Identification of Peptide Substrates for Human MMP-11 (Stromelysin-3) Using Phage Display
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m304436200
ISSN1083-351X
AutoresWeijun Pan, Marc R. Arnone, Marvin Kendall, Robert H. Grafström, Steven P. Seitz, Zelda R. Wasserman, Charles F. Albright,
Tópico(s)Signaling Pathways in Disease
ResumoThe MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A)↓(L/Y)(T/V/M/R)(R/K), where ↓ denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1′, but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E↓LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest k cat/K m values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower k cat/K m values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer. The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A)↓(L/Y)(T/V/M/R)(R/K), where ↓ denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1′, but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E↓LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest k cat/K m values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower k cat/K m values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer. MMP-11, also known as stromelysin-3, is one of more than 20 matrix metalloproteinases (MMP) 1The abbreviations used are: MMP, matrix metalloproteinase; HPLC, high pressure liquid chromatography.1The abbreviations used are: MMP, matrix metalloproteinase; HPLC, high pressure liquid chromatography. (reviewed in Ref. 1Woessner J. Nagase H. Matrix Metalloproteinases and TIMPs. 1st ed. Oxford University Press, New York2000Google Scholar). The MMP-11 gene was originally identified by screening a breast cancer cDNA library for genes that were expressed at higher levels in invasive carcinomas than in breast fibroadenomas (2Basset P. Bellocq J. Wolf C. Stoll I. Hutin P. Limacher J. Podhajcer O. Chenard M. Rio M. Chambon P. Nature. 1990; 348: 699-704Crossref PubMed Scopus (1008) Google Scholar). Additional work has shown that MMP-11 is usually overexpressed in many human carcinomas, including breast, non-small cell lung, and colorectal carcinomas, but is rarely expressed in normal tissue, including the normal tissue surrounding the tumor (3Rouyer N. Wolf C. Chenard M. Rio M. Chambon P. Bellocq J. Basset P. Inv. Metastasis. 1994; 14: 269-275PubMed Google Scholar, 4Kossakowska A. Huchcroft S. Urbanski S. Edwards D. Br. J. Cancer. 1996; 73: 1401-1408Crossref PubMed Scopus (125) Google Scholar, 5Tetu B. Brisson J. Lapointe H. Bernard P. Hum. Pathol. 1998; 29: 979-985Crossref PubMed Scopus (60) Google Scholar). In fact, adults only express MMP-11 in tumors and regenerating or healing tissues (reviewed in Ref. 6Basset P. Bellocq J. Lefebvre O. Noel A. Chenard M. Wolf C. Anglard P. Rio M. Crit. Rev. Oncol. Hemotol. 1997; 26: 43-53Crossref PubMed Scopus (69) Google Scholar). Furthermore, MMP-11 expression correlated with a shorter recurrence-free survival for breast cancer patients, providing further support for an important role for MMP-11 in breast cancer (5Tetu B. Brisson J. Lapointe H. Bernard P. Hum. Pathol. 1998; 29: 979-985Crossref PubMed Scopus (60) Google Scholar, 7Chenard M. O'Siorain L. Shering S. Rouyer N. Lutz Y. Wolf C. Baset P. Bellocq J. Duffy M. Int. J. Cancer. 1996; 69: 448-451Crossref PubMed Scopus (96) Google Scholar, 8Ahmad A. Hanby A. Dublin E. Poulsom R. Smith P. Barnes D. Rubens R. Anglard P. Hart I. Am. J. Pathol. 1998; 152: 721-728PubMed Google Scholar).Experiments with mice or fibroblasts lacking MMP-11 also suggest that MMP-11 plays an important role in human cancer. First, mice lacking MMP-11 have a reduced incidence and size of tumors induced by 7,12-dimethylbenzanthracene compared with mice with a functional MMP-11 (9Masson R. Lefebvere O. Noel A. El Fahime M. Chenard M. Wendling C. Kebers F. Le Meur M. Dierich A. Foidart J. Basset P. Rio M. J. Cell Biol. 1998; 140: 1535-1541Crossref PubMed Scopus (257) Google Scholar). Second, MMP-11 null fibroblasts, unlike wild type fibroblasts, do not stimulate the implantation rate of MCF7 xenografts in nude mice (9Masson R. Lefebvere O. Noel A. El Fahime M. Chenard M. Wendling C. Kebers F. Le Meur M. Dierich A. Foidart J. Basset P. Rio M. J. Cell Biol. 1998; 140: 1535-1541Crossref PubMed Scopus (257) Google Scholar). Third, syngenic tumor cells had a higher rate of apoptosis when injected into MMP-11 null mice compared with wild type mice (10Boulay A. Masson R. Chenard M. El Fahime M. Cassard L. Bellocq G. Sautes-Fridman C. Basset P. Rio M. Cancer Res. 2001; 61: 2189-2193PubMed Google Scholar). Hence, the frequent overexpression of MMP-11 in human tumors and the effect of MMP-11 levels on tumor formation in mice both suggest that MMP-11 plays an important role in human cancer.Relatively little is known about the physiologic substrates for MMP-11. In particular, human MMP-11, unlike most MMPs, does not readily cleave extracellular matrix components, such as collagen, laminin, fibronectin, or elastin (11Pei D. Majmudar G. Weiss S. J. Biol. Chem. 1994; 269: 25849-25855Abstract Full Text PDF PubMed Google Scholar, 12Noel A. Santavicca M. Stoll I. L'Hoir C. Staub A. Murphy G. Rio M. Basset P. J. Biol. Chem. 1995; 270: 22866-22872Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In vitro studies showed, however, that MMP-11 cleaves α1-proteinase inhibitor, α2-macroglobulin, and insulin-like growth factor-binding protein-1, although the physiologic significance of these reactions is unknown (11Pei D. Majmudar G. Weiss S. J. Biol. Chem. 1994; 269: 25849-25855Abstract Full Text PDF PubMed Google Scholar, 13Manes S. Mira E. Barbacid M. Cipres A. Fernandez-Resa P. Buesa J. Merida I. Aracil M. Marquez G. Martinez C. J. Biol. Chem. 1997; 272: 25706-25712Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). More importantly, recent experiments showed that MMP-11 mutants without proteolytic activity did not stimulate the implantation of MCF7 cells into nude mice (14Noel A. Boulay A. Kebers F. Kannan R. Hajitou A. Calberg-Bacq C. Basset P. Rio M. Foidart J. Oncogene. 2000; 19: 1605-1612Crossref PubMed Scopus (65) Google Scholar). These studies strongly suggest that MMP-11 has proteolytic activity that is important for its role in cancer.In addition to the poor cleavage of extracellular matrix components, human MMP-11 differs from other MMPs in that it contains alanine at residue 235, whereas mouse MMP-11 and other MMPs contain proline at the corresponding residue (12Noel A. Santavicca M. Stoll I. L'Hoir C. Staub A. Murphy G. Rio M. Basset P. J. Biol. Chem. 1995; 270: 22866-22872Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This alanine residue affects catalytic activity because the human protein MMP-11-A235P, but not MMP-11, readily cleaved laminin and type IV collagen. In fact, human MMP-11-A235P had activity similar to that of mouse MMP-11. It is unclear whether the alanine 235 to proline mutation in human MMP-11 affects catalytic activity, substrate dependence, or both.In this study, we used phage display libraries to select peptide substrates for human MMP-11. Preferred sequences for MMP-11 cleavage could identify physiologic substrates for MMP-11. Such substrates would be interesting because they are likely to be important for human cancer.EXPERIMENTAL PROCEDURESConstruction of Phage Display Library—A phage display library was constructed containing a random hexamer peptide sequence expressed between a hexahistidine tag and the mature gene III protein of M13. To construct this library, the following two oligonucleotides were annealed, elongated with the Klenow fragment in the presence of nucleotide triphosphates, and then digested with KpnI and BamHI (first and second underlined segments, respectively): 5′-gttcggtacctttctattctcactccgctcaccatcaccaccatcacggtgtggtagtggtggtagtgccaccgnnsnnsnnsnnsnnsnnsccgccgcctaggtgtag-5′, where n indicates equimolar a, t, c and g and s indicates equimolar c and g. The digested DNA fragment was then ligated with the vector M13PL9, which was similarly digested with KpnI and BamHI, and the ligation mixture was transformed into Escherichia coli strain K91 (thi/HfrC) yielding 4.4 × 108 independent transformants. The resulting phage mixture was amplified in K91 cells and concentrated by polyethyleneglycol precipitation to yield 4.7 × 1013 plaque-forming units/ml. The resulting phage expressed a protein containing: gene III leader sequence-Ala-His6-Gly2-Xaa6-Gly2-gene III mature protein.Construction of Control Phage Clones—Positive and negative control phage clones were constructed to help develop methods for library screening. A positive control phage was constructed with oligonucleotides that expressed the hexapeptide PLG↓LYA because this sequence is known to be a substrate for MMP-14. Two clones containing the hexapeptides MTQMIS or TALSPQ were isolated from the library that were not cleaved by MMP-14.Expression Vectors for MMP-14 and MMP-11—Fragments of human MMP-14 and MMP-11 that encoded the catalytic domains were inserted into vectors for expression in E. coli. For MMP-11, a DNA fragment encoding residues 98–272 was amplified by PCR from IMAGE clone 2425546 (Research Genetics, Birmingham, AL) with a 5′ oligonucleotide that contained NdeI and a 3′ oligonucleotide that contained BglII. The amplified DNA fragment was digested with NdeI and BglII and then ligated with pET3b (Novagen, Madison, WI) that was digested with NdeI and BglII. MMP-11 mutants with increased activity, MMP-11-A235P, or decreased activity, MMP-11-E216A, were generated by site-directed mutagenesis. DNA sequencing confirmed the authenticity of the preceding plasmids. For MMP-14, a vector that directed the expression of MMP-11 residues 111–298 was created as previously described (15Kannan R. Ruff M. Kochins J. Manly S. Stoll I. El Fahime M. Noel A. Foidart J. Rio M. Dive V. Basset P. Protein Expression Purif. 1999; 16: 76-83Crossref PubMed Scopus (31) Google Scholar).Purification of MMP-14 and MMP-11 Catalytic Protein Fragments— Plasmids that expressed the catalytic regions of MMP-11 or MMP-14 were transformed into BL21pLysS (Novagen). Recombinant proteins were induced and purified based on published methods (15Kannan R. Ruff M. Kochins J. Manly S. Stoll I. El Fahime M. Noel A. Foidart J. Rio M. Dive V. Basset P. Protein Expression Purif. 1999; 16: 76-83Crossref PubMed Scopus (31) Google Scholar). Briefly, inclusion bodies were purified from E. coli that were induced to express the desired recombinant protein for 3 h at 37 °C. The purified inclusion bodies were dissolved in buffer with 8 m urea, diluted, and dialyzed against buffer with decreasing amounts of urea to facilitate refolding. Insoluble protein was then removed by centrifugation, and the resulting proteins were stored at –80 °C. The identity of the MMP-11 was confirmed by Western blotting with antibody SL305 (Oncogene Sci.). The concentration of the soluble protein was determined by comparison with bovine serum albumin following Coomassie Blue staining of SDS-poly-acrylamide gels.Screening the Phage Display Library with MMP-14, MMP-11, or MMP-11-A235P—0.1 ml of phosphate-buffered saline containing ∼109 phage was added to each well of a 96-well Reacti-Bind Metal Chelate Plate (Pierce), and the resulting plate was shaken gently overnight at room temperature. Unbound phage were removed by washing six times with 0.2 ml of phosphate-buffered saline containing 0.05% Tween 20. This procedure yielded ∼2–5% of the input phage bound to the plate based on titering phage that were eluted from the plate with 0.1 ml of 0.02 m EDTA for 10 min. To enrich for MMP-14 substrates, the bound phage were digested in 0.1 ml of 50 mm Hepes, 10 mm CaCl2, 100 mm NaCl, 0.1% Brij 30, 10 nm MMP-14 (pH 7.5) for 6 h at 37 °C with gentle shaking. The resulting supernatants were then collected and amplified, and the enrichment cycle was repeated as above. Individual phage were cloned and analyzed as described under "Results." Likewise, bound phage were digested in 0.1 ml of 50 mm Tris-HCl, 10 mm CaCl2, 100 mm NaCl (pH 7.5) with either 500 nm MMP-11 or 180 nm MMP-11-A235P for 6 h at 37 °C with gentle shaking to enrich for MMP-11 and MMP-11-A355P substrates. The resulting supernatants were then collected and amplified, and the enrichment cycle was repeated three more times. Individual phage were cloned, amplified, and analyzed as described under "Results." In some cases, the amount of phage released from the nickel plate was quantified by titering the reaction supernatant and/or phage eluted from the plate after a 10-min treatment with 0.1 ml of 0.02 m EDTA.Enzyme Assays with Peptides—2 ml of recombinant enzyme were dialyzed twice against 1 liter of 50 mm Hepes, 100 mm NaCl, 5 mm CaCl2, 1 μm ZnCl2, 5 mm β-mercaptoethanol (pH 7.6) (reaction buffer plus β-mercaptoethanol) to remove amines that would react with fluorescamine. Peptides (Research Genetics, Huntsville, AL) were dissolved in Me2SO at 10 mm. The peptide at concentrations ranging from 0.016 to 1 mm was diluted into reaction buffer for a final volume of 0.1 ml and equilibrated to 37 °C. Enzyme at concentrations from 1 to 65 nm was then added, and the reaction was continued for 1–20 h. To quantify the extent of hydrolysis, 0.05 ml of 200 mm potassium borate (pH 10.0) was added followed by 0.05 ml of 1 mg/ml fluorescamine in acetonitrile and gentle mixing. The fluorescent signal generated by the reaction of the fluorescamine with the primary amines created by enzymatic digestion of the peptides was measured using a plate reader with excitation and emission wavelengths of 390 and 490 nm, respectively. The molar amount of fluorescamine-peptide conjugate was determined using standards prepared from fluorescamine. These values were then used to calculate the amount of product formed (16Bantan-Polak T. Kassai M. Grant K. Anal. Biochem. 2001; 297: 128-136Crossref PubMed Scopus (100) Google Scholar). k cat/K m was calculated from nonlinear regression analysis of Michaelis-Menten plots using Enzyme Kinetics software (Trinity Software).As indicated under "Results," the digestion of some peptides was also monitored using reversed-phase HPLC separation. In these cases, the reaction was stopped with 10 mm EDTA, and the reaction products were separated using a C18 reversed-phase HPLC column and acetonitrile gradient. The peptide fragments were detected by their absorbance at 210 nm. The results obtained by the HPLC method were in excellent agreement with the results obtained with the fluorescamine method. Finally, analysis of the digestion of peptides MA15 and MA18 with either the wild type or the A235P mutant MMP11 was performed using a Waters Micromass ZQ mass spectrometer interfaced with a Shimadzu HPLC. Analysis was performed using either acetonitrile, water, 0.1 mm ammonium acetate or methanol, water, 0.1% trifluoroacetic acid gradients as mobile phase on a C18 reversed-phase HPLC column using electrospray ionization(+/–) ionization.Gel-based Activity Assay for MMP-11 Cleavage of α1-Proteinase Inhibitor—MMP-11 α1-antitrypsin inhibitor cleavage activity was measured by incubating 5 μgof α1-proteinase inhibitor (Calbiochem 178251) in 50 mm Tris-HCl (pH 7.5), 100 mm NaCl, 5 mm CaCl2, 1 μm ZnCl2, 26 mm MMP-11 or MMP-11-A235P in a total volume of 0.05 ml at 37 °C for the indicated times. The reactions were stopped by adding 10 μl of 3 m β-mercaptoethanol, 10% SDS, 50 mm Tris-HCl (pH 7.6), 25% glycerol. The reaction products (15 μl) were separated using an 18% Tris-HCl gel (Bio-Rad) and visualized with Coomassie Blue.RESULTSIdentification of MMP-14 Substrates Using Phage Display—A phage display library was constructed to identify peptide substrates for proteases. In this system, each phage expresses five copies of a hexahistidine peptide followed by a random hexapeptide on its surface. Proteolytic digestion within the hexapeptide removes the hexahistidine tag, thereby allowing separation of phage where the hexapeptide was cleaved from phage that were uncleaved.A screen for MMP-14 substrates was used to test this phage display system. A phage clone that contained a hexapeptide that was cleaved by MMP-14, PLG↓LYA, and two clones with hexapeptides that were not cleaved by MMP-14, MTQMIS and TALSPQ, were first used to develop methods for library screening. Based on these studies, conditions were identified where ∼75% of the positive control phage were released from the nickel plate, whereas less than 2% of the negative control phage were released from the nickel plate (see "Experimental Procedures" for details.) Using these conditions, two rounds of enrichment were performed with MMP-14. 52 random phage clones from this enriched pool were then individually digested with MMP-14 or buffer controls, and the fraction of the phage released by MMP-14 was determined. This analysis showed that 31 of 52 clones were released from nickel plates at levels equal to or greater than the level for the positive control phage. Some of these release rates are quantified in Table I. DNA from these 31 phage were sequenced, and the predicted hexapeptides were aligned (Table I). Based on this alignment, a consensus sequence of PL(G/P/A)↓L(R/M) was obtained (Table II). The individual peptide sequences and the consensus sequence were in excellent agreement with preceding identification of MMP-14 substrates using phage display and peptide methods (17Ohkubo S. Miyadera K. Sugimoto Y. Matsuo K. Wierzba K. Yamada Y. Biochem. Biophys. Res. Commun. 1999; 266: 308-313Crossref PubMed Scopus (45) Google Scholar, 18Ohkubo S. Miyadera K. Sugimoto Y. Matsuo K. Wierzba K. Yamada Y. Comb. Chem. High Throughput Screening. 2001; 4: 573-583Crossref PubMed Scopus (21) Google Scholar, 19Turk B. Huang L. Piro E. Cantley L. Nat. Biotech. 2001; 19: 661-667Crossref PubMed Scopus (456) Google Scholar). Hence, our studies substantiate the conclusions of other investigators and validate our phage display method for identifying peptide substrates.Table IPredicted peptide sequences selected by MMP-14 digestionPhage cloneaPhage clones isolated from screens using MMP-14. Predicted peptide sequences were grouped based on their putative position 1 residue.Putative subsiteHydrolysisbPercentage of phage released from nickel plate with 3 nM MMP-14.P4P3P2P1P1′P2′P3′%14-A40IPQGLL(G)c(G) encoded by vector sequences.7714-B4LPRGLQ(G)7014-B15(G)PIGLRL6414-A21QPMGLF(G)6514-B21KPIGLM(G)6014-B11(G)PLGMMS6114-A12(G)PRGLTA3314-B3LAMGLR(G)5914-A18KPSGVL(G)NDdNot determined.14-B7TPSGLW(G)ND14-B32(G)PAGLQRND14-A11(G)PLPMIA6714-B17(G)PLPLKS5414-A44QPLPMK(G)4914-A31FPLPLL(G)5814-A7FANPIR(G)ND14-B6SGEPLM(G)ND14-B18IPVPLR(G)ND14-B20QPQPLW(G)ND14-B25(G)PAPLREND14-A41IPRAIM(G)5814-A8SPLALL(G)6514-A20RPYAMS(G)3414-B31LPQALQ(G)3414-B8(G)PLALTTND14-B13QPQALT(G)ND14-B19(G)PAALVKND14-B22LPLALI(G)ND14-A15NPLSLR(G)8114-B14(G)PASLRLND14-B16HPLSLM(G)ND+ control(G)PLGLYA19- control(G)TALSPQ2- control(G)MTQMIS2a Phage clones isolated from screens using MMP-14. Predicted peptide sequences were grouped based on their putative position 1 residue.b Percentage of phage released from nickel plate with 3 nM MMP-14.c (G) encoded by vector sequences.d Not determined. Open table in a new tab Table IISubsite preferences from hexapeptides selected by MMP-14Putative subsiteResidue (%)aResidues occurring in more than 15% of 31 peptides in Table I were included in this analysis.P3P2P1P1′P2′P (90)L(36)G(39)L(86)R(23)P(32)M(16)A(29)a Residues occurring in more than 15% of 31 peptides in Table I were included in this analysis. Open table in a new tab Identification of MMP-11 Substrates Using Phage Display—A fragment of human MMP-11 containing residues 98–272 was expressed in E. coli. Two MMP-11 mutants were also expressed in E. coli to aid in the identification of MMP-11 substrates. One mutant, MMP-11-E216A, altered a residue required for catalytic function (14Noel A. Boulay A. Kebers F. Kannan R. Hajitou A. Calberg-Bacq C. Basset P. Rio M. Foidart J. Oncogene. 2000; 19: 1605-1612Crossref PubMed Scopus (65) Google Scholar), whereas a second mutant, MMP-11-A235P, had increased activity for cleavage of α1-proteinase inhibitor (12Noel A. Santavicca M. Stoll I. L'Hoir C. Staub A. Murphy G. Rio M. Basset P. J. Biol. Chem. 1995; 270: 22866-22872Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) (Fig. 1A). This analysis showed that both MMP-11 and MMP-11-A235P cleaved α1-proteinase inhibitor, yielding the expected products. Furthermore, MMP-11-A235P was about 10-fold more active than MMP-11, consistent with previous results (12Noel A. Santavicca M. Stoll I. L'Hoir C. Staub A. Murphy G. Rio M. Basset P. J. Biol. Chem. 1995; 270: 22866-22872Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Similar results were obtained using either β-casein, α2-macroglobulin, or insulin-like growth factor-binding protein-1 as a substrate (data not shown). The cleavage of α1-proteinase inhibitor was inhibited by AG3340, a broad spectrum MMP inhibitor (20Shalinsky D. Brekken J. Zou H. McDermott C. Forsyth P. Edwards D. Margosiak S. Bender S. Truitt G. Wood A. Varki N. Appelt K. Ann. N. Y. Acad. Sci. 1999; 878: 236-270Crossref PubMed Scopus (221) Google Scholar), with an IC50 of ∼6 nm (Fig. 1B). As previously described (14Noel A. Boulay A. Kebers F. Kannan R. Hajitou A. Calberg-Bacq C. Basset P. Rio M. Foidart J. Oncogene. 2000; 19: 1605-1612Crossref PubMed Scopus (65) Google Scholar), the active site mutant MMP-11-E216P was inactive in all of the assays tested (data not shown). Taken together, these findings are consistent with earlier findings and validate our MMP-11 preparations.MMP-11 and MMP-11-A235P were then used to enrich the phage display library for substrates. Unfortunately, neither the positive control phage clone for MMP-14 (PLG↓LYA) nor any of the phage clones selected with MMP-14 were good substrates for MMP-11 or MMP-11-A235P. In the absence of phage substrates to establish conditions for screening, enrichment conditions were chosen based on the published activity of MMP-11 and MMP-11-A235P (see "Experimental Procedures" for details). Using these conditions, ∼25-fold more phage were released from the pool of phage that had undergone four rounds of enrichment than from the nascent phage library. 60 phage from both the MMP-11 and MMP-11-A235P enrichments were then individually cloned and tested for release from nickel plates by MMP-11, MMP11-A235P, or buffer controls. This analysis showed that 7 of 60 phage clones from the MMP-11 enrichment and 34 of 60 phage clones from the MMP-11-A235P enrichment were released from nickel plates at 100-fold or higher levels than the negative control clones. This analysis also showed that all phage released by MMP-11 could be released by MMP-11-A235P and vice versa. An additional test was performed to ensure that phage clones were released by MMP-11 instead of potentially contaminating E. coli proteases. Putative MMP-11 or MMP-11-A235 substrate phage were first digested with MMP-11-A235P either in the presence or absence of 0.1 μm AG3340, a broad spectrum MMP inhibitor. This analysis showed that the release of 32 of 41 phage clones selected with either MMP-11 or MMP-11-A235P was inhibited 95% or more by AG3340. DNA from these 32 phage clones were then sequenced, and the predicted hexapeptides were aligned (Table III). Three groups of peptide substrates for MMP11 were readily identifiable. Based on this alignment, the four clones from Group B had hexapeptides that shared the sequence G(G/A)E↓LR, whereas two clones had hexapeptide sequences that were represented by neither Group A nor Group B. The majority of peptides belong to Group A. Separate analysis of these 26 peptides belonging to Group A indicated that the hexapeptides were well represented by the consensus sequence A(A/Q)(N/A)↓(L/Y)(T/V/M/R)(R/K) (Table IV). As a final test that these phage were released by MMP-11, 13 phage clones were treated with either MMP-11 or MMP-11-E216A. This analysis showed that at least 100-fold more phage were released with MMP-11 than with MMP-11-E216A. Hence, this phage screen isolated two classes of hexapeptides and two unique hexapeptides outside these classes that were specifically released from nickel plates by both MMP-11 and MMP-11-A235P but not by MMP-11-E216A.Table IIIPredicted peptide sequences selected by MMP-11 digestionPhage cloneaPhage clones selected with MMP-11 or MMP-11A235P are designated as 11-x or 11AP-x, respectively. The sequences are grouped based on their position 1 (P1) residues.Putative subsiteP4P3P2P1P1′P2′P3′P4′Group A11-19AQNLVK11-45AANLVK11-47AANLVK11-50SANYTM11-56AQNYTR11AP-6WANLTK11AP-7AANLVR11AP-27AANLVR11AP-15AANLLR11AP-16(G)b(G) encoded by vector sequences.ANYIVK11AP-28AGNLMM11AP-52(G)ANLILK11AP-B2MAANYV11AP-B11AANYMM11AP-B15AANLRL11AP-B16AQNLMR11AP-B5SANYIS11AP-11(G)AALTAK11AP-40(G)AALRMY11AP-60(G)AAYTKK11AP-B3AQALRI11AP-B4(G)AAMLMV11AP-B10AQAYTR11AP-B12ASALRM11AP-20(G)QSMTMP11AP-B14(G)ASMMKAGroup B11AP-35(G)(G)ELRTSK11AP-39(G)AELRQK11AP-41(G)(G)ELRLAP11AP-B7YAELRMOther sequences11-36QPRGVW11-38TDAWLSa Phage clones selected with MMP-11 or MMP-11A235P are designated as 11-x or 11AP-x, respectively. The sequences are grouped based on their position 1 (P1) residues.b (G) encoded by vector sequences. Open table in a new tab Table IVSubsite preferences from Group A hexapeptides selected by MMP-11Residue (%)aResidues occurring in more than 15% of 26 Group A peptides from Table III were included in this analysis. The residues encoded by vector sequences were not included in this compilation.Putative subsiteP3P2P1P1′P2′P3′A(58)A(69)N(65)L(58)T(27)R(23)(G)b(G) encoded by vector sequences. (31)Q(23)A(27)Y(31)V(23)K(23)M(15)R(15)a Residues occurring in more than 15% of 26 Group A peptides from Table III were included in this analysis. The residues encoded by vector sequences were not included in this compilation.b (G) encoded by vector sequences. Open table in a new tab Determination of Kinetic Parameters for MMP-11 Substrate Peptides—Several peptides were synthesized based on the hexapeptide sequences identified by phage display so that kinetic parameters could be determined and compared with known substrates. In particular, decapeptides were synthesized where the hexapeptide sequences identified by phage display were flanked by a pair of glycine residues at both the N and C termini as in the phage. The N terminus was blocked with an acetyl group to eliminate primary amines in the peptides. The peptides were then incubated with either MMP-14, MMP-11, MMP-11-A235P, or MMP11-E216A, and the extent of cleavage was determined using fluorescamine, which reacted with the primary amines created by peptide cleavage to generate fluorescent conjugates. Nonlinear regression analysis of Michaelis-Menten plots was used to determine K m and V max that were then used to calculate k cat/K m values (Table V). Although comparisons between the columns of Table V are subject to the vagaries of the percentages of active enzyme after refolding, certain conclusions are evident. For instance, peptide MA13, which contained the hexapeptide PLG↓LYA used for the positive control phage in the MMP-14 screen, was a good substrate for MMP-14 but was not cleaved at a detectable rate by MMP-11 or MMP-11 mutant proteins, as expected from the phage assays (Table V). MA15 and MA18 contained the hexapeptide
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