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• W2023302157 abstract "The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pz-peptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the full-length enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the full-length collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure. The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pz-peptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the full-length enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the full-length collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure. Collagens are the major protein constituents of the extracellular matrix and the most abundant proteins in all higher organisms (1Mayne R. Burgeson R.E. Structure and Function of Collagen Types. Academic Press, Orlando, FL1987Google Scholar). The tightly coiled triple helical collagen molecule assembles into water-insoluble fibers or sheets which are cleaved only by collagenases, and are resistant to other proteinases. Various types of collagenases, which differ in substrate specificity and molecular structure, have been identified and characterized. Bacterial collagenases differ from vertebrate collagenases in that they exhibit broader substrate specificity (2Peterkofsky B. Methods Enzymol. 1982; 82: 453-471Crossref Scopus (100) Google Scholar, 3Birkedal-Hansen H. Methods Enzymol. 1987; 144: 140-171Crossref PubMed Scopus (130) Google Scholar). Clostridium histolyticum collagenase is the best studied bacterial collagenase (4Mookhtiar K.A. Van Wart H.E. Matrix Suppl. 1992; 1: 116-126PubMed Google Scholar) and is widely used as a tissue-dispersing enzyme (5Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar, 6Worthington C.C. Worthington Enzyme Manual: Collagenase. Worthington Biochemical Co., Freehold, NJ1988Google Scholar). This enzyme is unique in that it can degrade both water-insoluble native collagens and water-soluble denatured ones, can attack almost all collagen types, and can make multiple cleavages within triple helical regions (4Mookhtiar K.A. Van Wart H.E. Matrix Suppl. 1992; 1: 116-126PubMed Google Scholar). Kinetic studies of collagenases have provided insight into the high-ordered structure of collagens (7French M.F. Bhown A. Van Wart H.E. J. Protein Chem. 1992; 11: 83-97Crossref PubMed Scopus (61) Google Scholar, 8Mallya S.K. Mookhtiar K.A. Van Wart H.E. J. Protein Chem. 1992; 11: 99-107Crossref PubMed Scopus (47) Google Scholar). However, the structure-function relationship of this unique enzyme is not known. Multiple forms of collagenase are produced by C. histolyticum. Seven different forms have been identified, and they are divided into two classes based on possible similarities in their amino acid sequences and their specificities toward peptide substrates (9Van Wart H.E. Steinbrink D.R. Biochemistry. 1985; 24: 6520-6526Crossref PubMed Scopus (64) Google Scholar). In a previous study (10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar) we have cloned and sequenced acolH gene encoding the 116-kDa collagenase (ColH), which is abundant in many commercial enzymes (11Steinbrink D.R. Bond M.D. Van Wart H.E. J. Biol. Chem. 1985; 260: 2771-2776Abstract Full Text PDF PubMed Google Scholar). Comparison of the predicted amino acid sequence of ColH with those of the Vibrio alginolyticus (12Takeuchi H. Shibano Y. Morihara K. Fukushima J. Inami S. Keil B. Gilles A.-M. Kawamoto S. Okuda K. Biochem. J. 1992; 281: 703-708Crossref PubMed Scopus (58) Google Scholar) and Clostridium perfringenscollagenases (13Matsushita O. Yoshihara K. Katayama S. Minami J. Okabe A. J. Bacteriol. 1994; 176: 149-156Crossref PubMed Google Scholar) revealed a segmental structure for these enzymes (10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar). ColH has been shown to consist of four segments, S1, S2a, S2b, and S3, and S2a and S2b are homologous. The molecular masses of S1, S2a, S2b, and S3 are 78.1, 10.0, 9.9, and 14.1 kDa, respectively (10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar). S1 contains the sequence HEXXH, a consensus motif located at the catalytic center of zinc-metalloproteases. A 98-kDa gelatinase that copurified with ColH hydrolyzed denatured but not native collagen. The two enzymes possessed identical N-terminal sequences and their peptide maps were almost identical. Therefore, the 98-kDa gelatinase is probably produced by cleaving off a C-terminal peptide from ColH (10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar). These observations led us to suspect that the C-terminal peptide forms a functional domain, which is involved in either providing accessibility to or binding of the enzyme to collagen. To gain insights into the structure-function relationships of C. histolyticum collagenases, we have attempted a molecular dissection of ColH by constructing recombinant derivatives of the enzyme. In this paper, we examined the collagen binding activities of various C-terminal peptides fused to the C terminus of glutathioneS-transferase (GST) 1The abbreviations used are: GST, glutathioneS-transferase; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; CB buffer, collagen binding buffer; FP, fusion protein; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. to localize the collagen-binding domain. We have also examined the enzymatic activities of C-terminal truncated species toward native type I collagen and a synthetic peptide substrate. C. histolyticum JCM 1403 (ATCC 19401) was obtained from the Institute of Physical and Chemical Research (Saitama, Japan). Bacillus subtilis DB104 (14Kawamura F. Doi R.H. J. Bacteriol. 1984; 160: 442-444Crossref PubMed Google Scholar) was used to produce the recombinant collagenases. Escherichia coli DH5α (15Bethesda Research Laboratories Focus. 1986; 8: 9Google Scholar) was used for the construction of all recombinant plasmids. Plasmid pCHC116Δ3 was generated by nested deletion of pCHC116 (10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar) from its 3′ end. The plasmid contains a colH gene fragment from nucleotide 2,719 to 3,391 (numbered as in Ref. 10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar), which encodes the C-terminal segments of ColH, S2b, and S3. E. coli BL21 and the pGEX-4T series plasmids (Pharmacia Biotech, Uppsala, Sweden) were used as a host-vector system for the production of GST fusion proteins. A ColH fraction containing the gelatinase, prepared from C. histolyticum cultures as described previously (10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar), was used as partially purified ColH. ColH purified from cultures of recombinantB. subtilis DB104 (16Jung C.-M. Matsushita O. Katayama S. Minami J. Ohhira I. Okabe A. Microbiol. Immunol. 1996; 40: 923-929Crossref PubMed Scopus (15) Google Scholar) was used as recombinant ColH (rColH). Recombinant B. subtilis strains were grown as described previously (16Jung C.-M. Matsushita O. Katayama S. Minami J. Ohhira I. Okabe A. Microbiol. Immunol. 1996; 40: 923-929Crossref PubMed Scopus (15) Google Scholar). For screening recombinant plasmids, E. coli DH5α transformants were grown in Luria-Bertani medium supplemented with 100 μg of ampicillin/ml. For the preparation of GST fusion proteins, all recombinant E. coli BL21 cells were grown in 2YT-G medium consisting of the following ingredients: 16 g of tryptone, 10 g of yeast extract, 5 g of NaCl, 20 g of glucose, and 100 mg of ampicillin/liter. The expression of fusion proteins was induced by the addition of 0.1 mmisopropyl-1-thio-β-d-galactopyranoside (Wako Pure Chemical Industries, Ltd., Osaka, Japan). An N-terminal peptide of ColH, consisting of S1 and S2a (S1S2a), was constructed as follows. A 2.9-kilobase HaeIII-PstI fragment containing the colH′ gene (10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar) was ligated into the EcoRV and PstI sites of Bluescript II KS(+). Into the downstreamSacI site of the resulting plasmid (pCHC200) a synthetic oligonucleotide, 5′-GCTTAATTAATTAAGCAGCT-3′, was inserted so that translation from a colH′ transcript terminates at the TAA codon. The resulting plasmid was designated as pCHC201. A 3.1-kilobaseBssHII fragment, which encodes amino acids 1 to 766, (numbered as in Ref. 10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar) corresponding to S1S2a plus an extra 5-amino acid stretch (Ala-Arg-Gly-Ile-His), was isolated from pCHC201 and then ligated into the SmaI site of pHY300PLK. The resulting plasmid was introduced into B. subtilis DB104. The transformant was grown, and the culture supernatant was subjected to ammonium sulfate precipitation and gel filtration as described elsewhere (16Jung C.-M. Matsushita O. Katayama S. Minami J. Ohhira I. Okabe A. Microbiol. Immunol. 1996; 40: 923-929Crossref PubMed Scopus (15) Google Scholar). The fractions with Pz-peptidase activity after gel filtration were applied to a hydrophobic interaction column (Ether-Toyopearl, bed volume 14 ml; Toso, Tokyo, Japan), which was pre-equilibrated with 50 mm Tris-HCl (pH 7.5) containing ammonium sulfate (40% saturation). Proteins were eluted by a 280-ml linear gradient from 40 to 0% ammonium sulfate in 50 mmTris-HCl (pH 7.5). The Pz-peptidase activity eluted at 29% ammonium sulfate. The active fraction was dialyzed against 50 mmTris-HCl (pH 8.5), and then applied to a MonoQ column. Chromatography was performed as described elsewhere (16Jung C.-M. Matsushita O. Katayama S. Minami J. Ohhira I. Okabe A. Microbiol. Immunol. 1996; 40: 923-929Crossref PubMed Scopus (15) Google Scholar) except that the pH was changed to 8.5. A recombinant N-terminal peptide containing only S1 was produced as follows. An oligonucleotide encoding the C-terminal peptide of S1 (Val564 to Ser683) followed by a stop codon was prepared by polymerase chain reaction using two primers (5′-GTGCCTTTTGTAGCTGATGA-3′ and 5′-CCGCGGTTAGGAATCACCTTCGTTTGGTA-3′) and pCHC201 plasmid DNA. The amplified fragment was cloned into the pT7Blue T-vector (Novagen, Madison, WI). An 0.33-kilobaseSphI-SacII fragment of this plasmid was substituted for a 0.61-kilobase SphI-SacII fragment of pCHC201. S1 was purified from cultures of recombinantB. subtilis as described above. An inactive ColH mutant was produced as follows. Replacement of the GAA codon encoding Glu416, one of the residues forming a putative catalytic center, with a GAT codon (Asp) was performed using pCHC201 plasmid DNA and a Transformer site-directed mutagenesis kit (CLONTECH Laboratories, Palo Alto, CA), according to the instruction of the manufacturer. Oligonucleotides, 5′-GCAAATAATGTGTATAATCATGTCTAAATAATTC-3′ and 5′-GTGACTGGTGAGGCCTCAACCAAGTC-3′, were used as a mutagenic primer and a selection primer, respectively. The mutant enzyme, designated as rColH(E416D), was produced using a shuttle vector, pAT19 (17Trieu-Cuot P. Carlier C. Poyart-Salmeron C. Courvalin P. Gene (Amst.). 1991; 102: 99-104Crossref PubMed Scopus (171) Google Scholar) as described elsewhere (16Jung C.-M. Matsushita O. Katayama S. Minami J. Ohhira I. Okabe A. Microbiol. Immunol. 1996; 40: 923-929Crossref PubMed Scopus (15) Google Scholar). The GST gene fusion system was employed to construct fusion proteins between GST and ColH C-terminal segments. A DNA fragment encoding segments 2a and 2b (S2aS2b, Pro678-Asp860) was obtained by polymerase chain reaction using pCHC11 plasmid DNA (10Yoshihara K. Matsushita O. Minami J. Okabe A. J. Bacteriol. 1994; 176: 6489-6496Crossref PubMed Google Scholar), a 5′-primer, 5′-CCCGGGCCAAACGAAGGTGATTCCAA-3′, and a 3′-primer, 5′-CTCGAGTTAATCTGTAATCTTAATCTTCA-3′. A DNA fragment encoding segment 2b (S2b, Glu767-Asp860) was also obtained in the same way using pCHC302 plasmid DNA, 5′-pGEX primer (Pharmacia), and the same 3′-primer as described above. These fragments were cloned into pT7Blue T-vector. They were inserted into pGEX-4T-2 vector DNA to express the enzyme fragments as GST fusion proteins. E. coliDH5α competent cells were transformed with each ligation mixture by electroporation. After the nucleotide sequence of each fusion gene was confirmed, E. coli BL21 was transformed with the recombinant plasmid. Each clone was grown in 100 ml of 2YT-G medium to an optical density at 600 nm of 2.5, and isopropyl-1-thio-β-d-galactopyranoside was added. Purification of the fusion protein was performed with a glutathione-Sepharose column (bed volume, 0.5-ml; Pharmacia) as described by the manufacturer. The fusion proteins for S2aS2b and S2b were designated as FP342 and FP344, respectively. GST fusion proteins for C-terminal fragments of various lengths were also constructed. The insert DNA fragment in pCHC116Δ3 (see above) encoding S2b plus S3 (S2bS3) was deleted from its 5′ end using appropriate restriction enzymes. Each fragment was inserted into the SmaI site of a suitable GST fusion vector of the pGEX-4T series (Pharmacia) so that the reading frame is intact. After the nucleotide sequence of each fusion gene was confirmed, fusion proteins carrying varying length C-terminal fragments were produced as described above, and designated as FP302–FP306. Restriction endonucleases were purchased from Takara Shuzo Co. (Kyoto, Japan), Toyobo (Osaka, Japan), and New England Biolabs (Beverly, MA). The DNA ligation kit was a product of Takara Shuzo. All recombinant DNA procedures were carried out as described by Sambrook et al. (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). All constructs were sequenced to confirm the reading frame on an automated nucleotide sequencer (model ABI PRISM 377, Perkin-Elmer, Foster City, CA). An ABI PRISM dye terminator cycle sequencing ready kit with AmpliTaq DNA polymerase, FS (Perkin-Elmer), and pGEX primers (Pharmacia) were used for sequencing the GST fusion constructs. A Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Japan Inc., Tokyo, Japan) and M13 dye primers (Perkin-Elmer) were used for sequencing all other constructs. The activities of the recombinant collagenases were determined using Pz-peptide (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, Sigma) or insoluble collagen from bovine achilles tendon (Worthington Biochemical Co., Freehold, NJ), as described elsewhere (16Jung C.-M. Matsushita O. Katayama S. Minami J. Ohhira I. Okabe A. Microbiol. Immunol. 1996; 40: 923-929Crossref PubMed Scopus (15) Google Scholar). The GST activity of GST fusion proteins was assayed using 1-chloro-2,4-dinitrobenzene as described in the supplier's protocol. Protein concentrations were determined by the Bradford method (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using the Bio-Rad protein assay reagent (Bio-Rad) with bovine serum albumin (BSA) as a standard. All the assays were carried out in triplicate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 7.5, 12.5, or 15% polyacryamide gels, and the gels were stained with Coomassie Brilliant Blue R as described previously (20Jin F. Matsushita O. Katayama S.-I. Jin S. Matsushita C. Minami J. Okabe A. Infect. Immun. 1996; 64: 230-237Crossref PubMed Google Scholar). Band intensity was determined using a flat bed scanner and the public domain computer program, NIH Image (developed at the National Institute of Health, Bethesda, MD, and available from the Internet by anonymous FTP from zippy.nimh.nih.gov). Two hundred picomoles of the isolated C-terminal fragments obtained from fusion proteins (FP302, FP305, FP342, and FP344) were blotted on a polyvinylidene difluoride membrane using ProSorb devices (Perkin-Elmer) as described by the supplier. Twenty amino acid residues from the N terminus were determined for each fragment on an automatic protein sequencer (Model 492, Perkin-Elmer). All the isolated fragments possessed the expected N-terminal amino acid sequences. A GST fusion protein containing S2bS3 (FP302, 1.1 mg) was cleaved by incubation with 10 units of thrombin for 5 h at room temperature. The reaction mixture was dialyzed twice against 2 liters of phosphate-buffered saline (PBS) at 4 °C to remove glutathione, and the cleaved N-terminal GST fragment was removed by adding glutathione-Sepharose beads (100 μl). After incubation at room temperature for 30 min, the suspension was centrifuged at 500 ×g for 5 min and the bead treatment was repeated three times. The supernatant was dialyzed against 2 liters of distilled water at 4 °C for 12 h with two changes, filtrated through an 0.45-μm filter, and lyophilized. The sample was dissolved in 100 μl of distilled water, mixed with 3,5-dimethoxy-4-hydroxycinnamic acid solution (10 mg/ml of 50% ethanol), and analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (model Kompact MALDI III, Kratos, Manchester, United Kingdom) with BSA as an internal standard. Collagen binding was assayed as follows, unless otherwise stated. Five milligrams of insoluble collagen (type I, C-9879; Sigma) were added to an Ultrafree microcentrifugal device with an 0.22-μm low-binding Durapore membrane (Millipore, Bedford, MA), which was placed in a microcentrifuge tube. All steps were carried out at room temperature. Two hundred microliters of collagen binding buffer (CB buffer: 50 mm Tris-HCl, 5 mm CaCl2, pH 7.5) were added to swell the collagen fibers. After incubation for 30 min, the tube was centrifuged at 15,000 × g for 15 min. Centrifugation was repeated after changing the direction of the tube in the rotor. The collagen precipitate was resuspended in 60 μl of CB buffer containing 100 pmol of enzyme and incubated for 30 min. The filtrate was collected by centrifugation at 15,000 × g for 15 min, and used for GST assay or analysis by SDS-PAGE. To determine the binding affinities of rColH(E416D) and various C-terminal segments by Scatchard plot analysis, the collagen binding assay was carried out with the following modification. Collagen was washed with 200 μl of CB buffer supplemented with 150 mm NaCl, and resuspended in 100 μl of the same buffer containing various concentrations (25–400 μg/ml) of the mutant enzyme or various fragments. A calibration curve was constructed for each sample by densitometory after SDS-PAGE, and was used to quantitate their amounts in the filtrates. The results obtained by triplicate assay were analyzed on a Scatchard plot, and the dissociation constant (Kd) and the number of binding sites on insoluble collagen (B max) for each protein were determined by the least-square method. To examine collagen binding, 6 μg of ColH in 60 μl of CB buffer was added to 5 mg of swollen collagen. After the mixture was incubated at room temperature for 30 min and centrifuged, a 20-μl sample of the supernatant was analyzed by SDS-PAGE. As shown in Fig. 1, the gelatinase but not ColH was detectable in the filtrate, suggesting that ColH but not the gelatinase binds to insoluble type I collagen. Various smaller polypeptides were also detected in the filtrate. These might have resulted from hydrolysis of insoluble collagen by ColH. To inhibit the collagenolytic activity of ColH, the incubation was carried out at 4 °C for 30 min, and then the filtrate was analyzed by SDS-PAGE (Fig. 1). The gelatinase was again detectable but neither ColH nor the smaller polypeptides were detected in the filtrate, indicating that ColH binds collagen without degrading it at 4 °C. Therefore, subsequent collagen binding assays of active collogenases were carried out at 4 °C. The difference in the collagen-binding capability of the two enzymes suggests that the C-terminal peptide which is absent in the gelatinase is involved in collagen binding. However, it has not yet been proved that the 98-kDa gelatinase is produced from ColH by cleaving off the C-terminal region. To test this possibility, two recombinant enzymes, 116-kDa rColH and its truncated form consisting of S1S2a were purified from cultures of recombinant B. subtilis cells. Twenty-five picomoles of each enzyme were mixed with 5 mg of swollen insoluble collagen, and the mixture was incubated at 4 °C for 30 min. A sample containing 1 μg of protein was analyzed by SDS-PAGE (Fig. 2). While the full-length rColH bound to insoluble collagen, the truncated form did not. Activities against insoluble collagen and Pz-peptide, a synthetic water-soluble substrate, were determined for the two recombinant polypeptides (Table I). The Pz-peptide hydrolyzing activity of the truncated form was 73% of that of rColH, while its collagenolytic activity was only 7%. This result suggests that the C-terminal peptide is required for binding and hydrolysis of insoluble collagen.Table ICollagenase and Pz-peptidase activities of recombinant collagenasesEnzymeCollagenasePz-peptidaseunits/pmolrColH0.164 ± 0.0060.0859 ± 0.0036rColH′(S1S2a)0.0116 ± 0.00590.0629 ± 0.0048rColH′(S1)0.00857 ± 0.001540.123 ± 0.006 Open table in a new tab The C-terminal peptide, S2bS3, was fused to the C terminus of GST to see if it conferred the ability to bind to collagen on GST. The apparent molecular mass of the fusion protein estimated by SDS-PAGE (Fig. 3), 51 kDa, agreed with the 50,526 Da value calculated from the nucleotide sequence of the corresponding gene. The fusion protein was cleaved by thrombin and SDS-PAGE showed two peptides. One migrated to the same position as the product generated by cleavage of GST alone (26 kDa), and the other (28 kDa) reacted with anti-collagenase antiserum (data not shown). The peptide was purified, its N-terminal amino acid sequence was determined, and it coincided with the predicted sequence. Its apparent molecular mass differed from the value (24,378 Da) calculated for the S2bS3 containing peptide. To determine the molecular mass more accurately, the peptide was analyzed by mass spectrometry. Its molecular mass was determined to be 24,370 Da and the reason for its abnormal mobility in SDS-PAGE is unknown. Samples of the GST fusion protein before and after cleavage with thrombin were combined, added to CB buffer containing BSA, chicken ovalbumin, and horse myoglobin (6 μg each) and then the final volume was adjusted with CB buffer to 60 μl. After incubation with 5 mg of insoluble collagen, and centrifugation, the supernatant was analyzed by SDS-PAGE (Fig. 3). All proteins except the fusion protein and S2bS3 were present in the filtrate, indicating that S2bS3 and the GST fusion protein specifically bound to collagen. To determine the collagen binding condition, the S2bS3 peptide was incubated with insoluble collagen under various conditions. When 20 μg of the peptide was incubated with varying amounts of insoluble collagen for 30 min at room temperature, it was almost completely bound to 10 mg or more collagen. When 20 μg of the peptide was incubated with 15 mg of collagen for varying times, approximately 70% of the peptide bound to collagen after 2 min, and the binding reached its maximal level after 30 min. It was shown that the binding capacity of collagen for the S2bS3 peptide is more than 2 μg/mg collagen, and that incubation at room temperature for 30 min is sufficient for completion of the binding reaction. The effect of pH on collagen binding was examined by determining the GST activity of the GST fusion protein in the filtrate after incubating using the standard assay except the buffer was changed to: 50 mm bis-Tris-HCl containing 5 mmCaCl2 (pH 5.0) or CB buffer (pH 7.0) or CB buffer (pH 9.0). Mean ± S.D. of collagen binding at pH 5, 7, and 9 were 97.9 ± 0.0, 97.5 ± 0.3, and 100 ± 0.0%, respectively. The Pz-peptidase activity of rColH determined in these buffers was 0.98 ± 0.98, 76.5 ± 2.4, and 60.1 ± 1.2 units/nmol, respectively. When 5 mm EDTA was added to the buffer in place of 5 mm CaCl2, the Pz-peptidase activity of rColH was completely inhibited, while the collagen binding activity of the GST fusion was 88.6 ± 1.2%. The addition of gelatin at a final concentration of 10 mg/ml to CB buffer did not affect binding of the GST fusion protein (96.0 ± 0.2%). The addition of sodium chloride at a final concentration of 1.5 m slightly affected binding (92.3 ± 0.3%). Two truncated peptides, S1S2a and S2bS3, exhibited Pz-peptidase and collagen binding activities, respectively. Given the homology between S2a and S2b, it appears that S3 is a collagen-binding domain, and that S2b and possibly S2a form an interdomain structure. To examine this possibility, S2bS3 was deleted to various lengths from its N terminus and fused to the C terminus of GST (Fig. 4 A). The fusion proteins were purified to homogeneity as shown in Fig. 4 B. These fusion proteins were incubated with insoluble collagen, and the GST activities in the filtrates were determined (Table II). Since GST activity differed slightly from one fusion protein to another, the collagen binding activity of each fusion protein was determined by calculating the difference of the GST activity in supernatants incubated with and without collagen. As the C-terminal peptide was shortened, collagen binding activity decreased. However, the binding activity of the fusion protein containing S3 (FP305) was still high, being more than 90% of that containing S2bS3 (FP302). When the C-terminal peptide was shorter than S3, binding decreased significantly. The fusion proteins were cleaved by thrombin, and the ability of the C-terminal peptides to bind insoluble collagen were tested by SDS-PAGE (Fig. 4 C). S3 alone bound collagen and the shorter peptide bound weakly, as was expected. The difference in band intensity between peptides incubated with and without collagen matched the binding activity of the corresponding fusion protein determined by its GST activity.Table IICollagen binding activities of the fusion proteins carrying various sizes of the ColH C-terminal fragmentFusion protein (fragment)Collagen binding%FP302 (Glu767–Arg981)98.7 ± 0.0FP303 (Gln811–Arg981)96.1 ± 0.3FP304 (Thr839–Arg981)85.3 ± 1.1FP305 (Pro861–Arg981)91.2 ± 0.6FP306 (Ile886–Arg981)64.8 ± 2.6GST1.1 ± 1.8 Open table in a new tab Since GST is known to exist as a dimer (21Henderson R.M. Schneider S. Li Q. Hornby D. White S.J. Oberleithner H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8756-8760Crossref PubMed Scopus (59) Google Scholar), GST fusion proteins may also exist as a dimer. Therefore, the GST fusion proteins are not suitable for the quantitative evaluation of the collagen binding affinity of various C-terminal segments. Thus, the fusion proteins were cleaved by thrombin, and the collagen binding assay was performed with varying concentrations of the purified C-terminal peptides. Scatchard analysis of the data (Fig. 5) showed that S3 alone bound to insoluble collagen with low but significant affinity (Kd = 1.59 × 10−5m) and high capacity (B max = 1.01 nmol/mg collagen), while neither S2a2b nor S2b bound. S2bS3 showed biphasic plots, one with higher affinity (Kd = 3.39 × 10−7m, B max = 0.201 nmol/mg collagen) and the other with low affinity (Kd = 2.11 × 10−6m, B max = 0.628 nmol/mg collagen). The affinity of the full-length enzyme was determined by using rColH(E416D), in which the Glu416 residue forming the putative catalytic center is replaced by an Asp residue. This replacement aboli" @default.
• W2023302157 created "2016-06-24" @default.
• W2023302157 creator A5022373532 @default.
• W2023302157 creator A5028501143 @default.
• W2023302157 creator A5032563346 @default.
• W2023302157 creator A5045343631 @default.
• W2023302157 creator A5045803775 @default.
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• W2023302157 date "1998-02-01" @default.
• W2023302157 modified "2023-09-28" @default.
• W2023302157 title "A Study of the Collagen-binding Domain of a 116-kDaClostridium histolyticum Collagenase" @default.
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