The conserved 19-residue N-terminal Gtf-P1 region, extending from aa 407 to 427 in GtfB (or aa 435 to 453 of GtfC), initially was identified due to an Eco
RI polymorphism in the gtfB
genes of clinical isolates of S. mutans
). This region is highly conserved among the GTFs of several streptococci, and DNA sequence variation did not affect the amino acid sequence of S. mutans
GtfB and -C (5
); this conservation suggests some biological importance of the domain. We have investigated this possibility and demonstrated previously that MAbs which reacted with Gtf-P1 were able to inhibit the synthesis of insoluble glucans by GtfC and the attachment of S. mutans
to glass surfaces (4
). The results of the present study provide further evidence that the 19-aa Gtf-P1 region is essential for both the sucrose hydrolysis and glucan synthesis activities of the GtfB and GtfC enzymes. In Gtf-P1, substitution of Asp residues, singly or in combination, appeared to be more critical for sucrase activity than substitutions at other positions in this region. Without crystallography data, we cannot exclude the possibility that substitution of Asp residues induces conformational changes, although the substitutions of the intervening valine residues (GtfB-V412I and GtfC-V438I) did not reduce either enzymatic activity or Km
, suggesting that the Asp mutations resulted in a functional, rather than structural, change. The finding that substitutions of amino acids adjacent to the Asp residues resulted in GtfB mutants (GtfB-ms2 and GtfB-ms3) exhibiting reduced but detectable sucrase activity with unaltered Km
s supported this hypothesis.
Consistent with our findings, mutagenesis of Asp413 to Thr in GtfB reported by another laboratory also resulted in a significant reduction of enzymatic activities, and this mutated GtfB exhibited a Km
similar to that of wild-type GtfB (32
). Nevertheless, in that study, conversion of Asp411 to Thr did not inhibit GtfB’s activity, whereas we found that the activity of GtfB-D411N was approximately 20% of wild-type enzyme activity. Further investigation is needed to determine whether substitution of Asp411 with other charged or uncharged residues may have similar effect on enzymatic activities. Functional analysis conducted by Funnae and coworkers (9
) suggested that both Asp and Glu residues in the Gtf-P1 region are directly involved in enzymatic catalysis by dextransucrase of L. mesenteroides
. Nevertheless, our data suggested that the Asp residues may be more important than Glu in catalysis by the GTFs, particularly in the case of GtfC. Specifically, substitution of Glu but not the Asp residues (GtfC-E448Q) did not result in loss of sucrase activity, whereas substitution of the Asp residues (GtfC-D437N and GtfC-D439N) resulted in complete loss of activity. Moreover, in the case of GtfB, the results of substitution analysis suggested that the functional role of Asp may not be replaced by Glu: GtfB-ms3, a GtfB mutant which contains three amino acid substitutions (L408S, W426F, and L427D), had detectable sucrase activity which was abolished when Asp 411 was additionally converted to Glu (in GtfB-ms4). Both GtfB and GtfC synthesize primarily insoluble glucan in a primer-independent manner, but both soluble and insoluble glucan synthesis could be enhanced in the presence of dextran (8
). Significant increases in soluble glucan synthesis by GtfB were found earlier when corresponding residues were converted to those in GtfD, either singly or in multiple combinations (27
). However, enhanced synthesis of both soluble and insoluble glucan as we found in GtfB-V412I and GtfC-V438I due to single amino acid substitution was an unexpected finding. Moreover, the results of two different assays confirmed that these two mutant also exhibited enhanced sucrase activity.
Another interesting finding was the discrepancy between GtfB and GtfC induced by substitutions of identical residues in Gtf-P1. This was observed initially when sucrase activity was assessed with a colorimetric method and subsequently confirmed by the use of a more sensitive method. Our results indicated that two Asp residues are indispensable for the activity of GtfC, whereas residual activity of GtfB remains when only one or the other Asp is converted to Asn. Both GtfB-D411N and GtfB-D413N can hydrolyze sucrose and synthesize glucan, although the rate of hydrolysis was about one-third of the wild-type GtfB rate at various substrate concentrations (79% reduction for GtfB-D411N and 81% reduction for GtfB-V413I). Differences between GtfB and GtfC also were observed for mutations with substitutions other than of Asp residues. For example, GtfB-ms2 and GtfB-ms3 both exhibited detectable sucrase and glucan synthesis activities, whereas identical substitution of corresponding residues in GtfC resulted in inactivation of the enzymes. These results confirmed that although they are closely related, GtfB and GtfC are distinct entities structurally and/or functionally. The inconsistency found between the GtfB and GtfC mutants also indicated that identical amino acid residues may play distinct roles, structurally or functionally, in these closely related enzymes. However, the results of mutagenesis alone are not sufficient to determine whether differences also exist in the catalytic mechanisms for the same sucrase reaction. Currently, we are also investigating the effects of mutagenesis on the enzymatic activities of GtfD to determine the characteristics of this third GTF of S. mutans. We have shown previously that although MAbs directed to this 19-aa peptide reacted with the three GTFs on when tested by enzyme-linked immunosorbent assay and Western blotting, unlike the activities of GtfB and GtfC, the enzymatic activity of GtfD was not inhibited even though the sequences of this region are almost identical. It will also be interesting to investigate the effect of mutations on other GTFs, such as those of S. sobrinus and S. salivarius. The 19-aa Gtf-P1 is well conserved in all GTFs, and they all contain two Asp residues at corresponding positions.
The GTFs are considered to be significant virulence factors for bacterial adherence and initiation of dental caries on smooth surfaces (20
). Details of the enzyme structure and function are important not only in evaluating the nature of virulence but also for the development of a vaccine against dental caries. Several lines of evidence supported the view that protection induced by immunization with GTFs may involve antibody-mediated inhibition of their catalytic and/or the glucan-binding activities. We demonstrated previously that polyclonal or monoclonal antibodies which inhibited the enzymatic activities of the GTFs also could inhibit adherence in vitro. However, polyclonal antibodies which failed to inhibit the enzymatic activities of the GTFs could not interfere with the adherence of S. mutans
). More recently, we found that this 19-aa Gtf-P1 peptide is one of the major B-cell epitopes in the human humoral immune response and that the anti-peptide immunoglobulin A antibody level in saliva correlated with disease activity (6
). Furthermore, Gtf-P1 has recently been demonstrated in an animal model to be able to induce protective immunity against dental caries (30
). Results of the present mutagenesis study confirmed the functional role of this peptide region in the GTFs. Together, our results suggest that peptides derived from functionally important regions in the GTFs, such as Gtf-P1, may serve as candidate subunit vaccines against S. mutans