A peptidase hydrolyzing synthetic substrates of collagenase was purified from cell extract of an oral spirochete, Treponema denticola by sequential procedures including anionic ionexchange chromatography, gel filtration, and hydrophobic interaction chromatography. The purified enzyme was most active at pH 8.0 and was inhibited by p-chloromercuribenzoate or EDTA. This enzyme was active on Pz-peptide and Z-Gly-L-Pro-L-Leu-Gly-L-Pro. It was observed that Gly-L-Pro was released from the latter substrate, but proteins, including collagens, were not found to be hydrolyzed by this enzyme

FUJIMURA SETSUO

NAKAMURA TAKESHI

SHIBATA YUKINAGA

English

application/pdfA peptidase hydrolyzing synthetic substrates of collagenase was purified from cell extract of an oral spirochete, Treponema denticola by sequential procedures including anionic ionexchange chromatography, gel filtration, and hydrophobic interaction chromatography. The purified enzyme was most active at pH 8.0 and was inhibited by p-chloromercuribenzoate or EDTA. This enzyme was active on Pz-peptide and Z-Gly-L-Pro-L-Leu-Gly-L-Pro. It was observed that Gly-L-Pro was released from the latter substrate, but proteins, including collagens, were not found to be hydrolyzed by this enzyme.journal articl

SHIBATA, YUKINAGA

FUJIMURA, SETSUO

NAKAMURA, TAKESHI

Matsumoto Dental University Repository

(Original) Matsumoto Shigaku 18 : 138-143, 1992             key words : T. denticola - oral spirochete - peptidasePurification and Characterization of an Enzyme Produced by TrePonenza denticola Hydrolyzing Synthetic Collagenase SubstratesYUKINAGA SHIBATA SETSUO FUJIMURA and TAKESHI NAKAMURA     Department of Oral Microbiology, Matsumoto Dental College                  (Chief:Prof. T. IVahamura)Summary       A peptidase hydrolyzing synthetic substrates of collagenase was purified from cell   extract of an oral spirochete, TrePonema denticola by sequential procedures including   anionic ionexchange chromatography, gel filtration, and hydrophobic interaction   chromatography. The purified enzyme was most active at pH 8.0 and was inhibited by   P-chloromercuribenzoate or EDTA. This enzyme was active on Pz-peptide and Z-Gly   -L-Pro-L-Leu-Gly-L-Pro. It was observed that Gly-L-Pro was released from the latter   substrate, but proteins, including collagens, were not found to be hydrolyzed by this   enzyme.                                   Introduction   Genus TrePonema spirochetes are commonly found in subgingival flora in mani•3•5År, and it hasbeen shown that spirochetes increase in numbers during periodontal diseases3•6). Troponemadenticola is a spirochete species frequently detected in periodontal lesions9). Thus, oral treponemesare thought to be associated with etiology of periodontal disease. However, little is known aboutpathogenic factors of this spirochete. Several reports have indicated that oral spirochetes includingT. denticola elaborate enzymes responsible for tissue degradation',S,'2"3,i5,'6). We have also isolatedT. denticola from adult periodontal lesions and investigated biological properties and diversity ofprotease production of these organisms'i). The aim of this study was to characterize the enzymehydrolyzing synthetic peptide substrates of microbial collagenase and to evaluate its enzymaticaction on collagens.                                     Methods    TrePonema denticola strain N-1-2 was isolated from the adult periodontal lesion in ourlaboratoryii). The spirochete was grown in TYGVS medium'3) in anaerobic glove box filled withgases (N2+ H2+C02;85: 10:5) at 37eC for 7 days.    Enzyme activity hydrolyzing Pz-peptide (4-phenylazobenzyloxy-carbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine, Sigma Chem. Co.), a synthetic substrate for col-lagenase, was assayed based on the method of Wunsch and Heidrichi'). Briefly, reaction mixture(accepted for publication on June 29, 1992)tE}Jzlsctwe+" 18(2) 1992 139containing enzyme sample (O.1 ml), 2.5 mM Pz-peptide solution in O.05 M Tris-hydrochloride buffer,(pH 7.2) (O.1 ml), and O.05 M Tris-hydrochloride buffer, (pH 7.2) (O.3 ml) was incubated at 37eC for 30min To stop reaction, O.5 ml of O.4 M citric acid was added and shaken. Then 2.0 ml of ethyl acetatewas introduced into this mixture and mixed exhaustively followed by low speed centrifugation. Theethyl acetate phase containing Pz-L-Pro-L-Leu generated by the hydrolytic action of the enzymewas taken carefully and absorbance at 320 nm was assayed to estimate Pz-L-Pro-L-Leu. One unitof enzyme was defined as the amount that increased O.OOI absorbance unit per min under theseconditions. Hydrolytic activity against other peptide substrates was assessed by ninhydrinreactioniO). Protein degradation by the purified enzyme was determined by SDS-PAGE method`)which was also employed to examine the purity of the enzyme samples and determination ofmolecular weight. Marker proteins used for molecular weight determination were phosphorylase b(94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsininhibitor (20.1 kDa), and a-Iactalbumin (14.4 kDa).   Crude extract of T. denticola N-1-2 was prepared by the following procedure ; the spirocheteswere harvested by centrifugation at 12,OOO G for 15 min. Cells (12 g) washed twice with O.05 MTris-hydrochloride buffer (pH 8.4) were suspended in the same buffer. Then cells were disrupted bysonication at 9 KHz for 15 min. The sonicate was centrifuged at 100,OOO G for 60 min and thesupernatant (crude extract) was collected and used for a starting material of purification.   Reversed-phase high performance liquid chromatography (HPLC), equipped with Shim-PackCLC-ODS column (Shimadzu Co. ), equilibrated with O.10/o trifluoroacetic acid-350/o acetonitrile wasused for detection of peptideS. The peptides jn the eluate were monitored by absorbance at 210 nm.Results    Pz-peptidase activity was found in crude extract. No significant amount of activity wasdetected jn culture supernatant or in 100,OOO G pellet of sonicate.    Crude extract was applied to a column (2.6 by 20 cm) of Q-Sepharose equilibrated with O.05 MTris-hydrochloride buffer (pH8.4). The column was washed thoroughly with this buffer and subse-quently eluted with linearly increasing concentration of NaCl in this buffer. The enzyme was foundto elute at about O.8M NaCl from the column. The active fractions were combined, concentrated,and dialyzed against O.05M Tris-hydrochloride buffer (pH7.2) containing O.2M NaCl. The concen-trated material was applied to a column (2.6 by 80cm) of Sephacryl S-300 and eluted with the samebuffer saline. The specific activity (enzyme activity units per lmg protein) rose to 52fold of that ofcrude extract, however, this sample showed several stained protein bands on SDS-PAGE. The NaClwas added to this sample up to 1.0M and subjected to hydrophobic interaction chromatography onPhenyl Sepharose CL-4B column (1.6 by 8cm). When the column was washed with O.05M Tris-hydrochloride buffer (pH72) containing 1.0M NaCl, about 700/o of the total protein of the appliedsample eluted accompanying only negligible amount of Pz-peptidase. The column was then elutedwith O.05M Tris-hydrochloride buffer (pH7.2). The enzyme eluted as a sharp peak. The activefractions were combined and applied to a column (1 by 5cm) of hydroxyapatite equilibrated with O.05M Na-phosphate buffer (pH7.0). When the column was eluted with stepwise increasing concentra-tion of Na-phosphate buffer (pH7.0), Pz-peptidase eluted with O.12M Na-phosphate buffer (pH7.0).The active fractions from the column were combined and dialyzed against O.05M Tris-hydrochloridebuffer (pH7.2). This sample was a purified Pz-peptidase. The enzyme was purified 93-fold comparedto crude extract by the purification procedure.140 Shibata, et aL:Peptidase of T. denticola   As shown in Fig. 1, the purified Pz-peptidase appeared to be homogeneous on SDS-PAGE, andmolecular weight was calculated to be 65kDa.   The optimum pH for the reaction of the enzyme was found at pH8.0. The activity could not bedetected below pH6.0, however, about 400% the maximum activity at pH8.0 was seen even thoughat pH9.5.   Effects of various group specific reagents and metal ions are summarized in Table 1. Theenzyme was inhibited completely by P-chloromercuribenzoate, indicating sulfhydry1 group(s) of thisenzyme molecule may be important for its hydrolytic activity, although the enzyme was notactivated by reducing agents such as mercaptoethanol or dithiothreitol. Inhibition by EDTA is alsoobvious.   Pz-peptide was incubated with some proteins and peptides to determine its substrate specificity.All the proteins including human collagens (Type I , Type M, and Type IV), gelatin, bovine albumin,bovine fibrinogen, human Ig A, and human Ig G were not hydrolyzed. Z-Gly-L-Pro-L-Leu-Gly-L-Prowas found to be hydrolyzed, but it could not hydrolyze Z-Gly-L-Pro-L-Leu-Gly and Z-Gly-L-Pro-L-Leu. Hydrolytic products of Z-Gly-L-Pro-L-Leu-Gly-L-Pro which had been digested for lh and 4h byPz-peptidase were analyzed by HPLC. As illustrated in Fig. 2, only Gly-L-Pro and Z-Gly-L-Pro-L-Leu were generated from Z-Gly-L-Pro-L-Leu-L-Pro. These results suggest that Pz-peptidase splitsL-Leu-Gly bond of Z-Gly-L-Pro-L-Leu-Gly-L-Pro.A B94k67k ut 65k43k neu'-30k -20.lk •-14.4kFig. 1: SDS-PAGE of the purified Pz-peptidase.Lane A : Marker proteinsLane B : Purified Pz-peptidasetl} zkta,:!l-, 18(2) 1992 141Table 1: Effects of group specific reagents and metal ions on Pz-peptidase activityReagents Conc. (mM) Activity(%)ControlPhenylmethylsulfonyl fluoridep-ChloromercuribenzoateTosyl-L-lysine chloromethyl ketoneTosyl-L-phenylalanine chloromethyl ketoneEDTAMercaptoethanolDithiothreitolCaC12MgC12 ------• O.2 O.2 O.2 O.2 2.0 2.0 2.0 2.0 2.0100 87 o 83 79 8 82108101 94A) 2 34(B)3{c)134oo 20 4o'4o 20      oRetention time (min.)20(D)40 O 20 40Fig. 2: Reversed-phase chromatography (HPLC) of the hydrolytic products by Pz-peptidase ofZ-Gly-L-Pro-L-Leu-Gly-L-Pro.Cal Standard peptides,(B) Z-Gly-L-Pro-L-Leu-Gly-L-Pro with Pz-peptidase (Oh)(C) Z-Gly-L-Pro-L-Leu Gly-L-Pro with Pz-peptidase (1 h)a])) Z-Gly-L-Pro-L-Leu-Gly-L-Pro with Pz-peptidase (4 h)1 : Gly-L-Pro2 : Z-Gly-L-Pro-L-Leu-Gly3 : Z-Gly-L-Pro-L-Leu-Gly-L-Pro4 : Z-Gly-L-Pro-L-LeuDiscussion    In the present investigation, we describe the purification and the characterization of Pz-peptidase of a strain of T. denticola. isolated from an adult periodontal Iesion. The molecular weightwas 65kDa and was characterized as a thiolmetallo-enzyme, judged from inhibition tests by variousgroup specific reagents. The purified Pz-peptidase may cleave L-Leu-Gly bond as other microbialcollagenase'•8,i`). This was confirmed by the analyses of hydrolyzed products of the other peptide,Z-Gly-L-Pro-L-Leu-Gly-L-Pro. The enzyme splits the peptide bonds of amino group side of Gly-L-Pro.    Up to date, some enzymes of treponemes which are thought to be associated with destructionof gingival connective tissue have been purified and characterized ; Ohta et al.'3} purified an enzyme142 Shibata, et al.: Peptidase of T. denticolahydrolyzing a trypsin synthetic substrates from T. denticola. This enzyme was a serine protease andinhibited by leupeptin. The enzyme, however, could not hydrolyze proteins such as casein, albumin,hemoglobin, and gelatin.   Chymotrypsinlike enzyme was isolated from cell extrat of T. denticola This enzyme was activenot only on chymotrypsin synthetic substrates but on natural proteins including transferrin, fi-brinogen, Ig A, Ig G, gelatin, and albumini6). Followingly, location of this enzyme within the cell wasstudied by means of imrnunohistochemical technique, which revealed the enzyme attached to theoutside the cell envelope2). Furthermore, this enzyme was shown to degrade basement membraneType IV collagen, indicating chymotrypsinlike protease of T. denticola may play an important rolein the invasion and destruction of basement membrane2).    Makinen et al. reported that TrePonema vincentii and unidentified species of Treponemaproduced collagenase which were found to cleave collagens as well as synthetic substrates ofcollagens7•8). However, Pz-peptidase in the present study did not show the hydrolytic activity towardcollagens, but this enzyme may contribute to destruction of periodontal tissue through degradationof collagen with the cooperation of collagenase in gingival fluid secreted from tissue or producedby other bacterial species.Referecnes1 ) Armitage, G., Dickson, W. R, Jenderseck, R. S., Levine, S. M. and Chambers D. W. (1982) : Relationship   between the percentage of subgingival spirochetes and the severity of periodontal disease. J. Per-   iodontol. 53 : 550-556.2 ) Grenier, D,. Uitto, V-J. and McBride, B. C. (1990) Cellular location of a Troponema denticola chymotryp-   sin protease and importance of the protease in migration through the basement membrane. Infect.   Immun. 58 : 347-3513 ) Hinrichs, J. E., Wolff, L. F., Philstrom, B. L., Schaffer, E. M., Lijemark, W. F. and C. L Bandt. (1985)   Effects of scaling and root planning on subgingival microbial proportions standardized in terms of their   naturally occurring distribution. J. Periodontol. 56 : 187-194.4 ) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage   T4. Nature (London) 227 : 680-686.5 ) Listgarten, M. A. (1976) structure of the microbial fiora associated with periodontal health and disease   in man. A light and electron microscopic study. J. Periodontol. 47 : 1-18.6) Loesche, W. J., Syed, S. A, Schmidt, E. and Morrison, E. C. (1985) Bacterial profiles of subgingival   plaques in periodontitis. J. Periodontol. 56 : 447-456.7 ) Makinen, K. K., Syed, S. A ., Loesche, W. J . and Makinen, P-L. (1988) Proteolytic profile of Treponema   vincentii ATCC 35580 with special reference to collagenolytic and arginine aminopeptidase activity.   0ral Microbiol. Imrnunol. 3 : 121-1288 ) Makinen, K. K., Syed, S. A., Salvador S. L. and Makinen, P-L. (1990) Hydrolysis of the Leu-Gly bond   of phenylazobenzyloxy carbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (a substrate of microbial collagenase)   by treponemes isolated from the subgingival plaque of periodontitis patients. Current Microbiol. 20 :   69-74.9) Moore, W. E. C., Holdeman, L. V., Cato, E. P., Smibert, R. M., Burneister, J. A., Palcanis, K. G. and   Ranney, R. (1985) Coniparative bacteriology of juvenile periodontitis. Infect. Immun. 48 : 507-519.10) Moore, S. and Stein, W. H. (1948) Photometric ninhyidrin method for use in the chromatography of   amino acids. J. Biol. Chem. 176 : 367.-388.11)Nakamura, T., Shimura, R., Nagasaki, M., Shibata, Y. and Fujimura, S. (1990) Isolation of oral   spirochetes and their biological properties and enzyme production (in Japanese). Matsumoto Shigaku   16 : 36212) Fiehn, N-E. (1986) Enzyme activities from eight small-sized oral spirochetes. Scand. J. Dent. Res. 94 :   132-140.M} zis ts nti! ;4 18(2) 1992 14313) Ohta, K., Makinen, K. K. and Loesche, W. J. (1986) Purification and characterization of an enzyme  produced by Troponema denticola capable of hydrolyzing synthetic trypsin substrates. Infect. Immun.  53 : 213-220.14) Stephen, K. G. and Fung, S. S. (1984) PZ-peptidase activities of some black-pigmented llacteroides  species. Can. J Microbiol. 30 : 1305-1308.15) Uitto, V-J., Chan, E. C. S. and Quee, T. C. (1986) Initial characterization of neutral proteinases from oral  spirochetes. J. Periodontal Res. 21 : 95-100.16) Uitto, V-J., Grenier, D., Chan, E. C. S. and McBride, B. C. (1988) Isolation of chymotrypsinlike enzyme  from Troponema denticola. Infect. Immun. 56 : 2717-2722.17) Wunsch, E. and Heidrich, H. G. (1963) Quantitative Bestimmung der Kollagenase. Z. Physiol. Chem.  333 : 149-15LV'st : Treponema denticola OkÅ}t6 = 7Jf'V--'t!fgR"neeskdingNttrijl;,lt.taN$,fi , etNemik, tpN rt (zaZscdeJJk( • pMrwpa) meex tf p A- pt (Treponema denticola) cD matzmb HI bj ve X O = i tr' v' - -tz'oantgE ft"nzKfiee-g`6 st* tr, l t '/ zxk ge 1 p ? F pt 7 7 i -, tr' iv wame st zK reaa p ? F pt i 7 4 - ec at o "C xx pa L te.xxrvptket pH8.0v(}'fi"s(JJS(ts{gligfiL, -{ 7 e p p =r - } a V -sÅq ;/ 'i' r l F, EDTA VeX D 'C 5fi$62zte.e cDbl*ot PZ--Åq 7"f F", Z-Gly-L-Leu-Gly-L-Pro vceFre L, paZgKJ b Gly-L-Pro ftveX'r6 c Eh:ib hso te. L thiL, u 7 - tr' y ftatr di eE et Jzl: pt* e= x D vc 5i)• ne $ 2•t tg h,o k.

Purification and Characterization of an Enzyme Produced by Treponema denticola Hydrolyzing Synthetic Collagenase Substrates

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Treponema denticolaの産生するコラゲナーゼ基質分解酵素の精製と性状

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