Báo cáo Y học: Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology docx

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology Sylvain M. Cloutier 1 , Jair Ribeiro Chagas 2 , Jean-Pierre Mach 3 , Christian M. Gygi 1 , Hans-Jurg Leisinger 1 and David Deperthes 1 1 Urology Research Unit, Department of Urology, Lausanne, Switzerland; 2 Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes, Brazil; 3 Institute of Biochemistry, University of Lausanne, Switzerland Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithe- lium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was gener- ated and then screened with purified hK2. Phages display- ing peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluores- cent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P¢1 position. The scissile bonds identified by phage display substrate selection correspond to those of the nat- ural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development. Keywords: cyan fluorescent protein; human kallikrein; phage display; prostate cancer; substrate. The human prostatic kallikreins hK3, or prostate specific antigen (PSA), is considered the gold standard for prostate cancer diagnosis and screening; however, hK2, the second prostatic kallikrein to be discovered [1], has recently emerged as a complementary marker for its positive correlation with prostate cancer grade and progression. PSA is more highly expressed in benign hyperplasia (BHP) than in cancer thus hK2 is helpful to further distinguish malignant from benign disease [2–4]. The recent discovery of 12 new members of the kallikrein family [5–7] could provide additional prostate cancer markers. In the seminal plasma, hK2 is mostly recovered com- plexed with protein C inhibitor [1]. Because hK2 cleaves, with trypsin-like specificity, certain components of the semen coagulum (fibronectin and semenogelins), it is possible that it has a role in the early stages of semen liquefaction, a biological process which immediately follows ejaculation [8]. In addition, in vitro studies have shown that hK2 can activate urokinase-type plasminogen activator [9] and inactivate plasminogen activator inhibitor-1 [10] leading to the activation of urokinase system. Moreover, hK2 degrades insulin-like growth factor binding proteins (IGF- BP) to release IGF, a putative local mitogenic signal for prostate cancer cells [11]. Despite the in vitro identification of its proteolytic activities as well as its potential substrates, our understand- ing of the true physiological role of hK2 remains sketchy. Much progress has been made toward the characterization of hK2s serine protease activity using synthetic substrates derived from reactive serpin loops [12]; however, this type of approach is limited to known targets and cannot advance the discovery of new biological substrates. A system using a monovalent phage library capable of displaying several million different substrates, which enabled simultaneous testing of proteolytic specificity, was developed by Matthews and Wells [13]. Several proteases including furin [14], PSA [15], membrane type-1 matrix metalloproteinase [16], and granzyme B [17] have already been characterized using this approach. We adapted this method by constructing a phage- displayed random library that included all possible amino acid combinations of pentapeptides, then screening it with hK2. Of the 44 individual phage clones selected and identified, 90% had Arg at the P1 site and 30% had Ser in the P¢1 position. Kinetic studies and sites of cleavage in substrates have been determined with a new system using cyan fluorescent protein (CFP), a variant of the green fluorescent protein system. A search in the SwissProt database with selected substrates identified three new putative hK2 substrates: ADAM-TS8 precursor, cadherin- related tumour suppressor homologue precursor, and collagen alpha (IX) chain precursor. Correspondence to D. Deperthes, Urology Research Unit, Department of Urology, CHUV, CH-1011 Lausanne, Switzerland. Fax: + 41 213142985, Tel.: + 41 213140120, E-mail: david.deperthes@urology-research.ch Abbreviations: PCI, protein C inhibitor; PSA, prostate specific antigen; CFP, cyan fluorescent protein; IPTG, isopropyl thio-b- D -galactoside. (Received 23 January 2002, revised 19 April 2002, accepted 19 April 2002) Eur. J. Biochem. 269, 2747–2754 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02960.x MATERIALS AND METHODS Materials Following known methods, hK2 was purified from human semen [18]; its active site was titrated using 4-methylumbelli- feryl-4-guanidinobenzoate [19]. The following materials were obtained from commercial sources: restriction enzymes (Roche Biosciences; Amersham Pharmacia), PWO DNA polymerase and shrimp alkaline phosphatase (Roche Bio- sciences), T4 DNA ligase (Invitrogen), T4 polynucleotide kinase (Promega), Ni 2+ -nitrilotriacetic acid agarose, anti- His antibody, Ni 2+ -nitrilotriacetic acid magnetic agarose beads and 96-well magnet type A (Qiagen). Mycrosynth GmbH carried out DNA sequencing and oligonucleotides synthesis. Construction of the substrate phage display library Substrate phage libraries were generated using a modified pH0508b phagemid [20]. The construction consists of a His 6 tag at either end of a Gly-Gly-Gly-Ser-repeat-rich region that precedes the carboxyl-terminal domain (codons 249– 406) of the M13 gene III. The random pentamers were generated by PCR extension of the template oligonucleo- tides with appropriate restriction sites positioned on both side of the degenerate codons: 5¢-TGAGCTAGTCTAGAT AGGTGGCGGTNNSNNSNNSNNSNNSGGGTCGAC GTCGGTCATAGCAGTCGCTGCA-3¢ (where N is any nucleotide and S is either G or C) using 5¢ biotinylated primers corresponding to the flanking regions: 5¢-TGAGC TAGTCTAGATAGGTG-3¢ and 5¢-TGCAGCGACTGC TATGA-3¢. PCR templates are digested and purified as described previously [21], inserted into XbaI/SalIdigested pH0508b vector, and electroporated into XL1-Blue (F – ). The extent of the library was estimated from the transfor- mation efficiency determined by plating a small portion of the transformed cells onto Luria–Bertani plates containing ampicillin and tetracycline (100 and 15 lgÆmL )1 , respect- ively). The rest of the transformed cells were used to prepare a phage library by incubating overnight by adding an M13K07 helper phage at a concentration giving a multipli- city of infection of 100 plaque forming units (p.f.u.) per mL. Phages were collected from the supernatant and purified by poly(ethylene glycol) precipitation. Of these, 200 clones were selected arbitrarily for sequencing to verify the randomiza- tion of the library. Phage-displayed pentapeptide library screening This new pentapeptide library was subjected to eight rounds of screening with hK2. One hundred microliters of Ni 2+ -nitrilotriacetic acid coupled to sepharose beads (Ni 2+ -nitrilotriacetic acid resin) was washed with 10 mL NaCl/P i containing 1 mgÆmL )1 BSA. Phage particles (10 11 ) were added to the equilibrated Ni 2+ -nitrilotriacetic acidresinandallowedtobindwithgentleagitationfor 3hat4°C. The resin was subsequently washed (NaCl/P i / BSA 1 mgÆmL )1 ,5m M imidazole, 0.1% Tween 20) to remove unbound phages and then equilibrated in NaCl/ Pi. The substrate phage was exposed to 27 n M (final concentration) of hK2 for 45 min at 37 °C. A control selection without protease was also performed. The cleaved phages released into the supernatant were ampli- fied using XL1-Blue Escherichia coli andthenusedfor subsequent rounds of selection. After eight rounds of panning, about 15 individual clones were picked from the fifth, sixth and eighth round of selection and plasmid DNA were isolated and sequenced in the region encoding for the substrate. Expression of CFP fluorescent substrate The construction CFP-X 5 -His contains the following amino acid sequences at the C-terminus of CFP fluorescent proteins: IGGGXXXXXGSTGGGS HHHHHH. The ran- dom substrate sequence (in bold) takes place between a His 6 tag (underlined) and the CFP protein, separated by the same linker as described previously for substrate phage library. The BamHIandXbaI/HindIII DNA recognition sites were introduced by PCR onto 5¢ and 3¢ ends, respectively, of the cDNA encoding the CFP fluorescent protein. The PCR product was subcloned into a pQE-16 (Qiagen) vector. A DNA duplex encoding the SalI recognition site, the linker, and the His tag was then inserted into the XbaI/HindIII digested vector. The resulting CFP-X 5 -His constructions were used to insert 30 randomly selected substrate genes directly excised from the phage using the XbaIandSalI recognition sites (Fig. 1). In addition, two additional recombinant CFP–X 5 -His proteins harbouring a peptide known to be either resistant (IKFFS) or sensitive (TFRSA) to hK2 cleavage [12] were constructed and named CFP–Rst and CFP–protein C inhibitor (PCI), respectively. To produce recombinant proteins, XL1-Blue cells were transformed with the corres- ponding constructions followed by growth in 50 mL 2 · TY (16 g tryptone, 10 g yeast extract, 5 g NaCl per L) with ampicillin (100 lgÆmL) and tetracycline (15 lgÆmL) antibiotics. Cells were then induced until D 600 ¼ 0.5 to express recombinant fluorescent substrate by addition of 1m M of isopropyl thio-b- D -galactoside (IPTG) for 16 h at 37 °C. After an additional 16 h of growth, the cells were harvested by centrifugation and resuspended for 2 h at room temperature in 6 mL denaturation buffer (6 M GdN– HCl in NaCl/P i at pH 8.0 containing 10 m M 2-mercapto- ethanol) to recover the soluble and insoluble fractions. All recombinant CFPs were purified in denaturing conditions to prevent substrate cleavage by endogenous bacterial proteases. After centrifugation, 100 lLNi 2+ -nitrilotriacetic acid resin was added to the bacterial cell supernatant and incubated to bind recombinant proteins. The resin was subsequently washed with 5 M urea in NaCl/P i at pH 8.0 containing 30 m M imidazole and 10 m M 2-mercaptoethanol and proteins were eluted with the same buffer containing 150 m M imidazole. The purified recombinant CFPs were diluted 100 · in refolding buffer (0.15 M Tris/HCl at pH 8.3, containing 0.1 M NaCl and 1 m M 2-mercaptoeth- anol) and the time course of refolding was followed by monitoring increasing fluorescence with a FL X 800 fluores- cence 96-well microplate reader, with excitation at 440 nm and emission at 485 nm. Once refolding was completed, recombinant CFPs were dialysed against refolding buffer for 14 h at 4 °C. The purity of each refolded proteins was analysed by SDS/PAGE [22] followed by Coomassie Blue staining and Western blot using a horseradish peroxidase- conjugated anti-His 6 Ig (Qiagen). 2748 S. M. Cloutier et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Direct determination of the k cat / K m using CFP fluorescent substrates Refolded CFP-X5-His proteins were fixed to Ni 2+ -nitrilo- triacetic acid magnetic beads for 2 h at room temperature and an aliquot was collected for eluting proteins to determine the specific activity (fluorescence/amount of protein) and initial substrate concentration [S 0 ] for each CFP. Concentrations were determined by Bradford assay (Biorad, USA). All of the kinetic assays were carried out at 37 °Cin50m M Tris/HCl buffer pH 8.3, containing 0.01% Tween 20, for 120 min The time course of substrate hydrolysis was followed by monitoring the fluorescence released from the beads as the CFPs were cleaved in their substrate linker. Percentage of hydrolysis was calculated as the ratio of released CFPs to the initial amount of CFPs bound to the beads which was quantified by elution with imidazole. Specificity constants (k cat /K m ) were determined under pseudo-first order conditions using a substrate concentration well below the K m [23]. Briefly, scissile bonds in substrates were identified by N-terminal sequencing of fragments remaining bound to the beads after complete hydrolysis. The final concentration of hK2 was 19 n M for each enzymatic reaction. RESULTS Construction of the substrate phage library The pH0508b monovalent phage vector [20] was modified to generate a new pentamer substrate library with a His tag at the N-terminus of the random pentapeptides fused to the minor coat protein pIII. In this way, the phage can be attached through binding to an immobile phase, in this case the Ni 2+ -nitrilotriacetic acid resin. The constructed library contained 1.8 · 10 8 independent transformants and could thus be considered complete because, in theory, all of the 3.2 · 10 6 possible random pentamer sequences were repre- sented. The sequencing of phages further confirmed the randomness of the pentamer inserts. Random selection of hK2 substrates Although the filamentous phages are considered to be generally protease resistant, we first verified that hK2 activity had indeed no effect on infectivity. Following eight rounds of exposure to hK2, 44 individual phage clones were selected from different rounds; the deduced amino acids corresponding to the substrate sequences are shown in Table 1. No phage was selected more than once, indicating that a large repertoire of susceptible substrates was present in the pentamer library. DNA sequence analysis reveal that an arginine appears in 40 clones at the P1 site and only one peptide is cleaved at a lysine. Among the substrates hydrolysed at an arginine, 11 different amino acids appeared at the P¢1 subsite. However, some amino acids were more frequently recovered at this position with 30% of selected peptides exhibiting serine and 12% methionine, alanine, or valine. Interestingly, an evolution of the representation of scissile bonds emerged during the selection (Fig. 2); the highest variation was observed for the Arg–Ser scissile bond with continuously increasing recovery of 14, 32, and 42%, respectively, for the fifth, sixth, and eighth rounds of selection. A slight increase was also observed in the Arg– Met and Arg–Ala motif, while an important decrease was observed for the Arg–Val motif through the selection, which completely disappeared after eight rounds. The positions surrounding the scissile bond at the P3, P2, and P¢2subsites predominantly favoured small or uncharged residues as seen by the 65, 55, and 70% recovery (Fig. 3). Of these small or uncharged residues, none in particular was observed more frequently at these positions. Hydrophobic residues also appeared in the P3 and P2 subsites in 20% of peptides whereas no aromatic residues were recovered in the P3 and P¢2 positions. CFP fluorescent substrate assay A simple and direct system has been developed to determine the kinetics of peptide substrate selection from a phage display library (Fig. 1). All CFP recombinant proteins can be produced with good yields in bacteria (1 mg per 50 mL of culture) with  75% being refolded in stable conformations. To generate the substrate phage, the CFP–substrate molecule is attached by a His 6 tail to Ni 2+ - Fig. 1. Schematic outline of the approach used to select substrates for kallikrein hK2. (1) Phage displaying random peptides fused to a his- tidine tail (His) are immobilized on an affinity support (Ni 2+ -nitrilo- triacetic acid sepharose beads). (2) After treatment with kallikrein hK2, phages expressing sensitive substrates are released from the solid phase, (3) and are then used to infect F-positive bacteria (4) to be amplified for a next step of selection (5). Phages from the last round of selection are cloned by plating onto Petri dishes (6) and DNA of individual phages are amplified in region encoding for the substrate to determine the sequences cleaved by the enzyme. (7) Gene encoding the random substrate was subcloned into an expression vector, in order to be produced as a fusion protein between the CFP protein and a histidine tag. (8) The CFP-X5-his protein was fixed to Ni 2+ -nitrilotriacetic acid magnetic beads and (9) treated by the protease hK2. The released CFP fluorescent protein was measured with a fluorescence reader (10) which permitted to determine the percentage of hydrolysis, the specificity constant and the site of cleavage (11). Ó FEBS 2002 Substrate specificity of kallikrein hK2 (Eur. J. Biochem. 269) 2749 nitrilotriacetic acid beads; the substrate can then be released by hydrolysis only. By using two CFP–substrates harbour- ing a substrate that is either cleavable or resistant to hK2, we showed that the CFP recombinant protein is cleaved only in the substrate region and not within the CFP sequence as no fluorescence was detectable with CFP- resistant. On the other hand, CFP–PCI was efficiently cleaved with a first-order curve for the product generation (data not shown) and the specificity constant k cat /K m was  20 000 M )1 Æs )1 . Under the same conditions, hK2 cleaved the other 30 peptides constructed as CFP–substrates with catalytic efficiencies (k cat /K m ) ranging from 1.7 · 10 4 M )1 Æs )1 for LRSRA to 9.9 · 10 1 M )1 Æs )1 for peptide ERVSP. Thus, there is about a 170-fold difference in the efficacy of cleavage between the different substrates selected by phage Table 1. Alignment of translated amino acid sequences of random peptide clones selected by substrate phage display with hK2. Clone Scissile bond P5 P4 P3 P2 P1 P¢1 P¢2P¢3P¢4 6.1 RS M T R S N 6.6 K T R S N 6.8 I S P R S 6.11 G V F R S 6.19 G T V R S 5.5 E T K R S 5.2 L G R S L 8.3 R G R S E 6.2 R R S I D 8.11 V L R S P 8.20 L R S R A 8.5 RS GS V 8.9 A R A R S 8.18 RT S D R T A 6.7 K L R T T 8.13 RA R A A M M 5.3 T R A P M 8.17 P G R A P 6.9 V E S R A 6.20 A R A S E 5.19 RV T L Q R V 5.16 R L E R V 5.18 E R V S P 5.12 S S P R V 6.17 RVGP Y 6.4 RM P S A R M 6.14 R G R M A 6.5 T V R M P 8.12 L R M P T 8.14 H R M S S 5.11 RP R P Q E L 6.15 V R P L E 5.7 RL S G R L A 6.12 RF G T L R F 5.1 RN Q W R N S 5.14 RNDKL 6.13 M R N R A 8.19 RD T R D S R 5.4 T G S R D 5.10 RQ I M S R Q 6.3 KG L T T S K Fig. 2. Frequency of selection of the different scissile bonds. 2750 S. M. Cloutier et al. (Eur. J. Biochem. 269) Ó FEBS 2002 display substrate. The best substrate peptide, giving specif- icity constant approaching the PCI–peptide and a percent- age of hydrolysis superior to 90%, contained a serine residue in P¢1 subsite whereas the less sensitive peptide contained a valine, an observation that correlates with the evolution of the number of different scissile bonds during the selection. The only peptide cleaved at a Lys had a low specificity constant and gave a percentage of hydrolysis of only 20% confirming the preference for arginine in the P1 position. No cleavage was observed with the two peptides that did not contain either arginine or lysine suggesting a residual background among the selected substrates. All peptides having a k cat /K m superior to 5.7 · 10 3 M )1 Æs )1 possess two basic amino acids N-terminal to the scissile bond except for peptide LRSRA where the second basic residue was found at P¢2(Table2). Comparison with natural substrate When compared to previously reported substrates for hK2, the peptides selected here had scissile bonds containing the Arg–Ser motif, which is the same bond cleaved in PCI, a natural inhibitor of hK2 found in seminal plasma, as well as semenogelin I, antithrombin III, and kininogen. The Arg– Thr and Arg–Leu motifs are hydrolysed by hK2 in semenogelins I and II whereas the Arg–Met motif is cleaved in the plasminogen activator inhibitor-1 and the Arg–Gln motif is cleaved in IGF-BP-2. Using each of the 44 pentapeptides substrate sequences, FASTA and BLAST searches were done to look for new potential human protein substrates of hK2 (Table 3). Among the 11 identical matches (data not shown), three putative targets were identified for hK2: ADAM-TS 8 precursor, cadherin-rela- ted tumour suppressor homologue precursor, and collagen (IX) chain precursor matching peptides RGRSE, GVFRS and PGRAP, respectively. DISCUSSION A wide variety of critical processes depend on specific cleavage of targets by different enzymes so an ability to discriminate among many potential substrates is crucial to maintaining the fidelity of most biological functions. However, unnatural cleavage can occur through unpredict- able reactions between protease and substrate provoking unexpected biological events such as degradation of extra- cellular matrix, over-availability of growth factors, or degradation of tumor suppressor proteins. In the last 5 years, evidence has been mounting that support a role for hK2 in metastasis and cancer progression by virtue of its in vitro proteolysis of several biological substrates involved in cancer biology [8–11]. However, further investigation is needed to verify this hypothesis. Previously biochemical characterizations were incomplete due to the limit of classical iterative methods using already existing or modified substrates [12,24]. The unbiased approach used in this study clearly defined the preferential recognition sites for hK2 substrate hydro- lysis. The phage display substrate technique enables millions of substrates to be screen simultaneously in a single reaction [13,25]. Large biological libraries are constructed by displaying random sequences on the extremities of filamen- tous phages, then amplified and screened toward a protease to survey rapidly its specificity. Most reports using phage display substrate to character- ize proteases have not reported the extent of diversity of the library used in the screening, this being a direct product of the number of different combinations of amino acids displayed by the phage. Cloning substrates comprising more than six residues is limited by transformation efficacy, thus the ability to obtain completely adequate diversity with that number of amino acids is questionable [26]. Phage display does generate libraries that are many times more diverse,however, than those using other methods such as combinatorial chemistry [17] or immobilized positional peptide libraries [27]. In our experiments, randomised pentapeptides were fused to a truncated form of g3p to produce a library of 1.8 · 10 8 independent recombinant phages where all possible combi- nations of sequences even the rarest polypeptides, are represented. The screening of this library with hK2 showed that no phage was in duplicate which is in contrast to selections with other types of phage display libraries (antibody fragments, ligands, or peptide binders) where selections often identified only the best clones with highest reactivity [28,29]. Our results are consistent with other studies using phage display that reported a broad diversity but good enrichment in the selection of specific enzyme substrates [13,15,25]. The determination of the specificity constants of the substrates showed a positive tendency during the selection. Most of the better substrates were taken the last rounds. However, this does not preclude that bad substrates could be conserved throughout the screening process despite selection pressure. Therefore, selected substrates need to be further tested in other configuration than that of fused to a phage. The CFP system developed in the present work enabled direct determination of specificity constants and the site of cleavage of the substrate selected by phage display, an improvement over the previously described semiquantitative method [13,25] and chemical synthesis of substrates [15,30]. The effectiveness of our system was validated through a recombinant CFP carrying a PCI-derived peptide, a substrate efficiently cleaved by hK2. The k cat /K m of the peptide fused to CFP was significantly lower than that obtained with the same sequence as synthetic fluorogenic form [12]; this difference could be explained by the Fig. 3. P3-P¢2 substrate specificity profile of hK2 from selected peptides tested as CFP fusion protein. Alignment of translated amino-acid sequences of random peptide clones selected by substrate phage display with hK2. Ó FEBS 2002 Substrate specificity of kallikrein hK2 (Eur. J. Biochem. 269) 2751 modification in the Km caused by the peptide being linked to a fairly large protein (30 kDa). In addition, hydrophobic fluorophores used to make intramolecularly quenched fluorogenic substrates are known to modify the affinity of peptide for the active site of enzyme, increasing the Km [31]. Our results showing that hK2 cleaves quite selectively after an arginine residue, concurs with previous reports [12,24]. Nearly one-third of all selected peptides are cleaved at the Arg–Ser bond despite the large variety of residues being recovered at the P¢1 position. This result shows that hK2 can accommodate a broad range of amino acids, except for basic residues, in the P¢1 position. The strong preference for small or noncharged residues is also observed in P3, P2, and P¢2 subsites but no consensus could be deduced among the amino acids from the selected sequences. Despite this observation, hK2 seems to be dependent on a more extended site of binding than R–S bond for an efficient catalysis as some Arg–Ser peptides possess lower specificity constants. Nonetheless, the observation that the best three peptides are cleaved as efficiently as the sequence of PCI– peptide obtained by the classic iterative methods indicates the impressive ability of substrate phage technology to elucidate optimal subsite occupancy for proteases from large banks of randomly selected candidates. Interestingly, the Arg–Ser scissile bond found in numer- ous natural substrates like PCI, semenogelins I and II, fibronectin and kininogen as well as other preferential cleavage sites like Arg–Thr or Arg–Met in seminal coagu- lum proteins and in plasminogen activator inhibitor-1, respectively; is also preferentially selected by hK2 using phage display substrates thus confirming the success of phage display substrate selection. Finally, a SwissProt database search with selected sequences identified three potential human protein sub- strates for hK2. Regions identified in different substrates are extracellular and thus accessible to proteases. These poten- tial substrates are not yet well characterized, but are suspected to be involved in cancer progression. For example, the desintegrin-like and metalloprotease domain with thrombospondin type I modules 8 (ADAM-TS8) Table 2. Comparaison of specificity constant (k cat /K m ) values and the percentage hydrolysis of CFP-X5-his based on selected substrates with hK2. (Scissile bonds are designated by fl.) Clone Sequence K obs (s )1 ) Hydrolysis (%) k cat /K m ( M )1 Æs )1 ) PCI TFRflSA 3.66 E-04 84.4 19 284 8.20 LRflSRA 3.22 E-04 89.7 16 926 6.2 RRflSID 2.85 E-04 97.9 14 982 8.3 RGRflSE 2.77 E-04 96.2 14 605 6.7 KLRflTT 1.83 E-04 57.1 9646 8.9 ARARflS 1.46 E-04 61.9 7659 6.14 RGRflMA 1.10 E-04 55.8 5765 6.4 PSARflM 1.02 E-04 47.2 5389 8.12 LRflMPT 9.80 E-05 33.1 5158 6.12 GTLRflF 9.54 E-05 43.4 5020 5.5 ETKRflS 8.28 E-05 33.1 4358 6.19 GVFRflS 6.74 E-05 23.2 3545 5.19 TLQRflV 6.53 E-05 24.7 3436 5.3 TRflAPM 6.27 E-05 32.4 3299 8.17 PGRflAP 6.24 E-05 35.1 3282 5.7 SGRflLA 5.78 E-05 30.5 3042 8.11 VLRflSP 5.24 E-05 33.1 2756 8.19 TRDSR 4.84 E-05 30.9 2548 5.10 IMSRflQ 4.77 E-05 27.1 2512 6.5 TVRflMP 4.35 E-05 24.6 2289 6.20 ARflASE 4.10 E-05 23.5 2158 6.6 KTRflSN 3.64 E-05 26.2 1917 6.1 MTRflSN 3.37 E-05 22.9 1772 6.3 LTTSKfl 3.24 E-05 19.6 1705 5.4 TGSRflD 2.69 E-05 16.1 1417 6.15 VRflPLE 2.37 E-05 13.1 1248 5.14 RflNDKL 2.27 E-05 19.1 1196 5.18 ERflVSP 1.89 E-05 11.2 99 MTMQS ND ND ND QTSLS ND ND ND Rst AIKFF ND ND ND Table 3. Identification of potential physiological substrate of hK2 using the SwissProt data base. HK2 selected peptides Sequences Potential protein substrate (residues) 8.3 RGRflSE ADAM-TS 8 precursor (646–50) 6.19 GVFRflS Cadherin-related tumour suppressor homologue precursor (2473–77) 8.17 PGRflAP Collagen alpha (IX) chain precursor (753–57) 2752 S. M. Cloutier et al. (Eur. J. Biochem. 269) Ó FEBS 2002 could act as a tumour suppressor through its antiangiogenic activity [32,33]. Cadherin-related tumour suppressor homo- logue precursor [34] and collagen alpha (IX) chain precur- sor, a minor cartilage nonfibrillar collagen associated with type II collagen fibrils [35], are the two other potential protein substrates for hK2 that could also have a role in cancer progression. In conclusion, we developed an effective phage display system that enabled rapid and fruitful investigation of hK2 substrate specificity. 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Biochem. 26 9) 27 51 modification in the Km caused by the peptide being linked to a fairly large. percentage of hydrolysis, the specificity constant and the site of cleavage (11). Ó FEBS 20 02 Substrate specificity of kallikrein hK2 (Eur. J. Biochem. 26 9) 27 49 nitrilotriacetic acid beads; the substrate