Molecular analysis of the inhibitory effects of oolong tea polyphenols on glucan-binding domain of recombinant glucosyltransferases from Streptococcus mutans MT8148

FEMS Microbiology Letters 228 (2003) 73^80

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Molecular analysis of the inhibitory e¡ects of oolong tea polyphenols on glucan-binding domain of recombinant glucosyltransferases from Streptococcus mutans MT8148 M. Matsumoto a , S. Hamada b , T. Ooshima b

a;

a Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamada-oka, Suita, Osaka 565-0871, Japan Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, 1-8 Yamada-oka, Suita, Osaka 565-0871, Japan

Received 16 July 2003; received in revised form 17 September 2003; accepted 17 September 2003 First published online 8 October 2003

Abstract An oolong tea polyphenol (OTF6) has been shown to possess a strong anti-glucosyltransferase (GTF) activity and inhibit experimental dental caries in rats infected with mutans streptococci. The effects of OTF6 on the functional domains of GTFs of Streptococcus mutans, an N-terminal catalytic domain (CAT), and a C-terminal glucan-binding domain (GBD), were examined. The maximum velocity of glucan synthesis by recombinant GTFB (rGTFB) and GTFD (rGTFD) became significantly slower in the presence of OTF6, however, Km values remained stable when compared in their absence. These results suggest that OTF6 reduces glucan synthesis by non-competitively inhibiting the GBD of S. mutans GTFB and GTFD. Further, the recombinant proteins of CAT (rCAT) and GBD (rGBD) were expressed using Escherichia coli, and purified by affinity column chromatography. rGBD but not rCAT was found to possess dextran-binding activity, which was shown to be inhibited by OTF6. These results indicate that OTF6, a polymeric polyphenol specific for oolong tea is able to reduce glucan synthesis by inhibiting the GBD of S. mutans GTFB. 4 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Dental caries ; Glucan-binding domain ; Streptococcus mutans; Oolong tea; Polyphenol ; Recombinant glucosyltransferase

1. Introduction Streptococcus mutans is known to be a primary causative agent of dental caries in humans. The organism synthesizes adhesive glucan from sucrose by the action of glucosyltransferases (GTFs), and glucans mediate the ¢rm adherence of its cells to tooth surfaces [1]. S. mutans produces three types of GTFs (GTFB, GTFC, and GTFD), whose cooperative action is essential for this cellular adherence [2]. Each enzyme is composed of two functional domains, an amino-terminal catalytic domain (CAT), which binds and hydrolyzes the substrate of sucrose, and a carboxyl-terminal glucan-binding domain (GBD), which functions as an acceptor for binding glucan

* Corresponding author. Tel. : +81 (6) 6879 2961; Fax : +81 (6) 6879 2965. E-mail address : [email protected] (T. Ooshima).

and also plays an important role in determining the nature of the glucan synthesized by a GTF [3^5]. GTFB and GTFC, which mainly synthesize water-insoluble glucans rich in K-1,3-glucosidic linkages, are located on the cell surface, and encoded by the gtfB and gtfC genes, respectively [6,7]. On the other hand, GTFD, which synthesizes water-soluble glucans rich in K-1,6-glucosidic linkages, is released into culture supernatant and encoded by the gtfD gene [8]. Recombinant GTF (rGTF) samples can be prepared from Escherichia coli cells, into which the recombinant plasmids carrying the gtf gene can be transformed. Using this method, GTFB can be di¡erentiated from GTFC, though native GTFB and GTFC are very similar in terms of their biological and immunochemical properties. In our previous studies, a polymeric polyphenol compound prepared from a water^ethanol extract of oolong tea leaves (OTF6) was shown to possess a strong antiGTF activity [9] and inhibit experimental dental caries in speci¢c pathogen-free rats infected with S. mutans MT8148R [10]. The purpose of the present study was to

0378-1097 / 03 / $22.00 4 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00723-7

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examine the mode of inhibition by OTF6 against GTFs of S. mutans, using rGTFs as well as the two recombinant domains in GTFB.

swine anti-rabbit immunoglobulin conjugated with alkaline phosphatase (Dako, Glostrup, Denmark). 2.4. Preparation of rGTFC

2. Materials and methods 2.1. Microorganisms and plasmids S. mutans MT8148 (serotype c) was the primary organism used in the present study. Recombinant plasmids, pSK6 carrying the gtfB gene of MT8148, pSK16 carrying the gtfC gene of MT8148 [11], and pYT104 carrying the gtfD gene of MT8148 [12], were kindly provided by Dr. S. Kawabata (Osaka University Graduate School of Dentistry, Osaka, Japan). 2.2. Oolong tea polyphenol Oolong tea leaves were pulverized and suspended in 45% (v/v) ethanol and kept at room temperature for 1 day. After ¢ltration and evaporation of the ethanol using an evaporator (RE200A, Yamato Scienti¢c Inc., Tokyo, Japan), the remaining extract was lyophilized to give a powder that contained monomeric and polymeric polyphenols, as well as ca¡eine, and other components. OTF6 used in the present experiments was prepared from this extract using successive adsorption chromatography with a Diaion HP-21 and HP-20 (Mitsubishi Chemicals Industries, Tokyo, Japan), and was composed of polymeric polyphenols speci¢c for oolong tea [9]. 2.3. Preparation of recombinant GTFB (rGTFB) and GTFD (rGTFD) E. coli XL-2 specimens harboring either recombinant plasmid pSK6 or pYT104 were cultured in Luria^Bertani (LB) broth containing ampicillin (100 Wg ml31 ) and tetracycline (7.5 Wg ml31 ) at 37‡C for 16 h. After the cells were harvested by centrifugation and suspended in 10 mM of potassium phosphate bu¡er (KPB, pH 6.5), the suspension was sonicated with an ultrasonic disrupter (UD201, Tomy Seiko, Tokyo, Japan). A supernatant of the lysate was obtained by centrifugation and then passed through an Epoxy-activated Sepharose 6B column (Amersham Biosciences, Uppsala, Sweden) with dextran. Bound rGTFB or rGTFD was collected by eluting with 0.5^4 M of guanidine hydrochloride. The rGTFs were then dialyzed with KPB (pH6.5) and stored separately as aliquots at 380‡C. Sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) and Western blot analyses were carried out to determine the purity of the rGTFs and their expression of the gtf gene, according to a method previously described by Aoki et al. [6]. rGTFB and rGTFD were detected using rabbit anti-GTF antibody [13] as well as

The DNA fragment encoding gtfC from pSK16 was ampli¢ed by polymerase chain reaction (PCR) using LA Taq0 (Takara Biomedicals, Otsu, Japan). PCR primers were constructed based on a sequence that added a restriction enzyme site at the 5P and 3P ends (NcoI and BglII, respectively). The presence of the insert was con¢rmed by NcoI and BamHI digestion, followed by agarose gel electrophoresis and puri¢cation with a QIAEX gel extraction kit (Qiagen, Chatsworth, CA, USA). The puri¢ed fragments were subcloned into expression vector pET-32a (+) (Novagen, Darmstadt, Germany), and the resultant plasmid, named pMM301, was transformed into E. coli BL21 (DE3). E. coli BL21 (DE3) carrying pMM301 was grown in LB broth at 37‡C to the mid-exponential phase. Isopropylthio-L-D-galactoside (IPTG, Wako Pure Chemical Industries, Osaka, Japan) was then added to make a ¢nal concentration of 1.0 mM, and the cultures were incubated for an additional 3 h to induce protein synthesis, after which the cells were harvested by centrifugation. Pelleted cells were suspended in 20 mM of imidazole bu¡er (10 mM Na2 HPO4 , 10 mM NaH2 PO4 , 0.5 M NaCl, 20 mM imidazole, pH 7.4) and sonicated on ice. Insoluble recombinant proteins were collected by centrifugation and extracted with 20 mM of imidazole bu¡er containing 2% Tween 20 at 37‡C for 1 h, after which soluble protein was obtained by additional centrifugation. Insoluble protein was further extracted with 20 mM of imidazole bu¡er containing 8 M of urea at 37‡C for 1 h. The supernatant was then obtained by centrifugation and urea removed by dialysis. Supernatants obtained by Tween 20 alone and a combination of Tween 20 and urea were mixed, then applied to a HiTrap chelating a⁄nity chromatography column (Pharmacia), and eluted with 0.5 M of imidazole bu¡er (10 mM Na2 HPO4 , 10 mM NaH2 PO4 , 0.5 M NaCl, 0.5 M imidazole, pH 7.4). The rGTFC was dialyzed with KPB (pH 6.5) and stored as an aliquot at 380‡C. 2.5. Genetic construction to prepare recombinant GBD (rGBD) and recombinant CAT (rCAT) DNA fragments encoding CAT and GBD in gtfB from pSK6 were ampli¢ed by PCR using AmpliTaq Gold1 (Applied Biosystems, Foster City, CA, USA) (Fig. 1). The PCR primers were chosen according to the published nucleotide sequence ([14], GenBank accession number M17361), and appropriate restriction sites were introduced for subcloning (CAT : EcoRI at the 5P end of the upper primer and XhoI at the 5P end of the lower primer, GBD : BamHI at the 5P end of the upper primer and EcoRI at the 5P end of the lower primer). CAT and GBD fragments,

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Fig. 1. Genetic construction of two plasmids carrying functional domains of S. mutans GTFB. pSK6: gtfB-containing plasmid. CAT: catalytic domain of GTFB, which binds and hydrolyzes the substrate of sucrose. GBD: glucan-binding domain, which functions as an acceptor for glucan binding.

representing 2293^2989 bp and 4645^5526 bp, respectively, were cloned into plasmid pGEM0 -T (Promega, Madison, WI, USA) and then transformed into E. coli JM109. Transformed colonies were screened by blue^white selection on LB agar plates containing IPTG, 5-bromo-4chloro-3-indolyl-L-D-galactoside, and 50 Wg ml31 of ampicillin. Plasmid preparations were made from selected white colonies using a Wizard Miniprep DNA Puri¢cation System (Promega), and the presence of the insert was con¢rmed by EcoRI and XhoI, as well as BamHI and EcoRI digestions, followed by agarose gel electrophoresis and puri¢cation with a QIAEX gel extraction kit. The puri¢ed CAT fragment was subcloned into the expression vector pGEX6p-1 (Amersham Biosciences) and the resulting plasmid was named pMMN7, while the puri¢ed GBD fragment was subcloned into pET42a (+) (Novagen) and the resulting plasmid was named pMMN10. Both pGEX6p-1 and pET42a (+) were the vectors used for expression of the glutathione S-transferase (GST) fusion protein. pMMN7 and pMMN10 were transformed into E. coli BL21(DE3), and transformed colonies were selected on LB agar plates containing 50 Wg ml31 of ampicillin or 30 Wg ml31 of kanamycin. 2.6. Expression and puri¢cation of rCAT and rGBD proteins of GTFB E. coli BL21(DE3) organisms carrying pMMN7 and

pMMN10 were grown in LB broth (800 ml) to the midexponential phase at 30‡C. IPTG was added to produce a ¢nal concentration of 1.0 mM, and the cultures were incubated for an additional 3 h to induce GST fusion protein synthesis, after which the cells were harvested by centrifugation. The pelleted cells were suspended in PBS bu¡er (10 mM Na2 HPO4 , 1.8 mM KH2 PO4 , 140 mM NaCl, 2.7 mM KCl, pH 7.3) and sonicated on ice. Soluble proteins were obtained by centrifugation, applied to a glutathione Sepharose 4B column (Amersham Biosciences) and eluted with 10 mM of glutathione bu¡er (50 mM Tris^HCl, 10 mM glutathione, pH 8.0). The puri¢ed proteins were added to milliQ and stored as aliquots at 380‡C. 2.7. E¡ect of OTF6 on glucan synthesis by rGTF Enzyme activity was determined using [14 C-glucose] sucrose as described previously [15]. One unit (U) of rGTF activity was de¢ned as the amount of enzyme required to incorporate 1.0 mM of glucose residue from a sucrose molecule into glucan in 1 min [5]. To examine the e¡ect of OTF6 on glucan synthesis by rGTFs, a reaction mixture containing rGTFs (20 mU), 10 mM of [14 C-glucose] sucrose (1.85 GBq/M), and OTF6 (0^1 mg ml31 ) was incubated at 37‡C for 1 h. The reaction mixture was spotted on small square ¢lter papers, washed three times by stirring with 100% methanol to remove

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non-polymerized sugars, and dried in air. Radioactivity remaining on the ¢lter paper was quanti¢ed with a liquid scintillation counter. Km values were calculated at concentrations of 0 to 300 mM of sucrose in the presence of OTF6 (1.0 mg ml31 : ¢nal concentration) after incubation for 2 h. At the same time, the ratios of insoluble glucan and soluble glucan synthesized by rGTFs were examined using [14 C-glucose] sucrose. Insoluble glucan was collected as a precipitate by centrifugation, and soluble glucan was precipitated by adding 100% methanol to the supernatant to produce a ¢nal concentration of 75% methanol. Pellets were suspended with scintillation £uid and radioactivity was measured using a scintillation counter. The Km value for the substrate, sucrose, was determined based on a Lineweaver^Burk plot. 2.8. Dextran-binding assay A dextran-binding assay was done by the method of Lis et al. [16]. Microtiter plates were ¢lled with rGTFs and incubated at 4‡C for 16 h. The wells were washed three times with distilled water (DW), then blocking bu¡er (0.5% bovine serum albumin in 10 mM sodium acetate bu¡er, pH 6.0) was added, and the plates were incubated at 37‡C for 1 to 2 h. After washing with DW, the wells were ¢lled with OTF6 (0^0.1 mg ml31 ) for 10 min at room temperature. After washing again with DW, the wells were ¢lled with blocking bu¡er supplemented with 0.5 Wg ml31 of biotin^dextran (molecular mass 70 000, Sigma, St. Louis, MO, USA) for 10 min at room temperature. After another washing with DW, streptoavidin^horseradish peroxidase conjugate (Gibco-BRL, Gaithesburg, MD, USA) was added to all of the wells and the mixtures were incubated for 5 min at room temperature. After a ¢nal washing with DW, a color detection solution was applied as recommended by the supplier, and the samples were incubated for various time periods depending on the glucanbinding abilities of the samples. The subsequent A490 results were determined using a microplate reader. All assays were carried out three times, with mean and standard deviation results presented.

Fig. 3. Changes in the quantity of glucan produced by rGTFs. GTF activity was measured with [14 C-glucose] sucrose. The OTF6 concentration in all displayed data was 1.0 mg ml31 . Data are given in counts per minute. GTFs and sucrose were reacted without (a) and with (b) OTF6.

2.9. Statistical analysis

Fig. 2. Western blotting of rGTF preparations. Western blot analysis of SDS^polyacrylamide gels was performed using anti-CA-GTF serum.

Intergroup di¡erences of various factors were estimated by a statistical analysis using one-way analysis of variance, followed by Fisher’s PLSD post-hoc test (StatView ; SAS

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institute Inc., Cary, NC, USA). A P-value of 6 0.05 was considered to be signi¢cant.

3. Results 3.1. Isolation and glucan-synthesizing activity of rGTFs GTF activity in the supernatant from a lysate of E. coli cells harboring recombinant plasmid pSK6 containing gtfB was equal to that of 320 mU mg31 of protein, whereas the a⁄nity chromatography procedure resulted in signi¢cantly fewer bands on SDS^PAGE pro¢les. GTFB was detected by Western blot analysis and found to be equivalent to 503 mU mg31 of protein (Fig. 2a). A supernatant from the lysate of E. coli cells harboring recombinant plasmid pYT104 containing gtfD was also prepared using a⁄nity chromatography, and a single band was found in the SDS^PAGE pro¢le. Further, GTFD was detected by Western blot analysis (Fig. 2c) and found to be in a puri¢ed state from 164 to 208 mU mg31 of protein. The active rGTFC of a sonic extract of E. coli cells harboring recombinant plasmid pSK16 containing gtfC was not recovered by a⁄nity chromatography on Sepharose 6B. Therefore, rGTFC was expressed with plasmid pMM301 carrying the gtfC gene, solubilized from insoluble proteins of sonicated cells with Tween 20 and urea, and isolated using HiTrap chelating a⁄nity chromatography. GTFC was detected by Western blot analysis (Fig. 2b) and found to be in a puri¢ed state from 6.3 to 22.3 mU mg31 of protein. 3.2. Inhibitory e¡ect on glucan synthesis by rGTF OTF6 reduced glucan synthesis by the rGTFs, with 50% inhibition found in rGTFB and rGTFD at concentrations of 60 Wg ml31 and 100 Wg ml31 , respectively. The inhibitory activity against rGTFC glucan synthesis was not as strong as against that of rGTFB or rGTFD, with a 50% inhibition found at a concentration of 850 Wg ml31 . The ratio of insoluble glucan to soluble glucan was not di¡erent with or without OTF6 in rGTFB and rGTFD (GTFB ; insoluble glucan:soluble glucan = 2:1, GTFD ; nearly all glucans were soluble.), though glucan synthesis was signi¢cantly inhibited by OTF6 at a concentration of 1 mg ml31 . In rGTFC, the ratio of soluble glucan was

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increased to 45% in the presence of OTF6, from 30% in its absence. Kinetic analyses showed that the quantity of glucan produced by rGTFs was reduced by the addition of OTF6 (Fig. 3). Further, the glucan-synthesizing activities of rGTFB and rGTFD reached a plateau in the presence of 1 mg ml31 of OTF6 when the sucrose concentration was 12.5 mM (Fig. 3a,c). In a Lineweaver^Burk plot of rGTFB, the Km value was 6.89 mM and Vmax was 11.89 nmol min31 (Table 1, Fig. 4a). In the presence of OTF6, the Km value changed slightly to 6.84 mM, while Vmax was reduced to 5.02 nmol min31 . Similar results were obtained with rGTFD, as the Km value in the absence of OTF6 was nearly the same as that in its presence, while Vmax was reduced from 13.13 nmol min31 in the absence of OTF6 to 5.97 nmol min31 in its presence (Table 1, Fig. 4c). The Km values of rGTFB and rGTFD were not signi¢cantly di¡erent in the presence and absence of OTF6, however, their Vmax values were signi¢cantly di¡erent (P 6 0.001, Fisher’s PLSD analysis). Further, both the Km and Vmax values of rGTFC were reduced in the presence of OTF6, though not signi¢cantly (Table 1, Fig. 4b). 3.3. Puri¢cation of rCAT and rGBD in GTFB To con¢rm the recombinant domains in GTFB, an af¢nity chromatography procedure was utilized, which resulted in a single band on the SDS^PAGE pro¢le (Fig. 5a). CAT and GBD were detected by Western blot analysis (Fig. 5b). The molecular sizes of rCAT and rGBD, calculated from their molecular markers, were 51 and 60 kDa, respectively, and were consistent with the predicted sizes of the CAT-GST and GBD-GST fusion proteins (49 and 57 kDa, respectively). 3.4. Inhibition of dextran binding Dextran binding to each rGTF was reduced by OTF6 in a concentration-dependent manner (Fig. 6). For rGTFB, signi¢cant reductions by OTF6 were found at concentrations greater than 5 Wg ml31 , with 70% inhibition found at a concentration of 12.5 Wg ml31 and 85% at 50 Wg ml31 . As for rGTFD, signi¢cant reductions were found at concentrations greater than 12.5 Wg ml31 , with an inhibition of 85% at 100 Wg ml31 . Signi¢cant reductions in rGTFC

Table 1 Kinetic analysis of the inhibitory e¡ects of OTF6 on rGTFB, rGTFC and rGTFD rGTF

rGTFB rGTFC rGTFD a b

With OTF6a

Without OTF6 Km

Vmax (nmol min

6.89 V 0.23 19.06 V 1.53 5.60 V 0.17

11.89 V 0.81 16.90 V 3.80 13.13 V 0.84

31

)

Km

Vmax (nmol min31 )

6.48 V 0.42 16.94 V 2.10 5.62 V 0.23

5.02 V 0.57b 16.17 V 2.19 5.97 V 0.27b

OTF6 concentration : 1.0 mg ml31 . There were statistical di¡erences with and without OTF6 (*P 6 0.001, Fisher’s PLSD analysis).

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ductions by OTF6 in the dextran binding of rGBD were found at concentrations of more than 6 Wg ml31 (Fig. 7).

4. Discussion In the present study, kinetic analyses showed that the glucan-producing activities of rGTFB and rGTFD reached a plateau at various OTF6 concentrations when the concentration of sucrose was changed (Fig. 3). In Lineweaver^Burk plots, the Km values of rGTFB and rGTFD did not alter, however, their Vmax values were reduced by more than 50% in the presence of OTF6 (Table 1). These ¢ndings suggest that the inhibitory e¡ect of OTF6 on rGTFB and rGTFD is non-competitive, and that it operates on the GBD of these GTFs, however, not on their CAT. On the other hand, an 85% reduction in dextran binding to rGTFB and rGTFD was found at concentrations of 50 Wg ml31 and 100 Wg ml31 , respectively (Fig. 6), and a 50% inhibition of glucan synthesis by OTF6 was found at concentrations of 50 Wg ml31 with rGTFB and 100 Wg ml31 with rGTFD. Further, in the experiment using rGBD and rCAT of GTFB, OTF6 signi¢cantly inhibited glucan binding to rGBD of GTFB at the same concentration (6 Wg ml31 ) as to rGTFB. In addition, OTF6 at a concentration of 12.5 Wg ml31 reduced the rates of glucan-binding activity to 65% in rGBD of GTFB and 70% in rGTFB. These results suggest that OTF6 inhibits glucan binding to GBD of GTFB, which is associated with the reduction in glucan synthesis by GTFB. It is possible that OTF6 changes the function of the GBD of GTFB by either contacting with it or combining with it. We assayed the rate of hydrolysis by measuring the concentration of free glucose using a TC D-glucose/ D-fructose kit (Roche Biochemicals, Basel, Switzerland), and found that free glucose in the presence of OTF6 was reduced by 38% as compared to in its absence, while the quantity of glucan was reduced by 78%, indicating that

Fig. 4. Kinetic analysis of inhibitory e¡ects of OTF6 on glucan synthesis by GTF. All assays were done in triplicate, and mean values used for determination of Km values. GTFs and sucrose were reacted without (a) and with (b) OTF6.

were found at concentrations greater than 25 Wg ml31 ; however, inhibition never reached 85%, and at 1000 Wg ml31 the inhibition rate was 60%. In an examination of dextran binding to each rCAT and rGBD, rCAT did not show any dextran-binding activity, whereas that of rGBD was high. However, signi¢cant re-

Fig. 5. SDS^PAGE and Western blot analyses of rCAT and rGBD in GTFB. Western blot analysis was performed using anti-CA-GTF serum. a: Gel stained with Coomassie brilliant blue R-250. b: Western blot analysis.

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OTF6 acts strongly on the GBD of GTFB. Moreover, it is possible that OTF6 also acts on CAT in the presence of surplus OTF6. An unexpected result was the low rate of inhibition by OTF6 against glucan synthesis by rGTFC. The genetic and biological properties of GTFB and GTFC enzymes show a very high homology. Hence, our ¢nding that the reduction in dextran binding to rGTFC by OTF6 was 50% at a concentration of 25 Wg ml31 was considered to be reasonable. However, in spite of the signi¢cant inhibition in dextran-binding activity to rGTFC, glucan synthesis by rGTFC was not inhibited by OTF6 as clearly as it was by rGTFB. In the present study, the ratio of insoluble to soluble glucan in rGTFB and rGTFD was not changed in the presence of OTF6, whereas for rGTFC, the content of soluble glucan was increased to 45% in the presence of OTF6 from 30% in its absence. The GTFC enzyme was reported to increase the formation of insoluble glucan under enzyme aggregation conditions and the addition of ammonium sulfate to a GTFC enzyme solution resulted in an increased amount of insoluble glucan [7]. Polyphenols generally have the ability to bind and precipitate macromolecules such as enzymes [17], and it may be impossible for OTF6 to induce rGTFC to disaggregation. Furthermore, the ratio of insoluble glucan to soluble glucan produced by rGTFC or rGTFB is reported to change in the presence of the soluble primer glucan [6,7], as its addition shifts the product glucans to a more water-soluble form. In GTFB, OTF6 binds to the GBD of the enzyme and inhibits enzyme activity, thus, the glucan production of GTFB was completely abrogated by OTF6. In contrast, with GTFC, OTF6 may bind to the con¢ned site of the enzyme and modify enzyme activity, thereby producing greater amounts of soluble glucan.

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Fig. 7. Inhibitory e¡ect of OTF6, an oolong tea polyphenol, on dextran binding of rCAT and rGBD. Equivalent amounts of protein from each rGTF were incubated with 0.5 Wg ml31 of biotin^dextran, and binding levels were determined after incubation of the color reagent for 15 min. Statistical analyses were carried out in the absence and presence of OTF6 (***P 6 0.001).

The C-terminal GBD of GTF in Streptococcus sobrinus is considered to be involved in the binding of synthesized glucan polymer and presumably the chain extension of growing glucan polymers [18]. Further, this GBD plays an important role in determining the nature of the glucan product synthesized by GTFs [19]. In several studies, antibodies against peptides representing the functional domains of GTFs have been shown to inhibit glucan synthesis by GTFs, while the antibody against GBD has been found to be much more e¡ective in the inhibition of glucan synthesis than anti-CAT [20]. On the other hand, subcutaneous immunization with synthetic peptides representing components of the GBD or CAT of a GTFB equally reduced the number of smooth-surface caries in rats infected with either S. mutans or S. sobrinus [14]. These ¢ndings indicate that inhibition of GBD by OTF6 can reduce glucan synthesis, thereby diminishing the incidence of dental caries associated with mutans streptococci. In the present study, we cloned two proposed functional regions of S. mutans GTFB, CAT and GBD (Fig. 1). The CAT fusion protein did not show dextran-binding activity, whereas that of GBD showed it clearly, which was inhibited by OTF6. Our results suggest that OTF6 can reduce glucan synthesis, mainly by inhibiting the GBDs of GTFB and GTFD of S. mutans.

Acknowledgements

Fig. 6. Inhibitory e¡ect of OTF6 on dextran binding of rGTF. To compare biotin^dextran binding with OTF6, equivalent amounts of protein from each rGTF were incubated with 0.5 Wg ml31 of biotin^dextran, and binding levels were determined after incubation with the color reagent for 15 min. (b) rGTFB, (R) rGTFC, (a) rGTFD. There were statistically signi¢cant di¡erences in the absence and presence of OTF6. *P 6 0.05, **P 6 0.01, ***P 6 0.001.

This study was supported by Grants-in-Aid for Scienti¢c Research (B) 14370693 and Young Scientists (B) from Japan Society for the Promotion of Science.

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