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The prostate cancer-associated kallikrein-related peptidase 4 (KLK4) activates matrix metalloproteinase-1 (MMP1) and thrombospondin-1 (TSP1) Ruth Anna Fuhrman-Luck, Scott Hunton Stansfield, Carson Ryan Stephens, Daniela Loessner, and Judith A. Clements J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b01148 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 10, 2016

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Journal of Proteome Research

The prostate cancer-associated kallikrein-related peptidase 4 (KLK4) activates matrix metalloproteinase-1 (MMP1) and thrombospondin-1 (TSP1) Ruth A. Fuhrman-Luck1,2, Scott H. Stansfield2, Carson R. Stephens1,2, Daniela Loessner2, Judith A. Clements1,2*. 1

Australian Prostate Cancer Research Centre- Queensland, Institute of Health and Biomedical

Innovation, Queensland University of Technology at the Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia. 2

Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk

Avenue, Kelvin Grove, Queensland, 4059, Australia.

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ACS Paragon Plus Environment


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ABSTRACT

Prostate cancer metastasis to bone is terminal; novel therapies are required to prevent end-stage disease. Kallikrein-related peptidase 4 (KLK4) is a serine protease that is over-produced in localized prostate cancer and is abundant in prostate cancer bone metastases. In vitro, KLK4 induces tumor-promoting phenotypes; however, the underlying proteolytic mechanism is undefined. The PROtein TOpography and Migration Analysis Platform (PROTOMAP) was used for high-depth identification of KLK4 substrates secreted by prostate cancer bone metastasisderived PC-3 cells, to delineate the mechanism of KLK4 action in advanced prostate cancer.

Thirty-six putative novel substrates were determined from the PROTOMAP analysis. In addition, KLK4 cleaved the established substrate, urokinase-type plasminogen activator (uPA), thus validating the approach. KLK4 activated matrix metalloproteinase-1 (MMP1), a protease which promotes prostate tumor growth and metastasis. MMP1 was produced in the tumor compartment of prostate cancer bone metastases, highlighting its accessibility to KLK4 at this site. KLK4 further liberated an N-terminal product, with purported angiogenic activity, from thrombospondin-1 (TSP1), and cleaved TSP1 in an osteoblast-derived matrix.

This is the most comprehensive analysis of the proteolytic action of KLK4 in an advanced prostate cancer model to date, highlighting KLK4 as a potential multi-functional regulator of prostate cancer progression.

KEYWORDS Prostate cancer; kallikrein-related peptidase; proteomics; substrate; bone metastases. 2


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INTRODUCTION Prostate cancer claims the lives of an estimated 307,000 men each year, with 1.1 million annual incidences of the disease worldwide 1. Only palliative treatments exist for end-stage disease, primarily bone metastases 2. Understanding the molecular mechanisms driving prostate cancer progression is essential for developing therapies to attenuate metastatic growth. Proteases are an area of increasing therapeutic research as they can be readily targeted by small molecule inhibitors 3, 4. The prostate-specific antigen (PSA)-related serine protease, KLK4, is over-produced in prostate cancer, versus benign tissue 5-9, and KLK4 expression is a positive predictor of prostate cancer risk and tumor stage 10. In vitro, KLK4 promotes prostate cancer cell migration 9, 11, proliferation 12 and epithelial-to-mesenchymal transition 9, each hallmark cancer cell phenotypes. KLK4 is also produced in prostate cancer bone metastases 11, with its ability to remodel mineralized dental enamel matrix 13, 14 suggesting a similar role within mineralized bone to facilitate metastasis. Thus, KLK4 is a promising therapeutic target in prostate cancer. While KLK4 induces hallmark cancer cell phenotypes, in vitro, the associated substrate intermediates are largely unknown. KLK4 cleaves selected recombinant proteins 15; however, this was determined by screening individual proteins as putative KLK4 substrates. For such assays, putative substrates are often selected for screening based on a priori association to cancer. Further, assay conditions do not include the milieu of proteins that regulate proteolysis, in vivo. Accordingly, there is a need to define the KLK4 substrate repertoire more broadly, using conditions better representative of the tumor milieu. 3



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PROTOMAP is a high-depth substrate profiling approach used to identify putative protease substrates by detecting the mass decrease incurred upon proteolytic processing 16. Unlike other degradomics approaches 17, PROTOMAP employs simple, robust front-end separation of intact and cleaved proteins by SDS-PAGE, coupling this with in-gel digestion and LC-MS/MS, to map the approximate size and sequence of protease-generated cleavage products. This study applied the PROTOMAP approach to identify putative KLK4 substrates produced by prostate cancer cells, to delineate its proteolytic action in prostate cancer progression.

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EXPERIMENTAL PROCEDURES Reagents and antibodies. Except where stated, reagents were from Life Technologies. Antibodies used for Western blot analysis were as follows: KLK4 (mid- and C-terminal epitopes 18

), V5 (R960-25, Life Technologies), MMP1 (ab89767, Abcam) and TSP1 (N-terminal epitope,

sc-12312, Santa Cruz Biotechnology; C-terminal epitope, ab1823, Abcam). Antibodies targeting MMP1 (ab38929, Abcam) and KLK4 (ab40950, Abcam) were used for immunohistochemistry (IHC). Cell lines and cell culture. Cell lines were purchased from the American Type Culture Collection. Prostate cancer PC-3 cells, an androgen-insensitive cell line derived from a prostate cancer bone metastases 19 and expressing negligible levels of KLK4 9, were maintained at 37 °C [5% (v/v) CO2] in RPMI-1640 media with 2 mM L-glutamine, supplemented with 100 U/ mL penicillin G sodium, 100 mg/ mL streptomycin sulfate and 10% (v/v) fetal bovine serum (SigmaAldrich). SF-9 insect cells were maintained at 27 °C in SF-900 II SFM media, supplemented with the above antibiotics. Generation of PC-3 cell lines stably expressing KLK4. Generation of PC-3 cells overexpressing KLK4 (PC-3:KLK4) or empty vector (PC-3:Vector) was performed as previously reported 9; however, splice by overlap extension PCR was used to substitute the KLK4 pre-proregion (M1-Q30) with that of PSA (M1-R24). PC-3 cells stably expressing catalytically inactive mutant KLK4 (mKLK4) were generated as for KLK4, however with a S207A substitution, produced by site-directed mutagenesis (PC-3:mKLK4). Protein production was confirmed by Western blot analysis and activity assessed as per below.

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Production of recombinant KLK4. Recombinant wild-type KLK4 and mKLK4 (S207A substitution) were produced in SF-9 cells, affinity purified, and activated with thermolysin as described 6. Briefly, SF-9 cells were transfected with KLK4-pIB/V5-His or mKLK4-pIB/V5-His (S207A substitution, as above) constructs using Cellfectin, selected with blasticidin, and (m)KLK4 purified from conditioned media using Ni2+-nitrilotriacetic acid superflow resin (Qiagen). Eluates containing (m)KLK4 were pooled, and concentrated using Vivaspin microconcentrators (3 kDa cut-off; Sartorius), before buffer-exchanging into PBS in the same devices. Total protein concentration was determined using a bicinchoninic acid (BCA) assay (Sigma) and purified buffer-exchanged protein incubated (37 °C, 1 h) with recombinant thermolysin (Calbiochem; 80:1 molar ratio of (m)KLK4: thermolysin). Self-activating KLK4 (PSA pre-proregion, as above) was similarly produced, without requirement for thermolysin activation. Products were dialyzed into 20 mM Na3PO4 buffer (pH 7.4) using a Slide-A-Lyzer dialysis cassette (Thermo Scientific; 10 kDa cut-off) and purified by anion exchange chromatography on a Resource Q column (GE Life Sciences), with a linear elution gradient to 1 M NaCl (5 min, 4 mL/ min). Purified products were exchanged into assay buffer [50 mM Tris-NaCl, 0.01% (v/v) Tween, pH 8.8] using Vivaspin micro-concentrators (3 kDa cut-off). Total protein concentration was determined using a BCA assay. Generating PC-3 cell conditioned media (CM). Cells were grown to 85-90% confluence, washed thrice in PBS (pH 7.4) and cultured in serum-free media (24 h). Washes were repeated and cells cultured serum-free for an additional 48 h. CM was harvested with 50 µM EDTA and 2 mM phenylmethylsulfonyl flouride, and concentrated and exchanged into assay buffer using Amicon Ultra-15 centrifugal filter units (3 kDa cut-off; Merck Millipore) and Vivaspin microconcentrators (3 kDa cut-off). Total protein concentration was determined using a BCA assay. 6


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Measurement of KLK4 activity. Recombinant KLK4 (or mKLK4), or KLK4-treated (or untreated) PC-3 cell CM was incubated with serial dilutions of the irreversible KLK4 inhibitor, α2-antiplasmin (R&D Systems; 37 °C, 15 min), before addition of the fluorescent substrate, DVal-Leu-Arg-7-amido-4-trifluoromethyl coumarin (50 µM; Sigma) in assay buffer. Activity [the change in relative fluorescence units (∆ RFU) over time] was measured in a PolarStar Optima microplate reader (BMG Labtech; Ex 400 nm, Em 505 nm; 37 °C) and the concentration of inhibitor synonymous with negligible KLK4 activity determined (1:1 stoichiometry of KLK4 inhibition by α2-antiplasmin). Active KLK4 concentrations or total mKLK4 protein concentrations are stated (where mKLK4 had negligible peptidolytic activity, data not shown). Screening for KLK4 substrates in PC-3 cell secretions using PROTOMAP. Concentrated CM from PC-3:KLK4, PC-3:mKLK4 and PC-3:Vector cells (2.6 µg/ µL) was treated with selfactivating KLK4 (859.9 nM), or equivalent total amounts of mKLK4 (134.3 ng/ µL) or assay buffer as controls (37 °C, 18 h). Reactions were stopped with NuPAGE LDS sample buffer, reduced with tris(2-carboxyethyl)phosphine hydrochloride solution (20.8 mM; 60 °C, 10 min; Sigma) and alkylated with iodoacetamide (21 mM, room temperature, 15 min; Sigma). To identify putative KLK4 substrates, the PROTOMAP approach was followed 16, where samples (35 µg) were analyzed by SDS-PAGE using a large-format (18 × 20 cm) gradient gel [10.5-14% (w/v) acrylamide; Bio-Rad Laboratories]. Gel lanes were partitioned into 32 slices and slices further sectioned into ~1 mm3 pieces, before drying with neat acetonitrile. In-gel trypsin digestion was performed as published 20, omitting in-gel reduction and alkylation. Concentrated peptide extracts were resuspended in 5% (v/v) formic acid for LC-MS/MS analysis.

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LC-MS/MS and bioinformatics. LC-MS/MS was performed using a QSTAR Elite Hybrid Quadrupole-Time of Flight mass spectrometer (Applied Biosystems) with a nanospray ion source (AB Sciex Instruments). Resuspended peptide was subject to reverse-phase liquid chromatography with the following elution gradient [shown following as percentage of 5% (v/v) formic acid/ percentage of 5% (v/v) formic acid with 80% (v/v) acetonitrile]: 98/ 2 (14 min), 50/ 50 (25 min), 20/ 80 (61 min) and 98/ 2 (40 min), with constant flow rates (4 µL/ min). Tandem mass spectrometry (MS/MS) was performed in information-dependent acquisition mode. Raw spectra for each gel slice were converted to .xml files using the embedded mzwiff converter in the Trans Proteomic Pipeline (TPP; v4.4 21). Precursor charge detection was performed in the R statistical environment 22, using an averagine model [output, ‘.mgf(a)’] and further MS/MS peak detection performed using the MassSpecWavelet package 23 for the R environment [signal-tonoise threshold of 2; output, ‘.mgf(b)’]. Spectra were searched using SpectraST (v3.5 24; .xml file input), X!Tandem [Tornado; 2009.04.01.1 25; .mgf(b) input] and the Open Mass Spectrometry Search Algorithm [OMSSA; v2.1.4 26; .mgf(b) input] embedded within the TPP, or Mascot [v2.2.06 27 available from the Australian Proteomics Computational Facility; .mgf(a) input], against the International Protein Index human database (v3.60 28) concatenated with the common Repository of Adventitious Proteins 29. Search databases incorporated reverse sequences to identify false positives. Search parameters were as follows: precursor ion mass (monoisotopic) tolerance ± 2.2 Da; number of 13C = 0; MS/MS tolerance ± 0.4 Da; semi-tryptic cleavage allowing for 4 missed cleavages. Resulting files from Mascot and X!Tandem searches were converted to .pep.xml format within the TPP, consistent with SpectraST and OMSSA search output. Each .pep.xml file was submitted to Peptide Prophet (TPP) to curate peptide identifications with false discovery rate ≤ 5%. Within the TPP, output from each search engine 8


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pertaining to each gel slice was integrated with the Interprophet Parser and submitted to Protein Prophet to generate the minimum list of proteins to describe all peptides in the entire gel (5% error tolerance). Peptograph generation and analysis. A pictorial representation of peptides (protein coverage on X-axis) identified (false discovery rate ≤ 1%) in sample lanes (denoted by color) and gel slices (Y-axis) was generated, similar to Dix et al.’s peptograph

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. To simplify peptograph

interpretation, the following groups were compiled in silico: (i) control condition, assay buffertreated and mKLK4-treated CM from PC-3:Vector and PC-3:mKLK4 cells; (ii) KLK4-treated condition, KLK4-treated CM from PC-3:Vector, PC-3:mKLK4 and PC-3:KLK4 cells; and (iii) KLK4-transfected condition, assay buffer-treated and mKLK4-treated PC-3:KLK4 cell CM (Figure S-1 A). For each identified protein, cellular component annotations within The Gene Ontology database 30 were obtained using the UniProt 31 Retrieve/ID mapping tool. Peptographs of proteins with cellular component annotations of ‘extracellular space/ region/ matrix’, ‘cell surface’ or ‘extrinsic component of external side of plasma membrane’, were interrogated for evidence of KLK4-mediated proteolysis, using the following criteria. A protein fragment visualized on a peptograph was defined as a theoretical protein or protein fragment of sequence including at least two peptides identified within adjacent gel slices from a single sample. The protein sequence between and including these peptides was considered to be part of the protein fragment, although 100% coverage of tryptic peptides along these fragments was not usually attained. For a protein to be considered a putative KLK4 substrate, there was to be: (i) at least one fragment of the protein identified in the control sample (control fragment), as the identification of a control fragment(s) is essential to provide a reference against which protein migration in the treated/ transfected sample may be compared; and (ii) at least one protein 9



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fragment in the treated/ transfected sample that migrated to at least two gel slices below (a) those peptides in the control sample which comprised of all, or part, of the same sequence or (b) any control fragment identified for the protein, where no peptides of similar sequence were identified in the control sample in gel slices above the putative KLK4-generated fragment. A theoretical protein fragment that met the criterion (ii) was a KLK4-generated fragment and proteins satisfying both of the above criteria [(i) and (ii)] deemed putative KLK4 substrates.

In silico functional analysis of putative KLK4 substrates in PC-3 cell secretions Gene ontology biological process annotations 30 significantly enriched (P ≤ 0.05) among the putative KLK4 substrates identified were determined using a modified Fisher Exact test embedded within the functional annotation tool from the Database for Annotation, Visualization and Integrated Discovery (DAVID) software (v6.7 32, 33), with a custom background of all identified proteins. The topography and size of all putative KLK4-generated substrate fragments was manually compared to that of cleavage product chains for proteins annotated in the UniProt Knowledgebase 31. While PROTOMAP does not identify precise cleavage sites, product identity may be inferred from the approximate size of identified fragments and the sequence of tryptic peptides retrieved from each fragment. Substrates for which KLK4 putatively generated products similar to annotated cleavage products were selected as (i) containing all or part of the sequence of the cleavage product chain; and (ii) migrating to a gel slice(s) expected for products of the annotated size. Validation of KLK4-mediated proteolysis of MMP1 and TSP1. PC-3 cell CM was treated with thermolysin-activated KLK4 in assay buffer (37 °C, 18 h), using CM: KLK4 ratios of 117:1 to 3,159:1 (w/w). Human recombinant substrates were digested with thermolysin-activated 10


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KLK4 in assay buffer (37 °C, 18 h) using molar ratios of pro-MMP1 (R&D): KLK4 of 2:1 to 1,250:1, and TSP1 (Merck): KLK4 of 1:1 to 1,000:1. Treatment with buffer only or mKLK4 (equivalent to the highest total concentration of KLK4) served as controls. Samples were denatured, reduced and alkylated (CM only) as per above, prior to Western blot analysis. Validation of KLK4-mediated activation of MMP1. Recombinant pro-MMP1 (412.1 nM) was incubated with thermolysin-activated KLK4 (20.5 or 4.1 nM) in assay buffer, or with mKLK4 (2.8 ng/ µL) or assay buffer as controls (18 h, 37 ºC). Samples were diluted 25-fold in MMP1 buffer [50 mM Tris, 10 mM CaCl, 150 mM NaCl, pH 7.5, 5 × 10-2% (w/v) brj-350] and selected samples incubated with the metal chelator and MMP inhibitor, EDTA (2 mM; 15 min, 37 ºC), prior to addition of fluorogenic peptide substrate [7-(Methoxycoumarin-4-yl)acetyPLGL-N-3-(2,4-Dinitrophenyl)-L-2,3,-diaminopropionyl-A-R-NH2; 10.5 µM; R&D]. RFU was measured over time in a PolarStar Optima microplate reader (Ex 320 nm, Em 405 nm; 37 ºC). IHC of prostate cancer bone metastasis tissue sections from patients and tumor xenografts. Paraffin-embedded tissue sections of prostate cancer bone metastases were obtained as previously reported 11. IHC was performed on paraffin-embedded sections obtained from PC3 cell metastases to a humanized tissue-engineered bone construct implanted in mice, as published 34, except for the use of the antigen retrieval solution (pH 9.0; Dako) for immunodetection of MMP1, with overnight incubation of primary antibody (4 µg/ mL). Immunodetection of KLK4 was performed similarly; sections were blocked with 5% non-fat milk in TBST buffer, antigen retrieval performed using 5% (w/v) urea in 0.1 M Tris buffer (pH 9.5) and primary antibody (2 µg/ mL) incubated overnight.

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KLK4-mediated hydrolysis of human osteoblast-derived matrix by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). A human osteoblast-derived matrix 35 was treated with thermolysin-activated KLK4 (58.6 nM) or equivalent total amounts of mKLK4 in PBS (37 ºC, 44 h). Digested matrix was further solubilized and analyzed by 2D-PAGE/ silver staining. Protein spots with differential staining intensity between conditions were excised and subject to in-gel digestion, followed by LC-MS/MS, as described 35.

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RESULTS Applying PROTOMAP to identify putative substrates of recombinant or over-expressed KLK4 The PROTOMAP approach was employed to identify putative KLK4 substrates produced by PC-3 cells. Only those proteins with Gene Ontology database cellular component annotation of ‘extracellular space/ region/ matrix’, ‘cell surface’ or ‘extrinsic component of external side of plasma membrane’ were considered, as KLK4 is a secreted protease likely acting on extracellular/ cell surface components. Peptographs were manually interrogated to identify evidence for KLK4-mediated proteolysis. This firstly required identification of a protein fragment in the control sample, to serve as a reference point for migration of the intact protein and/ or its endogenous fragments. KLK4-generated cleavage products were any protein fragment identified in KLK4-treated or KLK4-transfected conditions that migrated at least two gel slices further during SDS-PAGE than a protein/ protein fragment of similar sequence identified in the control condition. The appearance of such products following KLK4 treatment or transfection suggests KLK4 to have initiated proteolysis of these proteins. Thirty-seven putative KLK4 substrates were identified Thirty-seven proteins were determined to be putative KLK4 substrates in KLK4-treated and KLK4-transfected conditions, among a background of 187 extracellular or cell surface proteins detected within the 452 proteins collectively identified across all samples (Figure 1 A and Table 1). Among these was the established KLK4 substrate, uPA (Table 1), validating the approach. Greater than 50% of the identified putative KLK4 substrates were detected with protein sequence 13



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coverage above 29% and were identified by at least 14 unique peptides (Table 1), reinforcing confidence in the detection of proteins and proteolytic fragments. Twenty-five putative substrates were identified in the KLK4-treated condition, eight in the KLK4-transfected condition, and four in both conditions (Figure 1 B). The greater number of putative substrates identified in the KLK4-treated versus KLK4-transfected condition may have been due to elevated KLK4 activity in the former condition. KLK4 was confirmed to be produced by PC-3:KLK4 cells, with < 12 ng KLK4 produced per 50 µL of cell CM (< 240 ng/ mL; Figure S-1 B). However, activity against a fluorogenic peptide substrate was demonstrated only in KLK4-treated but not KLK4-transfected CM, after correcting for background endogenous proteolysis (data not shown). Similarly, mKLK4 produced in PC-3:mKLK4 cell CM (Figure S-1 B) had no activity (data not shown). Nonetheless, identification of 12 putative substrates in the KLK4-transfected condition suggests a low level of KLK4 activity therein. Notably, for most putative substrates identified uniquely in the KLK4-transfected condition, peptide recovery in the KLK4-treated condition was relatively poor (data not shown), potentially as a result of extensive degradation upon recombinant KLK4 treatment. KLK4 activates MMP1 Next we sought to identify putative KLK4-generated bioactive products, particularly those involved in cancer progression. Six of the 36 novel putative KLK4 substrates were potentially cleaved into bioactive products annotated in the UniProt Knowledgebase (Table 2). Among those with products possessing established roles in prostate cancer progression, KLK4 appeared to activate MMP1 and liberate saposin-C and/ or -D from prosaposin (Table 2; prosaposin peptograph shown in Figure S-2). Given the important role for MMP1 in multiple processes 14


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facilitating prostate cancer progression (see discussion), KLK4-mediated MMP1 hydrolysis was selected for further validation. PROTOMAP identified peptides specific to the KLK4-treated sample (red boxes) of sequence consistent with the 22 kDa active protease (light purple arrowhead and shaded box, Figure 1 C) that results from MMP1 autolysis 36. These peptides were derived from a putative KLK4generated cleavage product (red arrow, boxes and shading) that migrated to within one gel slice (slice 21) of that expected of a 22 kDa product (slice 20; Figure 1 C). The putative KLK4generated product, however, harbors an additional seven amino acids at its C-terminus (I270K276), compared to the canonical 22 kDa chain (residue annotations and bottom panel showing sequence information, Figure 1 C). Peptides with migration and topography consistent with 52 kDa pro-MMP1 (light gray arrowhead and shaded box) were uniquely detected in the control sample (yellow boxes and shading, gel slices 9 and 10, Figure 1 C). Notably, the 52 kDa proform could not be distinguished from the active mature 42 kDa protease (dark gray arrowhead and shaded box), as both forms would be expected to migrate to adjacent gel slices; thus, one or both of these species may have been identified in gel slices 9 and 10 in the control condition. While the 22 kDa active protease was not identified in the control condition, the residual 27 kDa inactive product of MMP1-induced autolysis (turquoise arrowhead and shaded box) appeared to be identified only in this sample (yellow boxes and shading, gel slice 18, Figure 1 C), suggesting active MMP1 (capable of autolysis) to be present in both KLK4-treated and control conditions. To validate the direct involvement of KLK4 in MMP1 hydrolysis, recombinant pro-MMP1 was treated with KLK4 and products visualized by Western blot analysis. KLK4 induced dosedependent production of the 42 kDa mature protease (dark gray arrowhead) and reduced 15



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intensity of the 52 kDa pro-form (light gray arrowhead; lanes 4-8; Figure 1 D). A 22 kDa product resulting from autolysis was not observed, where generation of this product potentially requires the involvement of endogenous factors present in PC-3 cell CM. Pro-MMP1 treated with mKLK4 (lane 3) migrated as per the untreated control (lane 2; Figure 1 D). To confirm that the 42 kDa product reflected active mature protease, a fluorogenic peptide substrate assay was performed. KLK4 induced a dose-dependent increase in MMP1 activity (Figure 1 E). Importantly, KLK4 or pro-MMP1 alone, or pro-MMP1 treated with mKLK4, did not induce peptide substrate hydrolysis, consistent with the blank sample (peptide substrate and buffer only; Figure 1 E). Further confirming that the measured activity was due to MMP1 activity and not background peptide substrate hydrolysis by KLK4, incubation of KLK4-treated pro-MMP1with the metal ion chelator and MMP inhibitor, EDTA, abolished peptide hydrolysis (Figure 1 E). Thus, while other factors in PC-3 cell CM may contribute toward MMP1 activation, and/ or mediate MMP1 being processed into 22 kDa and 27 kDa forms, KLK4 directly cleaved recombinant pro-MMP1 to generate a 42 kDa active protease. KLK4 liberates the N-terminus from TSP1 PROTOMAP found that KLK4 liberated purported pro-angiogenic fragment(s) from TSP1 that are not annotated in the UniProt Knowledgebase, but are well-documented in the literature (Table 2). Western blot analysis confirmed that KLK4 generated 45 kDa to 150 kDa products of TSP1 in PC-3 cell CM (filled arrowheads; lanes 4-6; Figure 2 A), as compared to TSP1 in untreated (lane 9) or mKLK4-treated (lane 8) PC-3 cell CM, which migrated at 160 kDa (open arrowhead; Figure 2 A). This was consistent with migration of recombinant TSP1 (lane 2; Figure 2 A). TSP1 was confirmed as a direct target of KLK4, where recombinant TSP1 digested with 16


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10:1 (lane 7) and 100:1 (lane 8) molar ratios of TSP1:KLK4 yielded 30 kDa and 15 kDa products, as detected by an antibody targeting the protein N-terminus (filled arrowheads; Figure 2 B). Corresponding respective 130 kDa and 145 kDa products were detected by the C-terminal targeting antibody (filled arrowheads; Figure 2 C). Faint 30 kDa and more prominent 15 kDa products were also detected in the mKLK4-treated TSP1 sample (lane 5; Figure 2 B), suggesting low levels of mKLK4-mediated TSP1 hydrolysis. TSP1 (open arrowhead) was degraded to beyond the detection limit of the antibody by the highest employed concentration of KLK4 (lane 6), and was not hydrolyzed at a 1,000:1 molar ratio of TSP1: KLK4 (lane 9; Figure 2 B-C). Collectively, these results indicate that, at select concentrations, KLK4 directly liberates 15 kDa and 30 kDa N-terminal products from TSP1, although degrading TSP1 when present in equimolar amounts. KLK4 cleaves TSP1 in human osteoblast-derived bone matrix As TSP1 regulates bone homeostasis 37, where bone is the primary site of prostate cancer metastasis, it was sought to determine whether bone matrix-derived TSP1 was also cleaved by KLK4. Upon 2D-PAGE analysis of KLK4-treated human osteoblast-derived bone matrix, a spot exhibiting reduced intensity (Figure 2 D, left), as compared to mKLK4 control treatment (Figure 2 D, right), was identified as TSP1. Thus, KLK4 cleaves TSP1 produced by human bone matrixforming osteoblasts, in addition to bone metastasis-derived prostate cancer cells. MMP1 is produced in patient-derived prostate cancer bone metastases and in a humanized in vivo bone metastasis model

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It was next was sought to confirm reports of MMP1 production in prostate cancer bone metastases 38, to demonstrate that MMP1 is accessible to KLK4-mediated hydrolysis at this site. IHC showed both MMP1 (Figure 3 A) and KLK4 (Figure 3 B) to be produced in the tumor (arrows) and bone compartments in patient tissue sections of prostate cancer bone metastases, with negligible or weak staining of osteocytes (black arrowheads) and osteoblasts (turquoise arrowheads) in the bone compartment. Similarly, MMP1 was produced specifically by PC-3 cells (arrow), but not in osteocytes (black arrowhead) or osteoblasts (turquoise arrowhead), in PC-3 cell metastases formed in ectopic human tissue-engineered bone constructs in mice (Figure 3 C). This in vivo model recapitulates the biological and morphological properties of human bone, to which PC-3 cells colonize following intracardiac inoculation 34. Thus, MMP1 appears to be accessible for KLK4-mediated activation in prostate cancer bone metastases. Putative KLK4 substrates participate in tumor-promoting processes Functional enrichment analysis indicated that the biological process most significantly associated with the identified putative KLK4 substrates was defense response (P = 5.15 × 10-3; 2.96-fold enrichment), involving ten of the 37 putative KLK4 substrates (column 5) or 24% of background PC-3 cell CM proteins associated with that process (column 2; Table 3). These ten included TSP1 and MMP1, which also function in other of the biological processes significantly associated with the putative KLK4 substrates identified, namely, regulation of cell motion (P = 1.23 × 10-2), adhesion (P = 1.55 × 10-2), muscle organ development (P = 3.65 × 10-2) and proliferation (P = 4.00 × 10-2; Table 3). Putative KLK4 substrates associated with regulation of cell motion and adhesion, processes in which KLK4 has been previously implicated 7, 9, 11, included ECM and matricellular proteins, proteins regulating cytoskeletal rearrangements, 18


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growth factors and peptidases, and their regulators (Table 3). Thus, the PROTOMAP approach successfully identified putative substrate intermediates of known KLK4-regulated processes, and highlighted potential novel functions for the protease.

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DISCUSSION This study aimed to identify KLK4 substrates in late-stage prostate cancer cell secretions, to begin to delineate the mechanism of KLK4 action in prostate cancer progression. Thirty-six novel putative KLK4 substrates and one established substrate were identified by PROTOMAP. The former included two substrates selected for biochemical validation, MMP1 and TSP1, cleaved by KLK4 into potentially bioactive products. Pathways regulated by the identified putative KLK4 substrates include cell defense response, motion, adhesion, muscle organ development and proliferation, identifying KLK4 as a potential multi-functional regulator of prostate cancer progression. KLK4 activated MMP1, a protease with elevated production in prostate cancer versus benign tissue 39, 40. Over-expression of MMP1 in prostate tumor xenografts increased tumor size and induced lung metastasis in vivo 41. Further, MMP1 induced prostate cancer cell migration and invasion in vitro 41. Herein, MMP1 was confirmed to be expressed by bone metastatic prostate cancer cells in patient samples and in a humanized in vivo bone metastasis model, indicating that MMP1 is likely susceptible to activation by KLK4 in prostate cancer bone metastases. MMP1 was integral for in vitro colony formation of prostate cancer cells on bone marrow stroma 38, together with the above suggesting KLK4-mediated activation of MMP1 in prostate cancer promotes lung and bone metastasis. KLK4 was validated to directly cleave the matricellular protein, TSP1. While TSP1 is often considered to be a negative regulator of angiogenesis, in vivo functional studies have yielded conflicting results regarding the impact of TSP1 on angiogenesis 42. Similar conflicts arise with attempts to correlate TSP1 expression with microvessel density and other 20


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clinicopathological features in patient tissues; therefore, it has been suggested that the inconsistent activities of TSP1 in various systems may be due to differential proteolysis of TSP1 yielding bioactive products with unique functions 42. Here we show KLK4 to liberate N-terminal fragments of TSP1, although degrading TSP1 when applied at equimolar amounts as the substrate. KLK4-generated products were of similar size and topography to those previously shown to induce angiogenesis in vitro and in vivo 43-46. As angiogenesis is a key cancer hallmark 47

, KLK4 may be among those proteases able to facilitate a tumor-promoting phenotype via

specific TSP1 hydrolysis, provided that the ratio of TSP1:KLK4 favors production of these Nterminal products. Whether KLK4-mediated TSP1 degradation may also favor tumor neoangiogenesis remains unclear. Strikingly, mKLK4 also liberated the above N-terminal fragments from TSP1, when applied to the substrate in equimolar amounts. This finding is not unanticipated, as active site S>A mutants of rat trypsin also yielded an active protease, albeit with 105-fold reduced efficiency than wildtype trypsin 48. Despite potential mKLK4 activity in the control condition, TSP1 was still identified as a putative KLK4 substrate using PROTOMAP, as KLK4 produced fragments of TSP1 that were not detected in the control. Thus, while mKLK4 may have low levels of activity against certain proteins in PC-3 cell CM, as with TSP1, this is likely not to have affected substrate identification by PROTOMAP. TSP1 was also identified as a putative KLK4 substrate in human osteoblast-derived bone matrix. Bone is the primary site of prostate cancer metastasis and prostatic metastases in bone presents with an overall osteoblastic phenotype 2. Notably, TSP1 activates transforming growth factor β1 49, a protein that inhibits osteoblast differentiation 50. Plasmin-mediated processing of 21



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TSP1, which yielded similar products as did KLK4 treatment 51, reduced the ability for TSP1 to activate transforming growth factor β1 49. Moreover, silencing TSP1 expression in mice resulted in increased bone deposition 37. Thus, either specific processing or degradation of TSP1 by KLK4 would likely favor bone deposition at the site of prostatic bone metastases. The present study identified a large number of novel putative KLK4 substrates, where previously only a handful had been determined through biochemical assays 15 or were predicted, based on sequence similarity with KLK4-preferred cleavage sites 52. Herein, both the established KLK4 substrate, uPA 53, and the predicted substrate, fibronectin 52, were identified as KLK4 substrates. All other established or predicted KLK4 substrates were not sufficiently retrieved from PROTOMAP samples to make assessments regarding proteolysis. One exception was the predicted KLK4 substrate, inhibin A 52, which was detected by PROTOMAP but was not cleaved by KLK4 (data not shown). PROTOMAP was utilized herein, in part due to its simple, robust and reproducible front-end workflow, compared to other degradomics strategies requiring complex chemistries 17. Further, PROTOMAP approximates the size and topography of cleavage products, helping to inform as to their putative function. Although some products will not be trypsin-cleavable and will not elute from the gel, the detection of only one trypsin-susceptible KLK4-generated product is required to indicate proteolysis, where additional products may be characterized using targeted techniques. PROTOMAP, however, does not identify precise protein cleavage sites; therefore, direct proteolysis is not readily differentiated from indirect proteolysis using this technique. Positional degradomics strategies, such as Terminal Amine Isotopic Labeling of Substrates (TAILS) 54, 55, COmbined FRActional DIagonal Chromatography (COFRADIC) 56 and Charge-based 22


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FRActional DIagonal Chromatography (ChaFRADIC) 57, may be employed to complement PROTOMAP findings by defining precise cleavage sites and better discriminate direct from indirect targets. Although some of the novel putative substrates identified in this study may not be direct KLK4 targets, it is also imperative to map indirect proteolytic action when developing protease inhibitors for cancer therapy 58. While the biological implications of the identified cleavage events must be validated, our findings have identified 36 novel putative KLK4 substrates, two of which were validated biochemically and implicate KLK4 in the promotion of prostate cancer progression through functions not before ascribed to this protease.

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FIGURE LEGENDS Figure 1. (A) Number of proteins and putative KLK4 substrates identified in PC-3 cell CM by PROTOMAP. Of the 452 proteins identified in at least one condition in PC-3 cell CM (white circle), 187 were annotated to be extracellular or cell surface proteins (light gray circle), and 37 of these were putative KLK4 substrates (dark gray circle). (B) Number of putative KLK4 substrates cleaved in KLK4-treated or KLK4-transfected conditions. Twenty-five putative substrates were cleaved only in the KLK4-treated condition, with eight cleaved only in the KLK4-transfected condition and four in both conditions. (C) Peptograph for the KLK4 substrate, pro-MMP1. Peptograph displays all significantly identified peptides (colored boxes) from gel slices (right Y-axis, 1-32) and corresponding size (kDa; left Y-axis), aligned with the corresponding protein amino acid residue number (X-axis). Peptides are colored according to the condition from which they were derived: KLK4-treated (Tr; red), KLK4-transfected (Tx; blue) or control (Ctr; yellow; color key, bottom left). A schematic of selected protein chains, based on annotations in the UniProt Knowledgebase, is beneath the X-axis (light gray, dark gray, light purple and turquoise boxes). The appearance of a KLK4-generated cleavage product (red arrow and shaded box, gel slice 21) of topography and migration consistent with the active 22 kDa collagenase chain (light purple arrowhead and shaded box) infers a putative role for KLK4 in activating pro-MMP1. Control samples contained fragments (yellow shaded boxes) identified by peptides aligned with pro-MMP1 (light gray arrowhead and shaded box) and/ or active mature MMP1 (dark gray arrowhead and shaded box), as well as the 27 kDa chain (turquoise arrowhead and shaded box). Amino acid residues are annotated for the N- and C-termini of canonical protein chains (italics) or the most N- and C-terminal residue identified from protein fragments putatively identified by PROTOMAP (not in italics). The bottom panel shows the amino acid 24


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sequence of pro-MMP1, with sequences identified by PROTOMAP colored according to the condition in which they were identified; the canonical sequence of active 22 kDa collagenase is shaded in light purple. (D) Western blot analysis of KLK4-treated recombinant pro-MMP1. KLK4 cleaved pro-MMP1 at molar ratios of pro-MMP1:KLK4 of 2:1 to 250:1 (lanes 4-7), but not 1,250:1 (lane 8), to produce a 42 kDa product (MMP1; dark gray arrowhead). Equivalent concentrations of mKLK4 did not induce pro-MMP1 hydrolysis (lane 3), with MMP1 identified at 52 kDa, consistent with untreated pro-MMP1 (light gray arrowhead, lane 2). Additional controls included only KLK4 (lane 9) or mKLK4 (lane 10). Protein molecular weight standard (10-100 kDa) was loaded in lane 1. (E) Activity analysis of KLK4-treated recombinant proMMP1. MMP1-mediated hydrolysis of a fluorogenic peptide substrate, 7-(Methoxycoumarin-4yl)acety-PLGL-N-3-(2,4-Dinitrophenyl)-L-2,3,-diaminopropionyl-A-R-NH2, was measured every 34 sec for 952 sec. Relative fluorescence units (RFU) for each condition was corrected against the blank sample (cor. RFU; black line, white circle). High (dark red line) or low (light red line) concentrations of KLK4 activated pro-MMP1 in a dose-dependent manner. Proteolytic activity was abrogated with addition of the metal ion chelator and MMP inhibitor, EDTA (dark gray line). KLK4 (black line, white diamond) or pro-MMP1 (black line, white square), alone, and mKLK4-treated pro-MMP1 (light gray line), did not exhibit activity, indicating assay specificity for active MMP1. Figure 2. (A) Western blot analysis of KLK4-mediated hydrolysis of TSP1 in PC-3 cell CM. Immunodetection with an antibody targeted to the C-terminus of TSP1 showed that KLK4 cleaved TSP1 into 45-150 kDa fragments (filled arrowheads) in PC-3 cell CM at ratios of PC-3 cell CM:KLK4 (w/w) of 117:1 (lane 4), 351:1 (lane 5) and 1,053:1 (lane 6). TSP1 migration upon KLK4 treatment at a 3,159:1 ratio (lane 7) was not that different to its migration in 25



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mKLK4-treated (lane 8) or untreated (lane 9) PC-3 cell CM, and with recombinant TSP1 (rTSP1; 160 kDa, open arrowhead; lane 2). Protein molecular weight standard (20-250 kDa) was loaded in lane 1. (B, C) Western blot analyses of KLK4-treated recombinant TSP1. The molar ratio of TSP1:KLK4 (or mKLK4, negative control) is shown, normalized to the amount of KLK4. TSP1 (open arrowhead) was degraded by KLK4 (lane 6; B; and lanes 6-7; C) or cleaved by KLK4 into 15 kDa and 30 kDa N-terminal fragments (filled arrowheads, lanes 7-8; B), and 145 kDa and 130 kDa C-terminal fragments (filled arrowheads, lane 8; C). Controls included untreated (lane 4) or mKLK4-treated TSP1 (lane 5), and lanes containing only mKLK4 (lane 2) or KLK4 (lane 3). Protein molecular weight standard (20-250 kDa) was loaded in lane 1. (D) 2DPAGE analysis of KLK4-induced hydrolysis of TSP1 in human osteoblast-derived bone matrix. A protein spot identified as TSP1 exhibited reduced intensity in KLK4-treated (left) versus mKLK4-treated (right) matrix. Regions of decreasing molecular weight (MW) and increasing pH are annotated on the Y- and X-axes, respectively. Bottom images show magnification of the indicated area. Figure 3. IHC of MMP1 (A) and KLK4 (B) in patient-derived prostate cancer bone metastases. IHC showed MMP1 and KLK4 to be produced in both tumor (arrows) and bone compartments, with negligible or weak staining of osteocytes (black arrowheads) and osteoblasts (turquoise arrowheads) within the bone compartment (scale bars, 100 µm). (C) Immunodetection of MMP1 in an in vivo prostate cancer bone metastasis model. MMP1 was localized to the cytoplasm of PC-3 cells (arrow) and was absent in both osteocytes (black arrowhead) and osteoblasts (turquoise arrowhead; scale bars, 100 µm).

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Table 1. Putative KLK4 substrates in PC-3 cell CM Description

Tr/Tx

UniProt accession

Loc

Length

Percent coverage

Unique peptides

Total spectra

Enzyme: 78 kDa glucose-regulated protein Tr/Tx P11021 CS 654 36.5 18 67 Actin, alpha cardiac muscle 1 Tx P68032 EC 377 36.3 2 9 Glucose-6-phosphate isomerase Tr P06744 EC 558 36.4 14 92 Matrix metalloproteinase-1 Tr P03956 EC 469 19 7 16 (interstitial collagenase) Peroxiredoxin-6 Tx P30041 EC 224 59.8 14 92 Plastin-2 Tr P13796 EC 627 44.7 18 91 Protein disulfide-isomerase Tr P07237 EC 508 42.5 17 73 Protein disulfide-isomerase A4 Tx P13667 CS 645 6.7 4 7 Pyruvate kinase PKM Tx P14618 EC 531 29.4 12 35 Sulfhydryl oxidase 1 Tr O00391 EC 747 39.5 29 346 Urokinase-type plasminogen Tr P00749 EC/CS 431 46.2 17 289 activator Enzyme regulator: 14-3-3 protein sigma Tx P31947 EC 248 44.8 6 27 14-3-3 protein zeta/delta Tr P63104 EC 245 69 14 234 Amyloid beta A4 protein Tr P05067 EC/CS 770 21.7 14 108 Complement C3 Tx P01024 EC 1663 12 13 33 Fibronectin Tr P02751 EC 2386 20.8 31 111 Glia-derived nexin Tr P07093 EC/CS 398 22.4 6 70 Metalloproteinase inhibitor 2 Tr P16035 EC/CS 220 23.6 8 50 Prosaposin Tr P07602 EC 524 24 12 229 Serpin B7 Tr O75635 EC 380 46.6 14 119 Stanniocalcin-2 Tr O76061 EC 302 19.9 5 39 Growth factor (regulator): Collagen alpha-1(VI) chain Tx P12109 EC 1028 25.9 21 227 Granulins Tr P28799 EC 593 18.2 12 133 Myeloid-derived growth factor Tr Q969H8 EC 173 27.2 4 30 Receptor: Galectin-3-binding protein Tr Q08380 EC 585 32.3 17 261 Integrin beta-1 Tr P05556 CS 798 12.5 8 57 Structural molecule (regulator): Agrin Tr/Tx O00468 EC/CS 2067 17.8 23 113 Alpha-actinin-1 Tr P12814 EC 892 48 25 121 Alpha-actinin-4 Tr O43707 EC 911 53.6 24 55 Keratin, type II cytoskeletal 1 Tr/Tx P04264 EC 644 20.3 10 218 Laminin subunit alpha-5 Tr O15230 EC 3695 4.7 12 22 Mucin-5B Tr Q9HC84 EC 5762 4.8 15 47 Thrombospondin-1 Tr/Tx P07996 EC/CS 1170 46.3 46 1270 Vinculin Tr P18206 EC 1134 37.4 33 144 Transporter: Chloride intracellular channel Tx O00299 EC 241 12 3 8 protein 1 Neutrophil gelatinase-associated Tr P80188 EC 198 72.2 14 472 lipocalin Other: Pentraxin-related protein PTX3 Tr P26022 EC 381 38.8 11 161 Description, UniProt Knowledgebase protein name; Tr/Tx, putative substrate in the KLK4-treated (Tr) and/or KLK4transfected (Tx) condition; Loc, subcellular localization (EC, extracellular; CS, cell surface); Length, number of amino acid residues per protein; Percent coverage, proportion of amino acid sequence identified by LC-MS/MS; Unique peptides, number of peptide sequences matched only to the respective protein; Total spectra, the number of independent spectra matched to the respective protein.

27 ACS Paragon Plus Environment


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Table 2. Putative bioactive cleavage products generated by KLK4 and their functional association to prostate cancer Description

UniProt accession

Putative cleavage product chains

General bioactivity of the cleavage product chain

Association of the intact protein with prostate cancer

Association of the cleavage product chain with prostate cancer

Agrin

O00468

Agrin C-terminal 110, 90 and 22 kDa subunits

Induction of dendritic filopodia

-

-

Amyloid beta A4 protein

P05067

Soluble APP-alpha and beta

Neural regulation

-

-

Fibronectin

P02751

Ugl-Y3

Not specified

May indirectly induce metastasis through up-regulating vascular endothelial growth factor-mediated angiogenesis 59

Matrix metalloproteinase-1

P03956

22 kDa interstitial collagenase

Peptidase (collagenase activator)

Produced in bone metastasis; Promotes migration, expression is induced in prostate invasion, tumor growth cancer cells co-cultured with bone 41 and colony formation cells, in vitro 41 on bone 38

Prosaposin

P07602

Propeptides, saposin-C and Glucosylceramide and -D galactosylceramide hydrolysis; sphingomyelin phosphodiesterase activator

Promotes proliferation, migration and invasion

18-28 kDa N-terminal peptidesa

-

Thrombospondin-1

P07996

Five amino acid sequence induces TSP1 expression in Gr1+ myeloid cells, which infiltrate the lung and make it less accommodating of metastasis 60 a 43Promotes migration and tumor Induction of angiogenesis 46 ; Reduction of transforming growth; inhibits angiogenesis 63 growth factor β1 activationa

61, 62

49

Description, UniProt Knowledgebase protein name; Putative cleavage product chains, previously reported protein product chains (annotated in the UniProt Knowledgebase or a reported in the literature) of similar size and sequence to at least one putative KLK4-generated fragment from the respective protein; General bioactivity of the cleavage product chain, putative bioactivity of respective cleavage products, as reported in the UniProt Knowledgebase or denoted literature references; Association of the intact protein with prostate cancer, functional role of the protein in prostate cancer, where reported in the literature (denoted references); Association of the cleavage product chain with prostate cancer, as per previous, for putative cleavage product chains.

Table 3. Biological processes enriched among putative KLK4 substrates in PC-3 cell CM Gene ontology (GO) biological process annotation

Percent associated

Foldenrichment

P-value

Associated substrates

Reference (if previously reported to be KLK4-regulated )


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GO:0006952~defense response

24

2.96

5.15E-03

GO:0051270~regulation of cell motion

16

3.83

1.23E-02

FINC, CLIC1, ITB1, PTX3, LG3BP, K2C1, CO3, TSP1, 1433Z, MMP1a ACTN1, VINC, LAMA5, GDN, ACTN4, TSP1, MMP1a

7, 9, 11

ACTN1, FINC, VINC, CO6A1, ITB1, LAMA5, 11 A4, MUC5B, LG3BP, TSP1, MMP1a 13 3.61 3.65E-02 ITB1, LAMA5, AGRIN, ACTC, A4, TSP1a, GO:0007517~muscle organ development MMP1a 24 2.12 4.00E-02 GRN, TIMP2, QSOX1, ITB1, LAMA5, 7, 9, 12 GO:0042127~regulation of cell MYDGF, 1433S, UROK, TSP1, MMP1a proliferation Gene ontology (GO) biological process annotation, representative GO biological process annotations are shown for annotations sharing similar associated putative substrates; Percent associated, the number of putative KLK4 substrates associated with the respective biological process as a percentage of all background molecules annotated to be associated with that process; Fold-enrichment and P-value, statistics respectively defining the degree and significance of enrichment; Associated substrates, putative substrates associated with the GO biological process, as annotated in the GO database or aevidenced in the literature (41, 64-68; note that these substrates were not considered in enrichment calculations). Reference (if previously reported to be KLK4-regulated), literature reference demonstrating KLK4-mediated regulation of the identified enriched biological process. FINC, Fibronectin; CLIC1, Chloride intracellular channel protein 1; ITB1, Integrin beta-1; PTX3, Pentraxin-related protein PTX3; LG3BP, Galectin-3-binding protein; K2C1, Keratin, type II cytoskeletal 1; CO3, Complement C3; TSP1, Thrombospondin-1; 1433Z, 14-3-3 protein zeta/delta; ACTN1, Alpha-actinin-1; VINC, Vinculin; LAMA5, Laminin subunit alpha-5; GDN, Glia-derived nexin; ACTN4, Alpha-actinin-4; CO6A1, Collagen alpha-1(VI) chain; A4, Amyloid beta A4 protein; MUC5B, Mucin-5B; AGRIN, Agrin; ACTC, Actin, alpha cardiac muscle 1; GRN, Granulins; TIMP2, Metalloproteinase inhibitor 2; QSOX1, Sulfhydryl oxidase 1; MYDGF, Myeloid-derived growth factor; 1433S, 14-3-3 protein sigma; UROK, Urokinase-type plasminogen activator; G6PI, Glucose-6-phosphate isomerase; PLSL, Plastin-2.

GO:0007155~cell adhesion

27

2.31

1.55E-02


29


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SUPPORTING INFORMATION Figure S-1. (A) Conditions for PROTOMAP identification of putative KLK4 substrates in PC-3 cell CM.

Three conditions were compiled in silico for plotting peptographs: (i) control, sample

buffer- or mKLK4-treated CM from PC-3:Vector and PC-3:mKLK4 cells (yellow); (ii) KLK4-treated, KLK4-treated CM from PC-3:Vector, PC-3:mKLK4 and PC-3:KLK4 cells (red) and; (iii) KLK4-transfected, sample buffer- or mKLK4-treated CM from PC-3:KLK4 cells (blue). (B) Western Blot analysis of PC-3:Vector, PC-3:mKLK4 and PC-3:KLK4 cell CM. PC-3:Vector cell CM (lane 2) was devoid of KLK4 immunoreactivity. PC3:mKLK4 (lane 3) and PC-3:KLK4 (lane 4) cell CM exhibited immunoreactivity at 30 kDa. Recombinant thermolysin-activated KLK4 (rKLK4) positive control exhibited strong immunoreactivity for KLK4 at 23 kDa (lanes 5-7). Protein molecular weight standard (10250 kDa) was loaded in lane 1. Figure S-2. Peptograph for the putative KLK4 substrate, prosaposin. Peptograph displays all significantly identified peptides (colored boxes) from gel slices (right Y-axis, 132) and corresponding size (kDa; left Y-axis), aligned with the corresponding protein amino acid residue number (X-axis). Peptides are colored according to the condition from which they were derived: KLK4-treated (Tr; red), KLK4-transfected (Tx; blue) or control (Ctr; yellow; color key, bottom left). A schematic of selected protein chains, based on annotations in the UniProt Knowledgebase, is beneath the X-axis (light purple boxes). Prosaposin appeared at its expected molecular weight (open arrowhead), as well as migrating as lower molecular weight fragments, across all conditions. KLK4 cleaved prosaposin into at least one putative 15 kDa product (red arrow and red boxes), of size and topography similar to saposinC and -D (light purple arrowhead and shaded boxes). Amino acid residues are annotated for the N- and C-termini of canonical protein chains (italics) or the most N- and C-terminal


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residue identified from putative KLK4-generated protein fragments identified by PROTOMAP (not in italics).



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AUTHOR INFORMATION Corresponding Author D/Prof Judith Clements. E: [email protected]; T: +61 734 43 7 241; +61 407 074 032; F: +61 734 437 779; A: Queensland University of Technology at the Translational Research Institute, Level 3 West, 37 Kent Street, Woolloongabba, QLD, 4102, Australia. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Funding was provided by the National Health and Medical Research Council of Australia (#1010144), and the Cancer Council Queensland (#614273, #1064484 – Morris Ronald Huggett Research Project). Distinguished Professor Judith Clements is a National Health and Medical Research Council Principal Research Fellow (#1005717). Ruth Fuhrman-Luck was a recipient of a Queensland Government Smart Futures PhD scholarship, Australian Postgraduate Award and scholarships from Queensland University of Technology (QUT) and the Institute of Health and Biomedical Innovation (QUT).


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ACKNOWLEDGMENT LC-MS/MS analysis was performed by Mr Alun Jones and staff at the Institute for Molecular Bioscience, University of Queensland. We acknowledge Dr Boris M. Holzapfel for providing paraffin-embedded tissue sections, Dr Johannes C. Reichert and Prof Dietmar W. Hutmacher for providing human osteoblast-derived matrix, as well as Dr Lez J. Burke for digesting osteoblast-derived matrix with KLK4. We thank Dr Ali Shokoohmand for his helpful review of the manuscript.



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ABBREVIATIONS KLK4, kallikrein-related peptidase 4; PROTOMAP, PROtein TOpography and Migration Analysis Platform; uPA , urokinase-type plasminogen activator; MMP1, matrix metalloproteinase-1; TSP1, thrombospondin-1; PSA, prostate-specific antigen; IHC, immunohistochemistry; mKLK4 , mutant KLK4; BCA, bicinchoninic acid; CM, conditioned media; RFU, relative fluorescence units; MS/MS, tandem mass spectrometry; TPP, Trans Proteomic Pipeline; OMSSA, Open Mass Spectrometry Search Algorithm; DAVID, Database for Annotation, Visualization and Integrated Discovery; 2D-PAGE, 2-dimensional polyacrylamide gel electrophoresis; TAILS, Terminal Amine Isotopic Labeling of Substrates; COFRADIC, COmbined FRActional DIagonal Chromatography; ChaFRADIC, Chargebased FRActional DIagonal Chromatography. REFERENCES 1.

Ferlay, J.; Soerjomataram, I.; Ervik, M.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.; Forman, D.; Bray, F. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. http://globocan.iarc.fr

2.

Schroder, F. H.; Hugosson, J.; Carlsson, S.; Tammela, T.; Maattanen, L.; Auvinen, A.; Kwiatkowski, M.; Recker, F.; Roobol, M. J., Screening for prostate cancer decreases the risk of developing metastatic disease: findings from the European Randomized Study of Screening for Prostate Cancer (ERSPC). Eur. Urol. 2012, 62, (5), 745-52.

3.

Turk, B., Targeting proteases: successes, failures and future prospects. Nat. Rev. Drug Discov. 2006, 5, (9), 785-99.

4.

Imai, K.; Takaoka, A., Comparing antibody and small-molecule therapies for cancer. Nat. Rev. Cancer 2006, 6, (9), 714-27.

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Seiz, L.; Kotzsch, M.; Grebenchtchikov, N. I.; Geurts-Moespot, A. J.; Fuessel, S.; Goettig, P.; Gkazepis, A.; Wirth, M. P.; Schmitt, M.; Lossnitzer, A.; Sweep, F. C.; Magdolen, V., Polyclonal antibodies against kallikrein-related peptidase 4 (KLK4): immunohistochemical assessment of KLK4 expression in healthy tissues and prostate cancer. Biol. Chem. 2010, 391, (4), 391-401.


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6.

Ramsay, A. J.; Dong, Y.; Hunt, M. L.; Linn, M.; Samaratunga, H.; Clements, J. A.; Hooper, J. D., Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression. J. Biol. Chem. 2008, 283, (18), 12293-304.

7.

Klokk, T. I.; Kilander, A.; Xi, Z.; Waehre, H.; Risberg, B.; Danielsen, H. E.; Saatcioglu, F., Kallikrein 4 is a proliferative factor that is overexpressed in prostate cancer. Cancer Res. 2007, 67, (11), 5221-30.

8.

Dong, Y.; Bui, L. T.; Odorico, D. M.; Tan, O. L.; Myers, S. A.; Samaratunga, H.; Gardiner, R. A.; Clements, J. A., Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms. Endocr. Relat. Cancer 2005, 12, (4), 875-89.

9.

Veveris-Lowe, T. L.; Lawrence, M. G.; Collard, R. L.; Bui, L.; Herington, A. C.; Nicol, D. L.; Clements, J. A., Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells. Endocr. Relat. Cancer 2005, 12, (3), 631-43.

10. Avgeris, M.; Stravodimos, K.; Scorilas, A., Kallikrein-related peptidase 4 gene (KLK4) in prostate tumors: quantitative expression analysis and evaluation of its clinical significance. Prostate 2011, 71, (16), 1780-9. 11. Gao, J.; Collard, R. L.; Bui, L.; Herington, A. C.; Nicol, D. L.; Clements, J. A., Kallikrein 4 is a potential mediator of cellular interactions between cancer cells and osteoblasts in metastatic prostate cancer. Prostate 2007, 67, (4), 348-60. 12. Mize, G. J.; Wang, W.; Takayama, T. K., Prostate-specific kallikreins-2 and -4 enhance the proliferation of DU-145 prostate cancer cells through protease-activated receptors-1 and -2. Mol. Cancer Res. 2008, 6, (6), 1043-51. 13. Wright, J. T.; Daly, B.; Simmons, D.; Hong, S.; Hart, S. P.; Hart, T. C.; Atsawasuwan, P.; Yamauchi, M., Human enamel phenotype associated with amelogenesis imperfecta and a kallikrein-4 (g.2142G>A) proteinase mutation. Eur. J. Oral Sci. 2006, 114 Suppl 1, 13-7; discussion 39-41, 379. 14. Hart, P. S.; Hart, T. C.; Michalec, M. D.; Ryu, O. H.; Simmons, D.; Hong, S.; Wright, J. T., Mutation in kallikrein 4 causes autosomal recessive hypomaturation amelogenesis imperfecta. J. Med. Genet. 2004, 41, (7), 545-49. 15. Lawrence, M. G.; Lai, J.; Clements, J. A., Kallikreins on Steroids: Structure, Function, and Hormonal Regulation of Prostate-Specific Antigen and the Extended Kallikrein Locus.



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28. Kersey, P. J.; Duarte, J.; Williams, A.; Karavidopoulou, Y.; Birney, E.; Apweiler, R., The International Protein Index: an integrated database for proteomics experiments. Proteomics 2004, 4, (7), 1985-8. 29. The Global Proteome Machine Organization. The common Repository of Adventitious Proteins [Internet]. ftp://ftp.thegpm.org/fasta/cRAP. 30. Ashburner, M.; Ball, C. A.; Blake, J. A.; Botstein, D.; Butler, H.; Cherry, J. M.; Davis, A. P.; Dolinski, K.; Dwight, S. S.; Eppig, J. T.; Harris, M. A.; Hill, D. P.; Issel-Tarver, L.; Kasarskis, A.; Lewis, S.; Matese, J. C.; Richardson, J. E.; Ringwald, M.; Rubin, G. M.; Sherlock, G., Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 2000, 25, (1), 25-9. 31. UniProt Cosortium, UniProt: a hub for protein information. Nucleic Acids Res. 2015, 43, (Database issue), D204-12. 32. Huang da, W.; Sherman, B. T.; Lempicki, R. A., Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009, 4, (1), 44-57. 33. Huang da, W.; Sherman, B. T.; Lempicki, R. A., Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009, 37, (1), 1-13. 34. Holzapfel, B. M.; Wagner, F.; Loessner, D.; Holzapfel, N. P.; Thibaudeau, L.; Crawford, R.; Ling, M. T.; Clements, J. A.; Russell, P. J.; Hutmacher, D. W., Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone. Biomaterials 2014, 35, (13), 4108-15. 35. Reichert, J. C.; Quent, V. M.; Burke, L. J.; Stansfield, S. H.; Clements, J. A.; Hutmacher, D. W., Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment. Biomaterials 2010, 31, (31), 7928-36. 36. Vallon, R.; Muller, R.; Moosmayer, D.; Gerlach, E.; Angel, P., The catalytic domain of activated collagenase I (MMP-1) is absolutely required for interaction with its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1). Eur. J. Biochem. 1997, 244, (1), 81-8. 37. Amend, S. R.; Uluckan, O.; Hurchla, M.; Leib, D.; Novack, D. V.; Silva, M.; Frazier, W.; Weilbaecher, K. N., Thrombospondin-1 regulates bone homeostasis through effects on bone matrix integrity and nitric oxide signaling in osteoclasts. J. Bone Miner. Res. 2015, 30, (1), 106-15.



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38. Hart, C. A.; Scott, L. J.; Bagley, S.; Bryden, A. A.; Clarke, N. W.; Lang, S. H., Role of proteolytic enzymes in human prostate bone metastasis formation: in vivo and in vitro studies. Br. J. Cancer 2002, 86, (7), 1136-42. 39. Zhong, W. D.; Han, Z. D.; He, H. C.; Bi, X. C.; Dai, Q. S.; Zhu, G.; Ye, Y. K.; Liang, Y. X.; Qin, W. J.; Zhang, Z.; Zeng, G. H.; Chen, Z. N., CD147, MMP-1, MMP-2 and MMP-9 protein expression as significant prognostic factors in human prostate cancer. Oncology 2008, 75, (3-4), 230-6. 40. Escaff, S.; Fernandez, J. M.; Gonzalez, L. O.; Suarez, A.; Gonzalez-Reyes, S.; Gonzalez, J. M.; Vizoso, F. J., Study of matrix metalloproteinases and their inhibitors in prostate cancer. Br. J. Cancer 2010, 102, (5), 922-9. 41. Pulukuri, S. M.; Rao, J. S., Matrix metalloproteinase-1 promotes prostate tumor growth and metastasis. Int. J. Oncol. 2008, 32, (4), 757-65. 42. Miyata, Y.; Sakai, H., Thrombospondin-1 in urological cancer: pathological role, clinical significance, and therapeutic prospects. Int. J. Mol. Sci. 2013, 14, (6), 12249-72. 43. Ferrari do Outeiro-Bernstein, M. A.; Nunes, S. S.; Andrade, A. C.; Alves, T. R.; Legrand, C.; Morandi, V., A recombinant NH(2)-terminal heparin-binding domain of the adhesive glycoprotein, thrombospondin-1, promotes endothelial tube formation and cell survival: a possible role for syndecan-4 proteoglycan. Matrix Biol. 2002, 21, (4), 311-24. 44. Nunes, S. S.; Outeiro-Bernstein, M. A.; Juliano, L.; Vardiero, F.; Nader, H. B.; Woods, A.; Legrand, C.; Morandi, V., Syndecan-4 contributes to endothelial tubulogenesis through interactions with two motifs inside the pro-angiogenic N-terminal domain of thrombospondin-1. J. Cell. Physiol. 2008, 214, (3), 828-37. 45. Chandrasekaran, L.; He, C. Z.; Al-Barazi, H.; Krutzsch, H. C.; Iruela-Arispe, M. L.; Roberts, D. D., Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1. Mol. Biol. Cell 2000, 11, (9), 2885-900. 46. Taraboletti, G.; Morbidelli, L.; Donnini, S.; Parenti, A.; Granger, H. J.; Giavazzi, R.; Ziche, M., The heparin binding 25 kDa fragment of thrombospondin-1 promotes angiogenesis and modulates gelatinase and TIMP-2 production in endothelial cells. FASEB J. 2000, 14, (12), 1674-6. 47. Hanahan, D.; Weinberg, R. A., Hallmarks of cancer: the next generation. Cell 2011, 144, (5), 646-74. 48. Corey, D. R.; Craik, C. S., An Investigation into the Minimum Requirements for Peptide Hydrolysis by Mutation of the Catalytic Triad of Trypsin. J. Am. Chem. Soc. 1992, 114,


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(5), 1784-90. 49. Venkatraman, L.; Chia, S. M.; Narmada, B. C.; White, J. K.; Bhowmick, S. S.; Forbes Dewey, C., Jr.; So, P. T.; Tucker-Kellogg, L.; Yu, H., Plasmin triggers a switch-like decrease in thrombospondin-dependent activation of TGF-beta1. Biophys. J. 2012, 103, (5), 1060-8. 50. Bailey Dubose, K.; Zayzafoon, M.; Murphy-Ullrich, J. E., Thrombospondin-1 inhibits osteogenic differentiation of human mesenchymal stem cells through latent TGF-beta activation. Biochem. Biophys. Res. Commun. 2012, 422, (3), 488-93. 51. Bonnefoy, A.; Legrand, C., Proteolysis of subendothelial adhesive glycoproteins (fibronectin, thrombospondin, and von Willebrand factor) by plasmin, leukocyte cathepsin G, and elastase. Thromb. Res. 2000, 98, (4), 323-. 52. Matsumura, M.; Bhatt, A. S.; Andress, D.; Clegg, N.; Takayama, T. K.; Craik, C. S.; Nelson, P. S., Substrates of the prostate-specific serine protease prostase/KLK4 defined by positional-scanning peptide libraries. Prostate 2005, 62, (1), 1-13. 53. Takayama, T. K.; McMullen, B. A.; Nelson, P. S.; Matsumura, M.; Fujikawa, K., Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase. Biochemistry 2001, 40, (50), 15341-8. 54. Kleifeld, O.; Doucet, A.; auf dem Keller, U.; Prudova, A.; Schilling, O.; Kainthan, R. K.; Starr, A. E.; Foster, L. J.; Kizhakkedathu, J. N.; Overall, C. M., Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat. Biotechnol. 2010, 28, (3), 281-8. 55. Kleifeld, O.; Doucet, A.; Prudova, A.; auf dem Keller, U.; Gioia, M.; Kizhakkedathu, J. N.; Overall, C. M., Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates. Nat. Protoc. 2011, 6, (10), 1578-611. 56. Staes, A.; Van Damme, P.; Helsens, K.; Demol, H.; Vandekerckhove, J.; Gevaert, K., Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC). Proteomics 2008, 8, (7), 1362-70. 57. Venne, A. S.; Vogtle, F. N.; Meisinger, C.; Sickmann, A.; Zahedi, R. P., Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55. J. Proteome Res. 2013,



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68. Stein, J. J.; Iwuchukwu, C.; Maier, K. G.; Gahtan, V., Thrombospondin-1-induced vascular smooth muscle cell migration and proliferation are functionally dependent on microRNA-21. Surgery 2014, 155, (2), 228-33.


A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 381 MHSFPPLLLL 101 VLTEGNPRWE 39 ERWTNNFREY 201 301 40 FYMRTNPFYP 401 41 DEYKRSMDPG 42 43 44 kDa 1 2 45 100 46 75 47 50 48 37 49 50 51 25 52 20 53 54 15 55 56 57 10 58 59 KLK4 60 mKLK4 -

452 Journal of Proteome Research Proteins identified

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B

187 Extracellular/ cell surface proteins

Thrombospondin-1 Keratin, type II cytoskeletal 1 Agrin 78 kDa glucose-regulated protein

KLK4treated

37 Putative KLK4 substrates

C

KLK4transfected

4

25

kDa N250 150 100

-C 2 Gel slice

4

75

6

50

8

V75-K396

37 F20-

F100I270-

25 L282Putative cleavage product

8

20

F100-

Pro-MMP1 (52 kDa) Mature protease chain (42 kDa)

12 -N469 14 -N469 16 Internally cleaved MMP1: 27 kDa collagenase chain (inactive) 18 20

-K276

I118-

15

-K396

10

22 kDa collagenase chain (active)

22

-P269

24 26

Condition

28

6

30

Tx

32 Ctr

100

Tr Pro-

LFWGVVSHSF QTHLTYRIEN NLHRVAAHEL EVELNFISVF YPKMIAHDFP

PATLETQEQD YTPDLPRADV GHSLGLSHST WPQLPNGLEA GIGHKVDAVF

200 Residue number

300

400

Mature protease (42 kDa) 27 kDa collagenase 22 kDa collagenase

VDLVQKYLEK DHAIEKAFQL DIGALMYPSY AYEFADRDEV MKDGFFYFFH

YYNLKNDGRQ WSNVTPLTFT TFSGDVQLAQ RFFKGNKYWA GTRQYKFDPK

D

VEKRRNSGPV KVSEGQADIM DDIDGIQAIY VQGQNVLHGY TKRILTLQKA

VEKLKQMQEF FGLKVTGKPD ISFVRGDHRD NSPFDGPGGN GRSQNPVQPI GPQTPKACDS PKDIYSSFGF PRTVKHIDAA NSWFNCRKN 469

AETLKVMKQP LAHAFQPGPG KLTFDAITTI LSEENTGKTY

RCGVPDVAQF IGGDAHFDED RGEVMFFKDR FFVANKYWRY

100 200 300 400

E 3

4

5

6

7

8

9

10

pro-MMP1+KLK4(high) pro-MMP1+KLK4(low) pro-MMP1+KLK4(high)+EDTA pro-MMP1+mKLK4 pro-MMP1 KLK4 Blank

30 Pro-MMP1 (52 kDa) Mature protease chain (42 kDa)

25

20 Cor. RFU 15 (x 104) 10 5 0 2

2 -

10 50 250 1250 + -

pro-MMP1

- :1 [pro-MMP1: (m)KLK4] +

0

200

400

600

-5


Figure 1

Time (sec)

800

1000

A

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 2 kDa 19 20 250 21 150 22 23 100 24 75 25 26 27 28 50 29 30 37 31 32 25 33 20 34 35KLK4 36 + mKLK4 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

B

D

kDa 250 150 100 75


1

2

3

4

5

6

7

8

9

TSP1 (160 kDa)

Products: 45-150 kDa

50 37 25 20 +

KLK4 mKLK4

+ -

117 351 1053 3159

-

-

rTSP1

-

-

1

- :1 [PC-3 CM: (m)KLK4 (w/w)] -

PC-3 cell CM C-terminal antibody

3

4

5

6

7

8

9

C

kDa 250 TSP1 (160 kDa) 150

1

2

3

4

5

6

7

8

9 TSP1 (160 kDa)

100 75

Products: 145 kDa 130 kDa

50 37

Products: 30 kDa

25 + -

-

1

1 -

15 kDa 10 100 1000:1 [TSP1: (m)KLK4] -

20 KLK4 mKLK4 +

+ -

-

TSP1

Low MW

KLK4

1 -

10 100 1000 :1 [TSP1: (m)KLK4] -

TSP1

N-terminal antibody

pH 3 High MW

1

C-terminal antibody

11

pH 3 High MW

mKLK4

11

Low MW

ACS Paragon Plus Environment TSP1

Figure 2

A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

B


100 μm

C

100 μm


Figure 3

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100 μm

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... for TOC only ... 129x50mm (96 x 96 DPI)


Prostate Cancer-Associated Kallikrein-Related ... - ACS Publications

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