overnight at 4 °C, KLK14 was active-site titrated as described in Kantyka et al. [
68].
4.4. Screening the CleavExproMMP Fusion Proteins with KLK14 and N-Terminal Sequencing of KLK14-Released Fragments
CleavEx proteins were incubated at a 1:1000 and 1:200 KLK14:CleavExproMMP molar ratio, corresponding to 50 and 250 nM KLK14, respectively, in 50 mM Tris pH 7.5. In addition, lower KLK14 concentrations (10, 25, and 50 nM) were tested on CleavEx proMMP11, 14–17, 24, 25, and 28. Samples were incubated at 37 °C for 1 h, after which the reactions were immediately stopped by the addition of 50 mM DTT-supplemented SDS sample buffer (1:1) and boiled. The obtained samples were resolved using 10% Tricine SDS-PAGE. The proteins were electrotransferred onto a PVDF membrane (Amersham™ Hybond™, GE Healthcare) in 25 mM Tris, 190 mM glycine, and 20% methanol at 100 V for 1 h in 4 °C. The membranes were blocked with 5% skim milk in TTBS (50 mM Tris-HCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5) and incubated with an anti-HisTag-HRP antibody (catalog no. A7058, Sigma-Aldrich, St. Louis, MO, USA) diluted 1:20 000 in TTBS. The Western blots were developed with Pierce® ECL Western blotting substrate (Thermo Fisher Scientific, Waltham, MA, USA) using Medical X-Ray-Film Blue (Agfa HealthCare, Mortsel, Belgium).
Furthermore, each hydrolyzed CleavEx
proMMP (1 µg) was incubated with 250 nM KLK14 for 1 h at 37 °C, the reactions were stopped, and the products were resolved on SDS-PAGE and electrotransferred onto a PVDF membrane [
69]. The membranes were stained with Coomassie Brilliant Blue R-250 (BioShop, Burlington, Canada) and the bands of interest were sequenced via automated Edman degradation using a PPSQ/31B protein sequencer (Shimadzu Biotech, Kyoto, Japan) equipped with an LC-20AT HPLC, CTO-20A column heater and SPD20A UV detector (Shimadzu Biotech) for on-line PTH analysis. Data was recorded using proprietary software (Shimadzu Biotech), and the sequence was determined by visual inspection of the UV 269 nm chromatograms.
4.5. SDS-PAGE Analysis of KLK14-Mediated Recombinant proMMP Processing
A total of 0.5 µg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 0.5 µg proMMP14 (catalog no. 918-MP-010, R&D Systems), 1 µg proMMP15 (catalog no. 916-MP-010, R&D Systems), 0.5 µg proMMP16 (catalog no. 1785-MP-010, R&D Systems), and 1 µg proMMP17 (catalog no. 7796-MP-010, R&D Systems) were separately incubated in 10 µL with a range of KLK14 concentrations (25–250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 µM batimastat (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS. As a positive control, a total of 1 µg native proMMP2 and 0.5 µg proMMP14 were separately incubated in 10 µL with a range of furin (catalog no. 1503-SE-010, R&D Systems) concentrations (0–250 nM, molar ratios from around 1:65 to 1:6). The reactions were stopped with the addition of 50 mM DTT-supplemented SDS sample buffer (1:1, v:v) and boiled. The samples were resolved using SDS-PAGE as described above and then visualized with Coomassie Brilliant Blue G-250 (Bioshop, Burlington, Canada) staining. Additionally, 50 nM KLK14 (proMMP14) or 100 nM KLK14 (proMMP15, proMMP16, and proMMP17) was incubated with each respective proMMP (0.5 µg proMMP14 and 16, 1 µg proMMP15 and 17) in the presence of 5 µM batimastat for specified periods of time (0–180 min). The final molar ratio for KLK14:MMP14 was 1:32, KLK14:MMP15 was 1:16, KLK14:MMP16 was 1:16, and KLK14:MMP17 was 1:18. The reaction in each sample was stopped as above and SDS-PAGE separation was visualized using Coomassie Brilliant Blue G-250.
4.6. N-Terminal Sequencing of KLK14 Processed Recombinant proMMPs
Each native proMMP (2 µg) was incubated with 50 nM (proMMP14) or 250 nM (proMMP15, proMMP16, proMMP17) KLK14 for 1 h at 37 °C and resolved by SDS-PAGE as described above. Proteins were then electrotransferred onto a PVDF membrane in 10 mM CAPS, 10% methanol, pH 11 using the Trans-blot semi-dry transfer cell (Bio-Rad, Hercules, CA, USA) at 15V for 30 min. The membrane was stained with Coomassie Brilliant Blue R-250 (BioShop, Burlington, Canada), and bands of interest were sequenced via Edman degradation as described above.
4.7. Zymogram Analysis of Recombinant proMMP by KLK14
Each native proMMP (0.5 µg) was incubated with 50 and 100 nM KLK14 (proMMP14) or 100 and 250 nM KLK14 (proMMP15, 16, and 17) for 1 h at 37 °C. After incubation, KLK14 was inhibited with 10 µM biotin-Tyr-Gly-Pro-Arg-CMK, a specific KLK14 inhibitor, and 1 µM serine protease inhibitor Kazal-type 6 (SPINK6) [
70] for 15 min at 37 °C. Next, non-reducing sample buffer (1:1) was added, and samples were incubated for 30 min at 37 °C. Then, samples were separated using a Tricine-SDS gel containing 0.1% gelatin at 4 °C and 40 mA. Subsequently, the gels were washed three times with 2.5% Triton X-100, followed by overnight incubation in assay buffer with the presence of the KLK14 inhibitors at 37 °C. The assay buffer used for each proMMP was 50 mM Tris, 10 mM CaCl
2, 150 mM NaCl, pH 7.5 (proMMP2), 50 mM Tris, 3 mM CaCl
2, 1 μM ZnCl
2, pH 8.5 (proMMP14 and 16), 50 mM Tris, 500 mM NaCl, 5 mM CaCl
2, 1 μM ZnCl
2, pH 8 (proMMP15) and 50 mM Tris, 10 mM CaCl
2, pH 7.5 (proMMP17). The next day, the zymogram was fixed by using 30% methanol with 10% acetic acid for 2 min and stained with 0.1% amido black in 10% acetic acid for 2 h at room temperature, followed by destaining with 10% acetic acid.
4.8. Functional Activation of Recombinant proMMP14 Using A Synthetic Substrate
ProMMP14 (10 nM) was separately incubated with KLK14 (3 nM) and furin (3 nM) in the presence of the fluorogenic substrate Mca-KPLGL-Dpa-AR-NH2 (catalog no. ES010, R&D Systems, Abingdon, United Kingdom). The final substrate concentration was 10 µM in 50 mM Tris, 3 mM CaCl2, 1 µM ZnCl2, pH 8.5. Hydrolysis was recorded for 180 min at 37 °C (λex320 nm; λem405 nm) using a microplate fluorescence reader (Molecular Devices Spectra Max GEMINI EM, Molecular Devices, San Jose, CA, USA). The measurement was performed in triplicate, and the initial velocities were determined via the built-in linear regression algorithm. The velocities were plotted in triplicates as mean ± SD using GraphPad Prism (GraphPad Software, La Jolla, CA, USA) using time points 0, 5, 15, 30, 60, 90, 120, 150, and 180 min.
4.9. Cell Surface Processing of proMMP14 (MT1-MMP) by KLK14
Timp 2-/- mouse embryonic fibroblast (MEF) cells stably expressing FLAG-tagged MT1-MMP transfected with the pGW1GH/hMT1-MMP expression vector [
71] were seeded in a 12-well plate (250,000 cells per well) and cultured in 1.5 mL selection medium DMEM (Gibco, Waltham, MA, USA) with 10% FBS (Gibco), 25 µg/mL mycophenolic acid (Sigma-Aldrich, St. Louis, MO, USA), 250 µg/mL xanthine (Sigma-Aldrich), and 1X HT supplement (Life Technologies, Carlsbad, CA, USA) for 2 days at 37 °C, 5% CO
2 to 80% confluency. The selection media was aspirated, and cells were gently washed with PBS and then treated with 250 µL DMEM containing either 50 nM KLK14, 50 nM furin and each enzyme preincubated (15 min at 37 °C) with its specific inhibitor: either 250 nM SPINK6 [
70] or 1 µM decanoyl-RVKR-CMK (Bachem, Bubendorf, Switzerland), respectively, and further incubated for 30 min at 37 °C, 5% CO
2. In addition, separate cell populations were incubated with increasing concentrations of KLK14 (50, 100, 250, and 500 nM) and compared to the appropriate control samples, which included: (i) untreated cells; (ii) cells treated with SPINK6 and (iii) dec-RVKR-CMK inhibitors; (iv) 50 nM furin; (v) 50 nM furin with 1 µM dec-RVKR-CMK; and (vi) 500 nM KLK14 in the presence of 1 µM SPINK6.
The supernatant was aspirated and the cells were rinsed with cold PBS three times and then treated with 0.1 mg/mL EZ-link™ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Next, the cells were washed three times with PBS containing 100 mM glycine and then lysed with 500 µL RIPA buffer (10 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% DOC, 2 mM EDTA) containing 25X cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche, Mannheim, Germany) and 5 mM EDTA. Cells were detached and spun down 16,000 rcf for 15 min at 4 °C. The protein concentration of the lysate was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. A total of 500 µg was loaded onto PureProteome streptavidin magnetic beads (Merck Millipore, Burlington, MA, USA) and incubated overnight at 4 °C. The beads were washed twice in RIPA buffer followed by a twice wash in PBS and then suspended in 50 mM Tris pH 7.5 and 6x reducing sample buffer to a final 3× reducing sample buffer (
v/
v) in a final 40 µL volume. Samples were boiled at 95 °C for 5 min, resolved on SDS-PAGE, and electrotransferred onto a PVDF membrane [
69].
The membrane was subsequently blocked with 5% skim milk in TTBS (50 mM Tris-HCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5) for 2 h at 37 °C. Next, primary antibody rabbit-anti-MMP14 (catalog no. MA5-32076, Thermo Fisher Scientific, Waltham, MA, USA) in 5% milk was added at 1:1000 overnight at 4 °C. The following day, the membrane was washed four times with TTBS, and secondary antibody goat-anti rabbit-HRP (catalog no. A7058, Sigma-Aldrich, St. Louis, MO, USA) was added at 1:60,000 in 5% milk for 2 h at RT. Lastly, the membrane was rinsed four times with TTBS and developed with the SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA) using Medical X-Ray-Film Blue (Agfa HealthCare, Mortsel, Belgium).