Purification and Characterization of a Protease fromPorphyromonas gingivalis Capable of Degrading
Salt-Solubilized CollagenHAKIMUDDIN T. SOJAR,* JIN-YONG LEE, GURRINDER S. BEDI, AND ROBERT J. GENCO
Department of Oral Biology, State University ofNew York at Buffalo,3435 Main Street, Buffalo, New York 14214
Received 9 November 1992/Accepted 19 March 1993
An enzyme capable of hydrolyzing the substrate 4-phenylazobenzyloxycarbonyl-L-prolyl-leucyl-glycyl-prolyl-D-arginine (pZ-peptide), pZ-peptidase, was purified from the oral bacterium Porphyromonas gingivalis.pZ-peptidase hydrolyzed salt-solublized type I collagen from rat skin, rat plasma low-molecular-weightkininogen, and transferrin at room temperature in the presence of calcium and dithiothreitol. pZ-peptidase didnot cleave acid-soluble type I calf skin collagen, type V placental collagen, lysozyme, albumin, or humanplasma fibrinogen. Furthermore, the purified enzyme did not hydrolyze N-ct-benzoyl-DL-Arg-p-nitroanilide,Gly-Pro-p-nitroanilide, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide, N-p-tosyl-Gly-Pro-Lys-p-nitroanilide, azoalbu-min, or azocasein. Under reducing conditions, the native enzyme migrated as a single band at 120 kDa on
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. However, when heated to 100°C for 10 minin SDS under reducing conditions, the enzyme migrated as a major band at 50 kDa and a minor band at 60 kDaon SDS-polyacrylamide gel electrophoresis. Zymography using calf skin gelatin revealed the gelatin-cleavingactivity of the enzyme as evidenced by a diffuse band in the range of 120 to 300 kDa under reducing conditionsat room temperature, suggesting that this is the native form of the enzyme. However, incubation at 50°C for10 min under reducing conditions showed gelatin-cleaving activity at a distinct band of 60 kDa. A minimumtemperature of 50°C was required to dissociate the 60-kDa chain from the native complex in active form on
gelatin zymography. The ability of the enzyme to cleave other proteins, including kininogen and transferrin,suggests that it has specificity for the Pro-X-Gly sequence found in several proteins, including collagen.
Various studies suggest that Porphyromonas gingivalis isan important etiologic agent of periodontitis (22, 36, 39, 45).This pathogen produces a number of potential virulencefactors in cell-associated, as well as secretory, form (7, 8, 10,15, 29, 33, 37, 39, 44, 46). A number of proteins are
hydrolyzed by this organism, including collagen (1, 31, 43),immunoglobulin (6, 25, 32), iron-binding proteins (4), andvarious complement factors (40). Proteases produced by P.gingivalis likely play an important role in the development ofperiodontal disease, as they have the potential to destroyperiodontal tissues (18, 34, 41) and to activate or degradehost inflammatory proteins. The demonstration of collageno-lytic activity of P. gingivalis has raised questions about themechanism of this activity in disease progression. Collage-nase activity, both extracellular and cell associated, hasbeen observed in P. gingivalis strains (16, 38), and 4-phenyl-azobenzyloxycarbonyl-L-prolyl-leucyl-glycyl-prolyl-D-argi-nine (pZ-peptide)-hydrolyzing pZ-peptidase activity of someblack-pigmented bacteroids was reported by Ng and Fung(27). However, the enzyme responsible for the degradationof pZ-peptide has not been purified. Recently, Kato et al.(12) sequenced a gene from P. gingivalis which exhibitedcollagenase activity and degraded soluble and reconstitutedfibril type I collagen only and not gelatin or the substratepZ-peptide.
In this report, the isolation and purification of a proteasefrom P. gingivalis which is capable of degrading salt-solubi-lized collagen of rat skin at room temperature is discussed.The enzyme was unable to cleave acid-soluble type I colla-
* Corresponding author.
gen from calf skin or type V placental collagen. Further-more, it did not cleave lysozyme, albumin, or fibrinogen.However, its ability to cleave other proteins, including ratplasma low-molecular-weight kininogen and transferrin, sug-
gests that it has specificity for peptide bands Pro-X-Gly,which are found in several proteins, including type I colla-gen.
MATERIALS AND METHODS
Bacterial strains and growth conditions. P. gingivalis 2561(ATCC 33277) was grown in brain heart infusion broth (DifcoChemical Co.) supplemented with 5 mg of yeast extract, 5 ,ugof hemin, and 0.2 jg of menadione per ml, pH 7.4, at 37°Cfor 2 days in a Forma anaerobic chamber (85% N2, 10% H2,5% C02).Enzyme isolation. P. gingivalis cells were harvested from 2
liters of culture by centrifugation and washed three timeswith 50 mM Tris-malate buffer, pH 8.0. The washed cellswere stirred with 1% Triton X-100 at 4°C for 30 min tosolubilize cell surface proteins. The cells were centrifuged at12,000 x g in a Sorvall centrifuge for 1 h, and the superna-tant was collected. The supernatant was made 20, 40, 60, or
80% saturated with ammonium sulfate and stirred overnightat 4°C. The mixture was centrifuged at 12,000 x g, and theresulting pellets and supernatants were collected. The pelletswere dissolved in a small volume of Tris-malate buffer. Thedissolved pellets, as well as the supernatants, were dialyzedagainst three 2-liter changes of Tris-malate buffer for 24 hand lyophilized.
pZ-peptide hydrolysis. The dialyzed materials were
checked for pZ-peptidase activity by using pZ-peptide (Sig-
ma Chemical Company) as the substrate. The method de-scribed by Morales et al. (24) was used to examine pZ-peptide hydrolysis. The pZ-peptide substrate was dissolvedat 5 mg/ml in 0.1 M Tris-malate buffer containing 10 mMCaCl2 and 10 mM NaCl. One hundred micrograms of pZ-peptide was used to assay the hydrolytic activity of 100 ,ug ofeach fraction by incubation at 37°C for 1 h. Aliquots of eachsample were boiled at 100°C and used as negative controls.The reaction was terminated by adding 1.0 ml of 5% citricacid. The hydrolysis product pZ-Pro-Leu was extracted in 2ml of ethyl acetate by vortexing for 15 s. The ethyl acetatelayer was transferred to tubes containing 0.3 g of anhydrousNa2SO4, and the A320 of the resulting extracts was measuredin a 2000 Spectronic Spectrophotometer (Bausch & Lomb,Inc.). The enzyme was also assayed in the presence andabsence of 5 mM dithiothreitol (DTI).
Analytical procedures. Protein was quantitated by theBradford method (3) with bovine serum albumin as thestandard. Samples for amino acid analysis were run on asodium dodecyl sulfate (SDS)-10% polyacrylamide gel andWestern (immunotransferred) transferred onto a ProBlottmembrane (Applied Biosystems) with CAPS buffer (20).After the membrane was stained with 0.1% Coomassiebrilliant blue R-250, the stained bands of 60 and 50 kDa werecut out, destained, washed extensively with distilled water,and hydrolyzed in 6 N HCI at 110°C for 22 h in evacuated,sealed tubes. The samples were dried in vacuo, and thehydrolysates were analyzed with a Beckman 121 MB aminoacid analyzer.
Purification of pZ-peptidase from the ammonium sulfateprecipitate. The supernatant obtained after 40% ammoniumsulfate precipitation, which showed the highest pZ-peptidaseactivity, was further concentrated by using a Centricon-30microconcentrator (Amicon Inc.). pZ-peptidase activity wasassayed in the concentrated fraction containing componentsof 30 kDa and higher, as well as in the filtered fractioncontaining components of lower than 30 kDa.
Sephadex G-200 gel filtration. The higher-molecular-massfraction (.30 kDa) was applied to a Sephadex G-200 (Phar-macia Chemicals) column (1 by 30 cm) equilibrated with 50mM Tris-malate buffer, pH 8.0. The column was eluted withthe same buffer, and 1-ml fractions were collected. The A280of each fraction was measured, and its pZ-peptidase activitywas checked by using 50 ,ug of pZ-peptide.
Sephadex G-100 gel filtration. The fractions from theSephadex G-200 column which showed pZ-peptidase activ-ity were pooled and incubated for 10 min at 50°C in thepresence of 10 mM DTT and 1% SDS. They were thenloaded onto a Sephadex G-100 (Pharmacia Chemicals) col-umn (1 by 30 cm) and eluted with Tris-malate buffer, and1-ml fractions were collected. The fractions containing pZ-peptidase activity were pooled.SDS-PAGE. SDS-polyacrylamide gel electrophoresis
(PAGE) was performed in a mini-tall gel system (HofferScientific Co.) with a 1.5-mm-thick 10% polyacrylamide gelas described by Laemmli (13). Samples were prepared in thepresence of 5% P-mercaptoethanol and 1% SDS by boiling at100°C for 10 min. The gels were stained with Coomassie blue(30) or silver (23).Zymography. Since most microbial collagenases also
cleave gelatin effectively, the gelatin-cleaving activity of thepurified enzyme was checked by zymography as describedby Heussen and Dowdle (11). An SDS-10% polyacrylamidegel was copolymerized with 200 ,ug of calf skin gelatin(Sigma Chemical Co.) or human plasma fibrinogen (SigmaChemical Co.) per ml. The purified enzyme, incubated at
room temperature or 50°C with the reducing sample buffer,was electrophoresed. After electrophoresis, the gel waswashed in 100 mM Tris-Cl, pH 8.0, containing 2% TritonX-100 for 30 min and rinsed twice with the same bufferwithout Triton X-100. The gel was then transferred to adevelopment buffer containing 100mM Tris-Cl, 5 mM CaCl2,and 10 mM DTT buffered at pH 8.0 to activate the enzyme.The gel was incubated at 37°C for 2 h and stained with 0.1%Coomassie blue in 30% methanol and 7% acetic acid tovisualize lytic bands.
Amino-terminal amino acid sequence. The chains of theisolated protease were separated by preparative SDS-PAGEon the minigel system. The proteins were transferred ontoProBlott membranes on the PolyBlot transfer system (Amer-ican Bionetics). After being stained with 0.1% Coomassiebrilliant blue for 2 min, the membranes were destained. Themembranes were washed twice with high-performance liquidchromatography grade distilled water. The respective bandswere excised with a clean razor blade, dried, and thensubjected to automated stepwise sequencing on an AppliedBiosystems 477A gas phase sequencer with an on-line 120Aphenylthiohydantoin analyzer.
Hydrolysis of synthetic chromogenic substrates. Proteaseactivity was assayed by using the synthetic chromogenicsubstrates benzoyl-L-Arg-p-nitroanilide (5), Gly-Pro-p-ni-troanilide (26), N-p-tosyl-Gly-Pro-Arg-p-nitroanilide, andN-p-tosyl-Gly-Pro-Lys-p-nitroanilide (19). The reaction mix-ture consisted of 2.5 ml of 1 mM substrate in 50 mMTris-malate buffer, pH 7.5, 0.4 ml of 50 mM Tris-malatebuffer, and 0.1 ml of the enzyme, with or without 10 mMDTT. After incubation at 37°C for 1 h, 0.5 ml of 5 N aceticacid was added to stop the reaction. The p-nitroanilinereleased was determined by measuring A410.
Hydrolysis of azoalbumin and azocasein. Proteolytic activ-ity against azoalbumin and azocasein was determined by themethod of Leighton et al. (17). The reaction mixture, con-taining 0.2 ml of 5% azocasein or azoalbumin dissolved in 50mM Tris-malate buffer, pH 7.5; 0.7 ml of the buffer; and 0.1ml of the enzyme with or without 10 mM DTT, was incu-bated at 37°C for 1 h. The reaction was stopped by adding 1ml of 10% trichloroacetic acid, and the resulting precipitatewas removed by centrifugation. A 0.2-ml volume of 1.8 NNaOH was added to 0.8 ml of the supernatant solutioncontaining the diazotized-trichloroacetic acid-soluble pep-tides. A420 was measured.
Cleavage of collagens, transferrin, and rat plasma low-molecular-weight kininogen. Purified type I salt-soluble ratskin collagen was obtained from Moon-II Cho (State Univer-sity of New York at Buffalo). Rat plasma low-molecular-weight kininogen was prepared in our laboratory. Bovineapotransferrin and transferrin were purchased from Boehr-inger Mannheim. Acid-soluble type I collagens from calf skinand human placenta, acid-soluble type III collagen fromhuman placenta, acid-soluble type IV collagen from humanplacenta, acid-soluble type V collagen from human placenta,lysozyme, and chicken albumin were purchased from SigmaChemical Company. Stock solutions of these collagens weresolubilized in 10 mM HCl and mixed with Tris-HCl buffer toyield final concentrations of 100 mM Tris-HCl, pH 7.5; 0.2 MNaCl; and 5 mM CaCl2. The collagen solutions were incu-bated with 5 to 10 ,Ig of purified pZ-peptidase at various timeintervals at room temperature and at 37°C in the presence of5 mM DTT. Lysozyme was incubated as a control to checkfor nonspecific protease activity of the enzyme. The reactionwas terminated by boiling the reaction mixture at 100°C for10 min in the presence of 2x sample buffer containing 2%
a The supematant of the Triton X-100 extract from P. gingivalis 2561 wasmade 20, 40, 60, and 80% saturated with ammonium sulfate. After centrifu-gation, the resulting pellets and supernatants were dialyzed and lyophilized.These materials were checked for pZ-peptidase activity by using pZ-peptideas the substrate. For the assays, 100 jig of pZ-peptide and 100 jig of eachlyophilized material were incubated at 37°C for 1 h. The boiled materials andbuffer were used as negative controls. After incubation, the product wasextracted with ethyl acetate and mixed with Na2SO4 and the A320 of themixture was measured.
SDS and 10% 3-mercaptoethanol. After termination of thereaction, the samples were run on SDS-10% polyacrylamidegels and the gels were stained with Coomassie blue.
Hemagglutination. Since P. gingivalis possesses stronghemagglutinating activity (28), the purified protein wastested for hemagglutination by the method described earlierby Slots and Genco (35). Hemagglutinating activity wasdetermined in round-bottom microtiter plates. The proteinwas diluted serially in phosphate-buffered saline, pH 7.2,and mixed with an equal volume of 2% washed sheeperythrocytes. The plates were gently shaken at room tem-perature for 30 min, left overnight at 4°C, and read on thebasis of the pattern of the settled erythrocytes.
RESULTS
Enzyme isolation. To isolate collagenase, the cells wereextracted with 1% Triton X-100, a nonionic detergent. Thesolubilized proteins were further fractionated at variousammonium sulfate concentrations. A synthetic pZ-peptidedeveloped to study clostridial collagenase was used to iden-tify collagenase-active fractions. The supernatant obtainedafter 40% ammonium sulfate precipitation of the TritonX-100 extract from P. gingivalis 2561 showed the highestpZ-peptidase activity (Table 1). The degradation was notenhanced by D1T.
Further fractionation of active samples. The supernatantobtained after 40% ammonium sulfate precipitation wasfurther concentrated with an Amicon Centricon-30 micro-concentrator and separated into fractions containing compo-nents with higher molecular masses (>30 kDa) and lowermolecular masses (<30 kDa). The pZ-peptidase activity wasfound mainly in the higher-molecular-mass fraction (data notshown).
Gel filtration. The higher-molecular-mass fraction wasfurther fractionated on Sephadex-G200, and the pZ-pepti-dase activities of the fractions were assayed. Most of thepZ-peptidase activity at 320 nm was present in the first halfof the major single protein peak at 280 nm (Fig. 1). Thefractions showing higher activity were pooled. The pooledfractions were dialyzed against 0.1% ammonium bicarbonateand lyophilized. The lyophilized material was run on anSDS-10% polyacrylamide gel under reducing conditionswith and without boiling and then stained with Coomassie
0.6
E
cocm
c~JC.)-C
0O
0
0.5
0.4
0.3
0.2
0.1
0.0
0.15
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0
c~Jcv)
0.13 ctsU)
00.12 c.-20
.0.11 .0
1.100 5 10 15 20
Fraction numbersFIG. 1. Sephadex G-200 gel filtration chromatography of the
higher-molecular-weight fraction. The fraction of higher-molecular-weight components separated by the microconcentrator was loadedon a Sephadex G-200 column (1 by 30 cm) and eluted with 50 mMTris-malate buffer, pH 8.0. One-milliliter fractions were collectedand monitored at 280 nm. Each fraction was assayed for pZ-peptidase activity at 320 nm.
blue. The unboiled sample showed a single band at 120 kDa;in contrast, on boiling at 100°C for 10 min there were twobands at 60 and 50 kDa (Fig. 2). The minimum temperaturefor partial dissociation of the 60-kDa chain from the nativeenzyme complex was 50°C. The enzyme activity was re-tained at this temperature. To obtain the active form ofindividual chains dissociated from the complex, first the poolof the active fractions from the Sephadex G-200 column wasfurther incubated at 50°C for 10 min in the presence of 10mM DTT and 1% SDS. The dissociated complex was thenloaded onto a Sephadex G-100 column. All of the fractionsfrom the Sephadex G-100 column had pZ-peptidase activity(Fig. 3). When these fractions were run on an SDS-10%polyacrylamide gel to check the purity, the fractions con-taining the first peak of pZ-peptidase activity showed twobands, one at 60 kDa and another at 50 kDa. The secondfractions with pZ-peptidase activity showed a single band at60 kDa on the zymogram when electrophoresed at 50°C.When the same fractions were heated to 100°C, the 60-kDachain was seen on the Coomassie blue-stained SDS-poly-acrylamide gel; however, the treatment rendered the enzymeinactive.SDS-PAGE and determination of molecular mass. On an
SDS-polyacrylamide gel under reducing conditions, the pu-rified enzyme in its native form showed a single, thin band at120 kDa. However, when heated to 100°C under reducingconditions the purified enzyme showed two nonidenticalchains of 60 and 50 kDa and a faint double in the region of 66kDa. At 50°C under reducing conditions, the 60-kDa chainstarted dissociating from the native enzyme, but for com-plete dissociation a temperature higher than 90°C was essen-tial. Under nonreducing conditions, even at 100°C, bothchains could not be dissociated. The molecular masses of thetwo chains were determined by comparing the migration ofthe native enzyme and the dissociated chains under reducingconditions with that of the standard molecular weight mark-ers on the gel.Zymography. Gelatin-cleaving activity in the native en-
zyme was observed in the range of 120 to 300 kDa on azymogram of an SDS-polyacrylamide gel (Fig. 4). When theenzyme was incubated at 50°C, most of the gelatin-cleavingactivity in the enzyme was found with the 60-kDa band on
Fraction numbers_1- FIG. 3. Sephadex G-100 gel filtration chromatography of the
purified enzyme treated at 50°C. The enzyme purified by SephadexG-200 gel filtration was treated at 50°C under reducing conditions todissociate the 60-kDa chain from the enzyme complex of 60- and50-kDa chains. The heat-treated enzyme was applied to a SephadexG-100 column (1 by 30 cm) and eluted with 50 mM Tris-malatebuffer, pH 8.0. One-milliliter fractions were collected and monitoredat 280 nm. Each fraction was assayed for pZ-peptidase activity at320 nm.
31 kDa-._
21.5 kDa-m2 3
FIG. 2. SDS-PAGE analysis of the enzyme purified by SephadexG-200 gel filtration. The purified enzyme (10 ,ug) was treated underreducing conditions with or without boiling at 100'C for 10 min,electrophoresed on an SDS-10% polyacrylamide gel, and thenstained with silver. Lane 1 contained the standard molecular weightmarkers rabbit muscle phosphorylase b (97.4 kDa), bovine serumalbumin (66.2 kDa), hen egg white ovalbumin (42.7 kDa), bovinecarbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5kDa). Lane 2 contained the purified enzyme treated under reducingconditions without boiling; note the faint band at 120 kDa. Lane 3contained the purified enzyme boiled at 100°C for 10 min underreducing conditions; note the two major bands at 60 and 50 kDa.
the zymogram. Also, the purified 60-kDa chain from theSephadex G-100 column showed the gelatin-cleaving activ-ity. However, under identical conditions human plasmafibrinogen was not hydrolyzed by the enzyme (data notshown).Amino-terminal amino acid sequence. The sequences of the
first 14 residues for both chains of the enzyme were eluci-dated by Edman degradation, and a search for proteinsequences similar to those of various known proteases was
conducted in GenBank. The protein sequences had nomarked similarity to known protease sequences. N-terminalHis-His-Ser and Met-Lys-Ser sequences were found for the60- and 50-kDa chains, respectively.
Hydrolysis of synthetic chromogenic substrates, azoalbu-min, and azocasein. No proteolytic activity against N-a-benzoyl-DL-Arg-p-nitroanilide, Gly-Pro-p-nitroanilide, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide, N-p-tosyl-Gly-Pro-Lys-p-nitroanilide, azoalbumin, or azocasein was found in thepurified enzyme preparation.
Cleavage of collagens, transferrins, and rat plasma low-molecular-weight kininogen. To evaluate the ability of thepurified pZ-peptidase to cleave various collagens, the en-zyme was incubated with salt-soluble rat skin collagen andacid-soluble collagens from calf skin and placenta at roomtemperature and at 37°C. At room temperature, the purifiedenzyme in the presence of CaCl2 and DTT selectivelycleaved the ao2 chain of collagen. When the enzyme wasincubated under these conditions for 16 h, both chains ofcollagen were cleaved (Fig. 5). No significant effect onacid-soluble collagen at room temperature was found (datanot shown), but at 37°C acid-soluble calf skin collagen wascleaved. No cleavage of type V placental acid-soluble colla-gen was found. At room temnperature, the purified enzymealso hydrolyzed apotransferrin, transferrin, and rat plasmalow-molecular-weight kininogen (Fig. 6).
Hemagglutination of the purified enzyme. The involvementof this purified enzyme in the agglutination of sheep eryth-rocytes was investigated, and none was detected.Amino acid composition. The amino acid compositions of
both the 60- and 50-kDa chains of the enzyme are shown inTable 2. The amino acid compositions shown are averages ofduplicates of the hydrolyzed sample at 22 h. Many identicalamino acids were found in both chains of the enzyme.Whether these two chains are different or autolytic productsof a single protein remains to be established.
FIG. 4. Zymography of the purified enzyme. An SDS-10% poly-
acrylamide gel was copolymerized with 200 pLg of calf skin gelatin
per ml. After electrophoresis, the enzyme in the gel was activated
for 2 h at 370C by development buffer containing 100 mM Tris-HCI,5 mM CaCl2, and 10 mM DTT, pH 8.0. The lytic bands were
visualized by staining with 0.1% Coomassie blue. Lane 1 contained
standard molecular 'Weight markers (see the legend to Fig. 2). Lane
2 contained the purified enzyme treated with reducing sample buffer
at room temperature for 10 min; note the lytic area at the top of the
gel in the 120- to 130-kDa region. Lane 3 contained the purified
enzyme heated at 500C with reducing sample buffer; note the lyticband at 60 kDa. Lane 4 contained sample buffer only.
DISCUSSION
P. gingivalis has been shown to possess several different
proteases in soluble form, as well as in cell-associated form.
These proteases differ in size, substrate specificity, and
sensitivity towards inhibitors. Various researchers (1, 12, 31,
42) have found collagenolytic activity in the outer membrane
of P. gingivalis. However, no collagenase-like protease has
been purified in native form from P. gingivalis. The present
study not only identified a unique protease molecule in the
detergent extract of P. gingivalis 2561 but also described the
method of its purific'ati'on in native form.
The purified protease has a molecular mass of 120 kDa in
its native form and showed a very thin band even after the
Ab'4i
I "_ . .sP. " 'i
sk,. * *~-i ,7 J j,,,41 2 3 4 5 6 7 8 9
FIG. 5. Cleavage of salt-soluble rat skin type I collagen by thepurified enzyme. The purified enzyme was incubated with salt-soluble rat skin type I collagen at various time intervals at roomtemperature in the presence of CaCl2 and DTT. Cleavage of thecollagen was determined by SDS-PAGE. Lane 1 contained standardmolecular weight markers (see the legend to Fig. 2). Lane 2contained, as a control, rat skin collagen incubated without thepurified enzyme. Lanes 3 to 9 contained the collagen incubated withthe enzyme for 1, 2, 3, 4, 5, 6, and 16 h, respectively.
protein concentration was increased on SDS-PAGE. It mightbe the nature of the native enzyme, or another proteincomponent(s) might be associated with the enzyme and didnot enter the gel prior to boiling of the enzyme. However,boiling of the enzyme with sample buffer showed twononidentical chains of 60 and 50 kDa along with a faintdoublet in the 66-kDa region. Most likely, the doublet at 66kDa is an artifact due to boiling of the sample buffer with2-mercaptoethanol that is stainable with silver, as it wasobserved in the lane without the enzyme also (42). Thelarger-molecular-size chain could be dissociated from theactive enzyme complex at 50°C in the presence of 5%,B-mercaptoethanol and 1% SDS, but this chain still showedgelatinase activity on zymography. Since complete dissoci-ation of both the 60- and 50-kDa chains required tempera-tures higher than 90°C under reducing conditions and theenzyme loses its activity above 50°C, the role of the 50-kDachain is not clear. The zymography profile shows that atleast some of the enzyme activity is associated with the60-kDa chain, and perhaps the 50-kDa chain helps in anchor-ing the enzyme to membranes, as seen with other enzymes.
Unlike other P. gingivalis proteases, the 120-kDa enzymewas solubilized with 1% Triton X-100, which is a gentle,nonionic detergent and solubilizes only cell surface proteins,causing very little cell breakage. As a result, the enzymeappears to be associated with the outer surface of cells. Theprecise localization of the enzyme, however, remains to bedetermined.The enzyme actively hydrolyzed a synthetic peptide con-
taining the Pro-Leu-Gly-Pro-Arg sequence but failed to
a The 60- and 50-kDa chains of the purified protease were separated bySDS-PAGE and electrophoretically transferred onto a ProBlott membrane.The respective bands of the chains were cut and hydrolyzed with 6 N HCI at110°C for 22 h.
1 2 3 4 5 6FIG. 6. Cleavage of apotransferrin, transferrin, and rat plasma
low-molecular-weight kininogen. The purified enzyme was incu-bated with various proteins in the presence of CaCI2 and DTT atroom temperature for 20 h. Lanes: 1, apotransferrin incubatedwithout the enzyme; 2, apotransferrin incubated with the enzyme; 3,transferrin incubated without the enzyme; 4, transferrin incubatedwith the enzyme; 5, rat plasma low-molecular-weight-kininogenincubated without the enzyme; 6, rat plasma low-molecular-weight-kininogen incubated with the enzyme.
hydrolyze a variety of arginine- and lysine-containing syn-thetic peptides. The enzyme did not hydrolyze azocasein orazoalbumin, suggesting that the enzyme hydrolyzes specificbonds.
Collagenase activity was demonstrated when rat skinsalt-solubilized collagen was used as a substrate at roomtemperature. The enzyme degraded the (t2 chain of salt-soluble rat skin collagen faster than the al chain. Forexample, in the first 5 h of incubation with the enzyme, thea2 chain was completely solubilized, and upon furtherincubation for 16 h at room temperature both chains ofcollagen were solubilized completely. However, denatured
collagen in the form of gelatin was also vulnerable tohydrolysis by the protease. It was surprising to find that theenzyme did not degrade acid-soluble collagen of calf skin ortype I and V collagens derived by acid solubilization anddigestion from human placenta. Possibly, the cleavage sitesin collagen are not accessible when the molecule is in itsnative form because of various degrees of cross-linking ofthe molecule in the acid-soluble state.The present study showed that the purified enzyme spe-
cifically hydrolyzed salt-solubilized collagen. Collagenaseactivity in oral bacteria was reported by Robertson et al. (31)and Mayrand and Grenier (21). Birkedal-Hansen et al. (1)reported obtaining from the cell envelope extract a 90-kDacomponent with collagenase activity, but the enzyme dis-cussed in the present study has a different molecular weight.A 58-kDa trypsinlike protease from culture supernatant ofthe W50 strain was reported by Smalley and Birss (37), butit hydrolyzed the synthetic substrate N-a-benzoyl-DL-Arg-p-nitroanilide, which the 60-kDa protease we purified did nothydrolyze. Recently, Grenier (9) isolated an 80-kDa proteasefrom an outer membrane vesicle preparation ofP. gingivalis.The enzyme was activated by reducing agents and hadtrypsin-like protease activity. In contrast, the enzyme puri-fied in this study did not show any N-a-benzoyl-DL-Arg-p-nitroanilide-hydrolytic activity. The protease activity re-ported by Grenier and McBride (10), from the outermembrane extract as well as extracellular vesicles of P.gingivalis in the absence of SDS, was found with the band ofnearly 130 kDa and in the presence of SDS at around 60 kDa.However, they identified the enzyme by zymography only.The enzyme purified in the present study may be related totheir reported protease. However, the enzyme reported byGrenier and McBride (10) had proteolytic activity against thesynthetic substrate N-a-benzoyl-DL-Arg-p-nitroanilide,whereas the enzyme purified in this study did not show anyprotease activity towards this synthetic substrate, maybebecause the enzyme was highly purified. The prtC geneisolated from P. gingivalis by Kato et al. (12), whichexpressed collagenase activity, degraded soluble and recon-
stituted fibril type I collagen only and not gelatin or thesynthetic substrate pZ-peptide. Moreover, this enzyme isnot a thiol protease, so the purified enzyme in the presentstudy appears also to be distinct from this enzyme. Bourgeauet al. (2) cloned, expressed, and sequenced a protease gene(tpr) from P. gingivalis W83 in Eschenchia coli. The clonecontained a 3.0-kb DNA fragment which revealed an openreading frame that encodes a protein of 482 amino acids witha molecular mass of 64 kDa. The molecular mass of thisprotease is close to that of our isolated 60-kDa chain, but theenzyme differs in specificity. The 64-kDa protease wasactive against the general protease substrates casein andbovine serum albumin but not against collagen. Lantz et al.(14) identified two major fibrinogen-degrading componentsof 120 and 150 kDa from P. gingivalis, but the enzymepurified in this study did not hydrolyze fibrinogen. Sorsa etal. (39) isolated a 70-kDa collagenase-like neutral proteasealong with a 35-kDa salt-activated trypsin-like protease.Since our enzyme hydrolyzed salt-solubilized collagen, theenzyme may also be a salt-activated enzyme. Whether 70-and 35-kDa neutral proteases and our 60-kDa protease areautolytic products of the 120-kDa native enzyme has yet tobe established. These possibilities are being explored in ourlaboratory.The highly purified enzyme will help us in understanding
the role of this enzyme and whether P. gingivalis collagenaseis a virulence factor and/or plays a role in the destruction ofgingival and periodontal collagen in human periodontal dis-ease. It can be postulated that the enzyme may help in thepenetration of the microorganism through connective tissueand function as a virulence factor. The presence of collage-nase on the outer surface of the bacterium may allow agreater interactive surface area for colonization and mayinactivate host molecules which bind to the surface of P.gingivalis. Further in-depth study of this enzyme is neededto determine the potential role this enzyme may have in themetabolism, nutrition, and virulence of the organism.
ACKNOWLEDGMENTS
This study was supported in part by U.S. Public Health Servicegrants DE08240, DE07034, DE04898, and DE06514.
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