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• W2012193516 abstract "A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (S treptomycescaespitosus neutralproteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K i value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN′ (K i = 1.4 nm) (EC3.4.21.62). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M r = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN′ was identified to be Met71–Tyr72 bond by specific cleavage. To identify the reactive site for ScNP, Tyr33 and Tyr72, which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN′ but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN′ was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN′. A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (S treptomycescaespitosus neutralproteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K i value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN′ (K i = 1.4 nm) (EC3.4.21.62). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M r = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN′ was identified to be Met71–Tyr72 bond by specific cleavage. To identify the reactive site for ScNP, Tyr33 and Tyr72, which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN′ but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN′ was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN′. S. caespitosus neutral proteinase S. caespitosusneutral proteinase inhibitor Streptomyces subtilisin inhibitor bicinchoninic acid digoxigenin subtilisin from Bacillus amyloliquefaciens 4-methylcoumaryl-7-amide (7-methoxycoumarin-4-yl) acetyl 2,4-dinitrophenyl polymerase chain reaction N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid polyacrylamide gel electrophoresis kilobase pair EcoRI Most metalloproteinases were divided into two groups, Gluzincins (HEXXH + E) and Metzincins (HEXXHXXGXXH) based on their zinc ligands (1Jiang W. Bond J.S. FEBS Lett. 1992; 312: 110-114Crossref PubMed Scopus (178) Google Scholar, 2Hooper N.M. FEBS Lett. 1994; 354: 1-6Crossref PubMed Scopus (671) Google Scholar, 3Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 183-228Crossref PubMed Scopus (699) Google Scholar). In 1969, Yokote and Noguchi (4Yokote Y. Kawasaki K. Nakajima J. Noguchi Y. Nippon Nogeikagaku Kaishi. 1969; 43: 125-131Crossref Scopus (16) Google Scholar, 5Yokote Y. Noguchi Y. Nippon Nogeikagaku Kaishi. 1969; 43: 132-138Crossref Scopus (14) Google Scholar) found a novel zinc metalloproteinase (ScNP)1 in the culture supernatant of Streptomyces caespitosus. ScNP is one of the smallest zinc metalloproteinase with a molecular weight of 14,376 (6Harada S. Kinoshita T. Kasai N. Tsunasawa S. Sakiyama F. Eur. J. Biochem. 1995; 233: 683-686Crossref PubMed Scopus (17) Google Scholar). ScNP specifically cleaves the peptide bond at the amino-terminal side of aromatic amino acid residues (7Kurisu G. Sugimoto A. Harada S. Takagi M. Imanaka T. Kai Y. J. Ferment. Bioeng. 1997; 83: 590-592Crossref Scopus (9) Google Scholar). ScNP has a common zinc ligand motif (HEXXH), but its third zinc ligand is not the conventional Glu or His but an Asp residue (8Kurisu G. Kinoshita T. Sugimoto A. Nagara A. Kai Y. Kasai N. Harada S. J. Biochem. (Tokyo). 1997; 121: 304-308Crossref PubMed Scopus (40) Google Scholar). Since ScNP carries a Met turn in its structure, which is a feature of Metzincins, it belongs to Metzincins superfamily (3Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 183-228Crossref PubMed Scopus (699) Google Scholar). Since proteinaceous proteinase inhibitors are very close to the natural substrate, it is very useful for studies of the structure and function of proteinases. Moreover, the studies of inhibitors can lead to efficient drug design that could ultimately lead to novel therapeutic interventions. Many proteinaceous proteinase inhibitors have been found in animals, plants, and microorganisms. However, natural inhibitors for metalloproteinases are very rare. Known examples includeStreptomyces metalloproteinase inhibitor (9Oda K. Koyama T. Murao S. Biochim. Biophys. Acta. 1979; 571: 147-156Crossref PubMed Scopus (37) Google Scholar), Erwinia chrythanthemi inhibitor (10Letoffe S. Delepelaire P. Wandersman C. Mol. Microbiol. 1989; 3: 79-86Crossref PubMed Scopus (60) Google Scholar), and tissue inhibitors of matrix metalloproteinases (11Osthues A. Knauper V. Oberhoff R. Reinke H. Tschesche H. FEBS Lett. 1992; 296: 16-20Crossref PubMed Scopus (26) Google Scholar, 12Apte S.S. Olsen B.R. Murphy G. J. Biol. Chem. 1995; 270: 14313-14318Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 13Leco K.J. Apte S.S. Taniguchi G.T. Hawkes S.P. Khokha R. Schultz G.A. Edwards D.R. FEBS Lett. 1997; 401: 213-217Crossref PubMed Scopus (180) Google Scholar). The structure-function relationship of these inhibitors has been well characterized. We have isolated a novel proteinaceous inhibitor for ScNP from a culture supernatant of Streptomyces sp. I-355 and named it S treptomyces caespitosus neutral proteinase inhibitor (ScNPI). ScNPI strongly inhibited not only ScNP (metalloproteinase) but also subtilisin BPN′ (serine proteinase). Unexpectedly, ScNPI had sequence homology to Streptomyces subtilisin inhibitor (SSI) family (15Ikenaka T. Odani S. Sakai M. Nabeshima Y. Sato S. Murao S. J. Biochem. (Tokyo). 1974; 76: 1191-1209Crossref PubMed Scopus (112) Google Scholar, 16Terabe M. Kojima S. Taguchi S. Momose H. Miura K. Eur. J. Biochem. 1994; 226: 627-632Crossref PubMed Scopus (15) Google Scholar, 17Ueda Y. Kojima S. Tsumoto K. Takeda S. Miura K. Kumagai I. J. Biochem. (Tokyo). 1992; 112: 204-211Crossref PubMed Scopus (23) Google Scholar, 18Tanabe M. Asano T. Moriya N. Sugino H. Kakinuma A. J. Biochem. (Tokyo). 1994; 115: 743-751Crossref PubMed Scopus (9) Google Scholar, 19Tanabe M. Kawahara K. Asano T. Kato K. Kakinuma A. J. Biochem. (Tokyo). 1994; 115: 752-761Crossref PubMed Scopus (6) Google Scholar). In order to clarify the function of ScNPI, the reactive sites for ScNP and subtilisin BPN′ were identified. DEAE-Sepharose fast flow, Sephadex G-75, Mono Q HR 5/5, and Superdex 200-HR-10/30 were purchased from Amersham Pharmacia Biotech. ScNP was purchased from Seikagaku Kogyo, Japan, and purified as described previously (6Harada S. Kinoshita T. Kasai N. Tsunasawa S. Sakiyama F. Eur. J. Biochem. 1995; 233: 683-686Crossref PubMed Scopus (17) Google Scholar). Subtilisin BPN′ was purchased from Nagase Biochemicals. Thermolysin was kindly donated by Daiwa Kasei, Japan.Pseudomonas aeruginosa elastase (20Morihara K. Tsuzuki H. Oka T. Inoue H. Ebata M. J. Biol. Chem. 1965; 240: 3295-3304Abstract Full Text PDF PubMed Google Scholar) was kindly donated by Dr. Kumazaki, Hokkaido University, Japan. Vimelysin (21Oda K. Okayama K. Okutomi K. Shimada M. Sato R. Takahashi S. Biosci. Biotechnol. Biochem. 1996; 60: 463-467Crossref PubMed Scopus (25) Google Scholar) and almelysin (22Shibata M. Takahashi S. Sato R. Oda K. Biosci. Biotechnol. Biochem. 1997; 61: 710-715Crossref PubMed Scopus (15) Google Scholar) were purified as described previously. BCA (bicinchoninic acid) Protein Assay Kit was purchased from Pierce. MOCAc-Ala-Arg-Gly-Tyr-Gln-Gly-Lys(Dnp)-NH2 was kindly synthesized by Prof. Ben M. Dunn and colleagues, University of Florida College of Medicine. Suc-Ala-Ala-Pro-Phe-MCA was purchased from Peptide Institute Inc., Osaka, Japan. Escherichia coli strain JM109, plasmid pUC18, and plasmid pIN-III-OmpA2 (provided by Dr. S. Taguchi, the Institute of Physical and Chemical Research) (23Ghrayeb J. Kimura H. Takahara M. Hsiung H. Masui Y. Inouye M. EMBO J. 1984; 3: 2437-2442Crossref PubMed Scopus (285) Google Scholar, 24Taguchi S. Yoshida Y. Matsumoto K. Momose H. Appl. Microbiol. Biotechnol. 1993; 39: 732-737Crossref PubMed Scopus (9) Google Scholar) were used as a host, cloning vector, and expression vector, respectively. Restriction enzymes and DNA-modifying enzymes were purchased from Nippon Gene (Toyama, Japan), New England Biolabs Inc., and Takara Shuzo (Kyoto, Japan). DIG (digoxigenin) DNA Labeling Kit and DIG Nucleic Acid Detection Kit were purchased from Roche Molecular Biochemicals. PCR kit and ABI PRISMTM DyeTerminator Cycle Sequencing Ready Reaction Kit were purchased from Perkin-Elmer. The mycelia of Streptomyces sp. I-355 were inoculated into 100 ml of a medium that consisted of 2% starch, 4% polypeptone, 0.1% NaCl, 0.1% K2HPO4, 0.1% yeast extract, and 0.05% MgSO4·H2O at pH 7.0 in a 500-ml flask at 30 °C for 72 h with shaking (100 strokes/min). After the cultivation, mycelia were removed by centrifugation (8,000 rpm, 40 min). The supernatant was adjusted to pH 4 with 1 n HCl and then the supernatant was treated at 80 °C for 5 min to inactivate endogenous proteinases. After the treatment, the supernatant was neutralized with 1 n NaOH and used for the purification of inhibitor. The precipitate from 80% saturation of ammonium sulfate was collected by filtration with Hyflo Super-Cel and dissolved in 20 mm Tris-HCl, pH 8.0 (Buffer A). Cold acetone was slowly added to the sample with stirring to give 33.3% saturation (v/v). After standing at −30 °C for 2 h, the precipitate was removed by centrifugation (8,000 rpm, 20 min). Cold acetone was slowly added to the supernatant with stirring to give 80% saturation (v/v). After standing at −80 °C for 2 h, the precipitate was collected by centrifugation (15,000 rpm, 10 min). The precipitate was dissolved in Buffer A and dialyzed against the same buffer. The dialysate was loaded on a column of DEAE-Sepharose fast flow (26 × 215 mm) equilibrated with Buffer A. The column was washed with the same buffer, and then the inhibitor was eluted with a 0–0.5 m NaCl linear gradient. Fractions containing inhibitory activity were pooled and precipitated with ammonium sulfate (80% saturation). The precipitate was collected by centrifugation (15,000 rpm, 20 min) and dissolved in Buffer A. The sample was loaded on a column of Sephadex G-75 (26 × 900 mm) equilibrated with 20 mm Tris-HCl, pH 7.5 (Buffer B), and then the inhibitor was eluted with the same buffer at a flow rate of 30 ml/h. Fractions containing inhibitory activity were pooled. The sample was loaded on a column of Mono Q HR 5/5 (5 × 50 mm) equilibrated with Buffer B. The column was washed with the same buffer, and then the inhibitor was eluted with a 0–0.2m NaCl linear gradient at a flow rate of 0.7 ml/min. Fractions containing inhibitory activity were pooled and stored at −80 °C until use. Molar concentration (as monomer) of ScNPI was measured by BCA Protein Assay Kit using bovine serum albumin as a standard. Concentrations of ScNP and subtilisin BPN′ were spectrophotometrically determined using E 1 cm,1% at 280 nm values of 15.5 and 11.7, respectively. Tricine SDS-PAGE was performed by the method of Schagger and Jagow (25Schagger H. Jagow G.V. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10494) Google Scholar) using 16.5% T, 3% C polyacrylamide gel. The following proteins were used as molecular weight standards: bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa). After the electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250. The purified ScNPI was loaded on a column of Superdex 200-HR-10/30 (10 × 300 mm) equilibrated with 50 mmTris-HCl, pH 7.5, containing 0.15 m NaCl, and then the inhibitor was eluted with the same buffer at a flow rate of 0.5 ml/min. Bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), chymotrypsinogen A (25.0 kDa), and cytochrome c (12.4 kDa) were used as molecular weight standards. In order to synthesize PCR primers, the partial amino acid sequence of ScNPI was determined. S-Pyridylethylated ScNPI was cleaved with 0.1% CNBr in 70% HCOOH and lyophilized. The resulting peptides were separated by Tricine SDS-PAGE. After electrophoresis, the peptides were electrophoretically transferred to polyvinylidene difluoride membrane and subjected to sequence analysis. Genomic DNA of Streptomyces sp. I-355 was prepared as described previously (26Tanaka T. Kuroda M. Sakaguchi K. J. Bacteriol. 1977; 129: 1487-1494Crossref PubMed Google Scholar) with slight modification. PCR was done with Ampli Taq Polymerase Stoffel fragment (Perkin-Elmer). The primers for PCR are shown in TableI.Table IPCR primers for cloning of scnpi gene and construction of ScNPI mutantsPrimerSequenceScNPI-85′-ATGGTGTTCACGGTGATCCAGGG-3′C C CScNPI-94R5′-AAGGTGTGCTTCCAGGCGACGC-3′C C C CER (+)5′-CCGGAATTCAGTGCACACGGCCCGTC-3′BH (−)5′-CGCGGATCCTCAGAACGCGTACACCGGG-3′Y33A (+)5′-CAGCTGCGCCGCTACGGCCGAAGGC-3′Y33A (−)5′-GGCCGTAGCGGCGCAGCTGAGGGTG-3′Y72A (+)5′-GCCCGATGGCCTTCGACCCCGTGAC-3′Y72A (−)5′-GGGGTCGAAGGCCATCGGGCAGA-3′ Open table in a new tab The amplified DNA fragment by PCR using ScNPI-8 as a sense primer and ScNPI-94R as an antisense primer (denaturation at 96 °C for 1 min, annealing at 62 °C for 1.5 min, and extension at 72 °C for 1.5 min, 30 cycles) was labeled with digoxigenin (DIG) and used as a probe for hybridization. The DNA fragments obtained bySalI digestion were ligated into SalI site of pUC18 and transformed into E. coli JM109 cells. DNA sequencing was carried out using ABI PRISMTM TaqDye Deoxy Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) with a Perkin-Elmer model 373A DNA sequencer. The plasmid pScNPI-4, containing the 2.5-kb scnpigene (in this paper), was digested with SmaI. The resulting 1.0-kb fragment was used as the template for PCR. The mature region ofscnpi gene was amplified by PCR (denaturation at 96 °C for 1 min, annealing at 55 °C for 1.5 min, and extension at 72 °C for 1.5 min, 25 cycles) using ER(+) as a sense primer and BH(−) as an antisense primer. The amplified DNA fragment was digested withEcoRI-BamHI and then ligated intoEcoRI-BamHI site of pIN-III-OmpA2 vector. The resulting plasmid was transformed into E. coli JM109 cells. Mutant ScNPIs were constructed by PCR using mutagenic primers (TableI). In a first PCR, mutant genes were constructed as separate halves by a combination of primer ER(+) and Y33A(−), primer BH(−), and Y33A(+) with the same PCR condition for the wild-type gene. Another combination was primer ER(+) and Y72A(−), primer BH(−), and Y72A(+). The PCR products were mixed together and used as templates in a second PCR using the flanking sequence primers, ER(+) and BH(−). The amplified fragment was digested with EcoRI-BamHI and then ligated into EcoRI-BamHI site of pIN-III-OmpA2 vector. The resulting plasmid was transformed into E. coliJM109 cells. The mutation site was confirmed by DNA sequence using ER (+) as a sequencing primer. E. coli JM109 cells harboring recombinant plasmid were inoculated into 100 ml of M9 medium (0.6% Na2HPO4, 0.3% KH2PO4, 1.0% NH4Cl, 0.2% glucose, 0.1 mmCaCl2, 1 mm MgCl2, and 0.2% casamino acid, pH 7.4) containing 50 μg/ml ampicillin and 1 mm isopropyl-1-thio-β-d-galactoside in a 500-ml flask with shaking (110 strokes/min) at 30 °C for 20 h. After the cultivation, cells were collected by centrifugation (6,000 rpm, 20 min) and suspended in 50 mm Tris-HCl, pH 7.5 (10 ml of buffer/1 g of cells). The suspension was sonicated and centrifuged (15,000 rpm, 10 min). Solid ammonium sulfate was slowly added to the supernatant with stirring to give 80% saturation. The precipitate was collected by centrifugation (15,000 rpm, 10 min) and dissolved in Buffer B and dialyzed against the same buffer. The subsequent purification steps were the same as described for the authentic ScNPI. 0.25 ml of 1.18 μm ScNP and 0.25 ml of inhibitor solution were incubated at 37 °C for 10 min. Then 1.5 ml of 4/3% Hammersten casein in 50 mm Tris-HCl, pH 7.5, was added and incubated at 37 °C for 60 min. After the incubation, the reaction was stopped by the addition of 2 ml of 0.44 mtrichloroacetic acid. The precipitate was then removed by filtration. 0.5 ml of the filtrate was neutralized with 2.5 ml of 0.44m sodium carbonate and incubated with 0.5 ml of 1n Folin-Ciocalteu's reagents solution at 37 °C for 20 min, and then the absorbance at 660 nm was measured. One inhibitor unit (IU) was defined as the amount of inhibitor that caused a 50% reduction of caseinolytic activity. In all of the reactions, enzyme was preincubated with 10 or 50 m excess of inhibitor at 37 °C for 10 min. 0.25 ml of enzyme (167 nm thermolysin, 104 nm vimelysin, 163 nm P. elastase, and 565 nmalmelysin) and 0.25 ml of inhibitor solution were incubated at 37 °C for 10 min. Then 1.5 ml of 4/3% Hammersten casein in 50 mmTris-HCl, pH 7.5, was added and incubated at 37 °C for 40 min. Residual enzyme activity was measured as described above. 0.25 ml of 1.18 μm subtilisin BPN′ and 0.25 ml of inhibitor solution were incubated at 37 °C for 10 min. Then 1.5 ml of Hammersten casein in 0.1 m borate buffer, pH 9.5, was added and incubated at 37 °C for 15 min. 0.25 ml of 2.85 μm pepsin and 0.25 ml of inhibitor solution were incubated at 37 °C for 10 min. Then 1.5 ml of Hammersten casein in 0.1 m lactate buffer, pH 3.0, was added and incubated at 37 °C for 30 min. 20 μl of 430 nm trypsin and 20 μl of inhibitor solution were incubated at 37 °C for 10 min. Then 360 μl of 0.1 mm Bz-Arg-MCA in 50 mm Tris-HCl, pH 8.0, containing 0.1 mNaCl, 10 mm CaCl2, and 0.01% Triton X-100 was added and incubated at 37 °C for 20 min. Reaction was stopped by the addition of 800 μl of 15% acetic acid. Fluorescence intensity was measured with a Hitachi F-2000 spectrophotometer at λex 380 nm and λem 460 nm. 25 μl of 5 nm chymotrypsin and 25 μl of inhibitor solution were incubated at 37 °C for 10 min. Then 200 μl of 0.1 mmSuc-Ala-Ala-Pro-Phe-MCA in 50 mm Tris-HCl, pH 8.0, containing 10 mm CaCl2 and 0.01% Triton X-100 was added and incubated at 37 °C for 20 min. Reaction was stopped by the addition of 750 μl of 15% acetic acid. 25 μl of 544 nm cathepsin B and 25 μl of inhibitor solution were incubated at 37 °C for 10 min. Then, 500 μl of 20 μm benzyloxycarbonyl-Phe-Arg-MCA in 0.4 mphosphate buffer, pH 6.0, containing 4 mm EDTA and 8 mm cysteine monohydrochloride was added and incubated at 37 °C for 30 min. The reaction was stopped by the addition of 1 ml of 0.1 m sodium acetate buffer, pH 5.0, containing 0.1m iodoacetic acid. Kinetic analysis for ScNP was performed by using a newly designed fluorogenic substrate, MOCAc-Ala-Arg-Gly-Tyr-Gln-Gly-Lys(Dnp)-NH2. All of the reactions were carried out in 50 mm TES-NaOH, pH 7.0, containing 10 mm CaCl2 and 0.01% Triton X-100 (v/v) (Buffer C) at 25 °C. Fluorescence intensity was measured with a Hitachi F-2000 fluorescence spectrophotometer at λex 328 nm and λem 393 nm. Substrate concentration was estimated from the fluorescence intensity of the perfectly cleaved substrate using MOCAc-Pro-Leu-Gly-OH as a standard compound. Initial velocity was calculated from the increase of fluorescence intensity for 1 min caused by the release of MOCAc-Ala-Arg-Gly. Kinetic parameters were determined from a Lineweaver-Burk plot. ScNP (final concentration of 5.9 nm) in 980 μl of Buffer C was incubated at 25 °C for 5 min. Then 20 μl of the fluorogenic substrate (final concentration of 4.7–16.5 μm) in the same buffer was added to the enzyme solution. Thek cat value was calculated by an equation ofV max = k cat[E]0, where [E]0 is the enzyme concentration. Inhibition constant (K i) was calculated from a Dixon plot. ScNP (final concentration of 5.9 nm) and various concentrations of ScNPIs (final concentration of 1.5–3.5 nm; in the case of authentic ScNPI, 2.1–6.2 nm) in 980 μl of Buffer C were incubated at 25 °C for 30 min. Then 20 μl of the substrate in the same buffer was added to give a final concentration of 2.6, 3.9, and 5.2 μm. K i values toward subtilisin BPN′ were also calculated from a Dixon plot. Subtilisin BPN′ (final concentration of 5.14 nm) and various concentrations of ScNPIs (final concentration of 0.51–2.09 nm) in 980 μl of 50 mm Tris-HCl, pH 8.0, containing 10 mmCaCl2 and 0.01% Triton X-100 (v/v) were incubated at 25 °C for 20 min. Then 20 μl of Suc-Ala-Ala-Pro-Phe-MCA in the same buffer was added to give a final concentration of 25, 50, and 100 μm. A fluorescence intensity was measured with a Hitachi F-2000 fluorescence spectrophotometer at λex 380 nm and λem 460 nm at 25 °C. Initial velocities were calculated from the liberated 7-amino-4-methylcoumarin (AMC) per min. Limited proteolysis by subtilisin BPN′ was performed according to the method of Hiromi et al. (14) with slight modification. ScNPI (1 nmol) and subtilisin BPN′ (0.65 nmol) were mixed in 50 μl of 50 mm Tris-HCl, pH 7.5, and incubated at 25 °C for 10 min. Then an equal volume of ice-chilled 0.5m glycine HCl, pH 2.5, was added to the mixture. Immediately, proteins were precipitated with trichloroacetic acid at a final concentration of 15%. The precipitate was subjected to Tricine SDS-PAGE. In the case of ScNP, ScNPI (1 nmol) was incubated with ScNP (1 nmol) in 100 μl of 50 mm Tris-HCl, pH 7.5, at 37 °C for 60 min. Then an equal volume of 0.5 m glycine HCl, pH 2.5, was added to the mixture and incubated at 4 °C for 60 min. The data were measured from the far-UV region (190–250 nm) to the near-UV region (250–350 nm) with a Jasco J-720 model CD spectropolarimeter using a 0.1-cm path length cell or a 1-cm path length cell, respectively. Complex formation between ScNPI and ScNP or subtilisin BPN′ was demonstrated by gel filtration on Superdex 200-HR-10/30 (10 × 300 mm) equilibrated with 50 mm Tris-HCl, pH 7.5, 10 mm CaCl2, 0.15 m NaCl, and 0.02% NaN3. ScNPI (1.2 nmol) and ScNP (1.2 nmol) or subtilisin BPN′ (1.2 nmol) were incubated at 25 °C for 30 min in 200 μl of the same buffer. After the incubation, the mixture was applied onto the column. Elution was carried out with the same buffer at a flow rate of 0.5 ml/min. A formation of the ternary complex was also analyzed by the same procedure. ScNPI (1.2 nmol), ScNP (1.2 nmol), and subtilisin BPN′ (1.2 nmol) were incubated at 25 °C for 30 min in the 200 μl of the same buffer. After the incubation, the mixture was applied onto the column. According to the high similarity of amino acid sequences between ScNPI and Streptomyces subtilisin inhibitor (SSI), we assumed that the structure of ScNPI is similar to that of SSI. Based on this, a hypothetical model was predicted. The locations of the reactive sites of ScNPI for subtilisin BPN′ and ScNP were predicted by superimposing the reactive sites on the crystal structure-based α-carbon framework of Streptomyces subtilisin inhibitor (SSI) (28Mitsui Y. Satow Y. Watanabe Y. Iitaka Y. J. Mol. Biol. 1979; 131: 697-724Crossref PubMed Scopus (99) Google Scholar). ScNPI was purified from a culture supernatant of Streptomyces sp. I-355 to electrophoretic homogeneity by three steps of column chromatography: DEAE-Sepharose fast flow, Sephadex G-75, and Mono Q. About 10 mg of the purified ScNPI was obtained from 2 liters of the culture supernatant (data not shown). The specific inhibitory activity of purified ScNPI was 340 IU/mg. The purified ScNPI showed a single protein band on Tricine SDS-PAGE with a molecular weight of 11,000 (Fig. 3). The molecular weight in the native state was also estimated to be 20,000 by gel filtration (data not shown), indicating that the inhibitor exists as dimers. The effects of ScNPI on several proteinase activities were investigated. Toward metalloproteinases, ScNPI strongly inhibited ScNP and slightly inhibited vimelysin. ScNPI did not show any inhibitory activity toward thermolysin, Pseudomonaselastase, and almelysin. In addition, ScNPI also inhibited subtilisin BPN′, trypsin, and chymotrypsin belonging to serine proteinase. ScNPI did not inhibit cysteine and aspartic proteinase (TableII).Table IIInhibition spectra of ScNPIProteinaseTypeSubstrate (pH)Molar ratioInhibitory activity(I/E)%ScNPMetalloCasein (7.5)10100ThermolysinMetalloCasein (7.5)500VimelysinMetalloCasein (7.5)103.95019.4P.elastase2-aP. elastase,Pseudomonas elastase.MetalloCasein (7.5)500AlmelysinMetalloCasein (7.5)500Subtilisin BPN′SerineCasein (9.5)10100TrypsinSerineBz2-bBz, benzoyl.-Arg-MCA (8.0)1042.15089.5ChymotrypsinSerineSuc2-cSuc, succinyl.-Ala-Ala-Pro-Phe-MCA1054.2 (8.0)5067.4Cathepsin BCysteineZ2-dZ, benzyloxycarbonyl.-Phe-Arg-MCA (6.0)500PepsinAsparticCasein (3.0)5002-a P. elastase,Pseudomonas elastase.2-b Bz, benzoyl.2-c Suc, succinyl.2-d Z, benzyloxycarbonyl. Open table in a new tab Kinetic analysis for ScNP was performed by using a newly designed fluorogenic substrate, MOCAc-Ala-Arg-Gly-Tyr-Gln-Gly-Lys(Dnp)-NH2. ScNP specifically cleaved this peptide at the Gly-Tyr bond. The kinetic parameters for cleavage by ScNP were calculated from a Lineweaver-Burk plot. The K m, k cat, andk cat/K m values of ScNP were determined to be 12.4 μm, 0.39 s−1, and 3.1 × 104m−1 s−1, respectively (data not shown). K i values of authentic ScNPI were determined from a Dixon plot (data not shown).K i values toward ScNP and subtilisin BPN′ were determined to be 1.6 and 1.4 nm, respectively. In order to synthesize oligonucleotide primers for PCR, the partial amino acid sequence of ScNPI was analyzed as described under “Experimental Procedures.” The amino-terminal amino acid sequence of the native ScNPI was identified as 1SAHGPSAMVTVIQGSGEPT20- and that of the CNBr-cleaved peptide fragment of ScNPI was identified as YFDPVTVTADGVLNGRRVAWKHTFS-. A 260-base pair DNA fragment containing the scnpi gene was amplified by PCR (data not shown). The fragment was labeled with DIG and used as a probe for hybridization. Genomic DNA of Streptomyces sp. I-355 was digested withBamHI, PstI, SacI, SalI,SphI, and XhoI and subjected to Southern blot analysis with the probe. A 2.5-kb fragment of SalI-digested DNA was hybridized with the probe (data not shown). A partial genomic library was then constructed. One colony was obtained from about 1,500E. coli transformants. The plasmid containing the full-length scnpi gene was named pScNPI-4. The location of the scnpi gene was determined by Southern blot analysis of pScNPI-4. A 1.0-kb SmaI fragment was subjected to sequence analysis (Fig. 1). An open reading frame consisted of 423 nucleotides. The deduced amino acid sequence consisted of 141 amino acid residues with a molecular weight of 14,656 (Fig. 1). All of the partial amino acid sequences of the ScNPI agreed with the predicted ScNPI gene product. A comparison of the NH2-terminal amino acid sequence of the authentic ScNPI and the deduced amino acid sequence of the scnpi gene revealed that ScNPI consisted of two regions, a signal region (28 amino acid residues) and a mature region (113 amino acid residues,M r = 11,857). The deduced amino acid sequence of ScNPI showed high similarity to those of Streptomycessubtilisin inhibitor (SSI) and its homologues (Fig.2). Thus, ScNPI was revealed to be a novel proteinase inhibitor that belongs to the SSI family.Figure 2Comparison of the amino acid sequence of ScNPI and those of SSI family inhibitors. ScNPI (this paper), SSI (15Ikenaka T. Odani S. Sakai M. Nabeshima Y. Sato S. Murao S. J. Biochem. (Tokyo). 1974; 76: 1191-1209Crossref PubMed Scopus (112) Google Scholar) (Streptomyces subtilisin inhibitor), SIL8 (16Terabe M. Kojima S. Taguchi S. Momose H. Miura K. Eur. J. Biochem. 1994; 226: 627-632Crossref PubMed Scopus (15) Google Scholar) (Streptomyces subtilisin inhibitor-like inhibitor 8), SLPI (17Ueda Y. Kojima S. Tsumoto K. Takeda S. Miura K. Kumagai I. J. Biochem. (Tokyo). 1992; 112: 204-211Crossref PubMed Scopus (23) Google Scholar) (Streptomyces lividans protease inhibitor), and SAC I (19Tanabe M. Kawahara K. Asano T. Kato K. Kakinuma A. J. Biochem. (Tokyo). 1994; 115: 752-761Crossref PubMed Scopus (6) Google Scholar) (Streptoverticillium anticoagulant I) are shown. Highly conserved amino acid residues that are common in more than four inhibitors among five aligned inhibitors are shaded. Thearrow indicates the deduced reactive site of ScNPI for subtilisin BPN′.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A high level expression system for ScNPI was constructed using pIN-III-OmpA2 vector. All the activity of the ScNPIs was found in the bacterial sonicate. Recombinant ScNPIs were purified as described un" @default.
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• W2012193516 title "A Novel Double-headed Proteinaceous Inhibitor for Metalloproteinase and Serine Proteinase" @default.
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