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Originally published In Press as doi:10.1074/jbc.M400820200 on April 1, 2004

J. Biol. Chem., Vol. 279, Issue 21, 22198-22203, May 21, 2004
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Peptide Mapping of a Novel Discontinuous Epitope of the Major Surface Adhesin from Streptococcus mutans*

Craig J. van Dolleweerd{ddagger}§, Charles G. Kelly{ddagger}, Daniel Chargelegue¶, and Julian K-C. Ma¶

From the {ddagger}Department of Oral Medicine and Pathology at Guy's, King's and St. Thomas' Hospital Medical Schools, Floor 28 Guy's Tower, Guy's Hospital, London SE1 9RT, United Kingdom and the Department of Infectious Diseases at St. George's Hospital Medical School, London SW17 ORE, United Kingdom

Received for publication, January 26, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Guy's 13 is a mouse monoclonal antibody that specifically recognizes the major cell-surface adhesion protein SA I/II of Streptococcus mutans, one of the major causative agents of dental caries. Passive immunization with Guy's 13 prevents bacterial colonization in humans. To help elucidate the mechanism of prevention of colonization conferred by this antibody, the SA I/II epitope recognized by Guy's 13 was investigated. It was previously established that the epitope is conformational, being assembled from two non-contiguous regions of SA I/II. In the current study, using recombinant fragments of SA I/II and, ultimately, synthetic peptides, the discontinuous epitope was localized to residues 170–218 and 956–969. This work describes the mapping of a novel discontinuous epitope that requires an interaction between each determinant in order for epitope assembly and recognition by antibody to take place. Guy's 13 binds to the assembled epitope but not to these individual epitope fragments. The assembled epitope results from the interaction between the individual antigenic determinants and can be formed by mixing together determinants present on separate polypeptide chains. The data are consistent with one of the epitope fragments adopting a polyproline II-like helical conformation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Gram-positive bacterium Streptococcus mutans is one of the main causative agents of dental caries (1). Adhesion of the bacterium to the tooth surface is mediated by a major bacterial cell surface protein, termed streptococcal antigen I/II (SA I/II)1 (2). This protein has been identified in a number of strains and serotypes of S. mutans and has also been called SR (3) or PAc (4).

Sequencing of the spaP gene encoding SA I/II (5) and alignment of the predicted amino acid sequence with the antigen I/II sequences from other streptococcal strains and species reveals many common features (6), and on this basis the proteins have been divided into a number of distinct regions (Fig. 1). These include an alanine-rich repeat (A) region (residues 121–447 of SA I/II from S. mutans NG5) consisting of four tandem repeats of an 82 amino acid (aa) sequence, a variable (V) region, where most of the sequence variation between antigen I/II homologues occurs and a proline-rich repeat (P) region (aa 840–983) consisting of three tandem repeats of a 39-aa sequence, together with a fourth, more degenerate repeat.



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  		FIG. 1.
Diagrammatic representation of the amino acid residues of SA I/II encoded by the recombinant polypeptides. The open reading frames expressed by each clone are aligned below the SA I/II sequence. Amino acid residues of SA I/II encoded by each clone are given in parentheses. Indicated are the signal sequence (ss) (aa 1–38), A-region, V-region (aa 448–839), P-region, C-terminal region (984–1463), and sequences associated with anchoring of the protein to the bacterial cell wall (CWA) (aa 1464–1561). The A-region (aa 121–447) consists of four tandem repeats: Ala1 (aa 121–201), Ala2 (202–283), Ala3 (284–365), and Ala4 (366–447), while the P-region (aa 840–983) comprises three tandem repeats: ProI (840–878), ProII (879–917), and ProIII (918–956) together with a fourth, more degenerate repeat, ProIV (957–983).

 
The mouse monoclonal antibody Guy's 13, which recognizes SA I/II of S. mutans and S. sobrinus (7), has been used successfully to prevent S. mutans colonization and the development of caries in non-human primates (8) and prevents bacterial colonization in human clinical trials (9, 10). Guy's 13 has been expressed, as a secretory immunoglobulin molecule in transgenic Nicotiana tabacum plants (11) and as a single chain Fv on the surface of the commensal bacterium Lactobacillus zeae (12), offering possible strategies for passive mucosal immunization against dental caries (13).

Previous work by our group has established that the epitope recognized by Guy's 13 monoclonal antibody is conformational, being assembled from two regions of SA I/II that are noncontiguous in sequence (14). Using immunoblotting, it was shown that Guy's 13 recognizes two non-overlapping recombinant polypeptides that show no sequence homology. One part of the discontinuous epitope was shown to reside in a recombinant polypeptide (fragment 114, see Fig. 1) spanning aa 45–457 of SA I/II. The partial epitope residing in this fragment, which includes the A-region of SA I/II, is designated the A-region binding site or A site. The other part of the epitope was localized to a fragment (Pro0-IV) spanning aa 816–983 of SA I/II. The partial epitope residing in this fragment (which spans the P-region of SA I/II) is called the P-region binding site or P site. In ELISA-based experiments, binding of Guy's 13 to these individual fragments could not be demonstrated unless a recombinant polypeptide containing the A site was incubated simultaneously with a recombinant fragment containing the P site. Additionally, we showed that the two binding sites are able to associate with each other, leading to a structure that can subsequently be recognized by Guy's 13. This suggested that the A and P sites, which are widely separated in the linear sequence, are situated in close proximity in the native SA I/II.

The aim of the current work was to map both binding sites in greater detail and, in the process, identify critical residues responsible for forming the discontinuous Guy's 13 epitope. This in turn may give a better understanding of the structure of SA I/II, the nature of the antibody-antigen interaction, and provide an insight into how the antibody prevents S. mutans colonization of the oral cavity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Antigen I/II—Escherichia coli BL21(DE3)-pLysS was from Novagen. SA I/II was prepared from S. mutans Guy's strain (serotype c) (7) as described previously (15).

Antibodies—Guy's 13 monoclonal antibody and all other primary and secondary antibodies were used as described previously (14).

Cloning and Expression of Recombinant spaP Gene Fragments— Genomic DNA of S. mutans was isolated according to the method of Bollet et al. (16). Amplification, cloning, and expression, using the pEXss{Delta}3 vector, of recombinant spaP gene fragments (see Fig. 1) was performed as described previously (14). The identity of the recombinant clones was confirmed by sequencing. Recombinant polypeptides are produced as a fusion with a C-terminal, 13-amino acid, E Tag peptide (17). An anti-E Tag antibody (Amersham Biosciences) was used to confirm expression of the recombinant polypeptides by immunoblotting (not shown). The residues of SA I/II encoded by the recombinant polypeptides are shown in Fig. 1.

Peptide Synthesis—Peptides, corresponding to the SA I/II sequence from S. mutans NG5 strain, were synthesized as peptide amides by manual procedure on a solid phase scaffold using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry (18) in a Biotech Instruments BT7400 peptide synthesis block (19), as described by Kelly et al. (20). Peptide compositions were determined by mass spectrometry.

Immunoassays—For ELISAs, Maxisorp 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated with SA I/II (2 µg/ml solution in PBS) or cell lysates from E. coli cultures expressing recombinant SA I/II fragments. The plates were incubated overnight at 4 °C and washed three times with H2O. Free protein binding sites were blocked with 2.5% (w/v) bovine serum albumin (Sigma, Fraction V) in PBS for 2 h at 37 °C. The plates were washed three times with H2O containing 0.1% (v/v) Tween 20, air-dried, and stored at -20 °C until required.

Competition ELISA to measure the ability of lysates or peptides to inhibit the binding of Guy's 13 IgG to solid phase SA I/II has been described previously (14). Essentially, 65 µl of a 1 µg/ml solution of Guy's 13 IgG in 5x DB (12.5% (w/v) bovine serum albumin, 0.5% (v/v) Tween 20 in PBS) was added to 130 µl of inhibitor-1 (either neat lysate, 250 µM peptide in PBS, or PBS) and 130 µl of inhibitor-2 (neat lysate or PBS). Aliquots of 100 µl were added in triplicate to the wells of SA I/II-coated ELISA plates. Following overnight incubation at 4 °C, the wells were washed six times with H2O containing 0.1% (v/v) Tween 20. Detection of bound Guy's 13 IgG was with a horseradish peroxidase (HRP)-conjugated secondary antibody and incubation at 37 °C for 2 h. HRP activity was quantitated by the addition of 3,3',5,5'-tetramethylbenzidine (Sigma). Enzyme activity was stopped with 1 M H2SO4 and the A450 measured.

Direct ELISA to measure the ability of peptides or lysates to promote the binding of Guy's 13 IgG to immobilized recombinant SA I/II fragments was described previously (14). Peptides were diluted to 100 µM in DB. Aliquots (240 µl) of peptide (100 µM), E. coli lysate, or DB were mixed with 10 µl of Guy's 13 IgG (5 µg/ml solution in DB). An isotype-matched mouse IgG1{kappa} monoclonal antibody (MOPC 31C) was used as a negative control. Aliquots of 100 µl were added in triplicate to the wells of an ELISA plate coated in advance with recombinant E. coli lysates. Following overnight incubation at 4 °C non-bound fluid phase components were removed and bound Guy's 13 IgG detected as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mapping of the A-region Binding Site Using Competition ELISA—We have previously demonstrated the simultaneous requirement for both the A and P sites in fluid phase assays (14). In a competition ELISA, Guy's 13 IgG binding to solid phase SA I/II was inhibited using a lysate from E. coli expressing a recombinant fragment of the P-region mixed with lysate from E. coli expressing an A-region fragment. A-region fragments 114, 115, 116, and 121 (Fig. 1) were mixed with recombinant P-region fragment Pro0-IV (Fig. 2A). When only one fragment was incubated with Guy's 13 (the "PBS" columns), antibody binding to solid phase SA I/II was unaffected compared with the control wells which received only Guy's 13 (the "no inhibitor" column). Mixing of 114 lysate with Pro0-IV lysate resulted in over 90% inhibition of Guy's 13 IgG binding to solid phase SA I/II (OD450 < 0.1), consistent with our previous report (14). When A-region fragments 115 or 121 (which both span the first alanine-rich repeat) were mixed with Pro0-IV lysate, >90% inhibition was also observed. In contrast, when A-region fragment 116 (representing the C-terminal half of 114) was mixed with Pro0-IV, inhibition of only 30% was observed.



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  		FIG. 2.
Inhibition of Guy's 13 binding to solid phase SA I/II using fluid phase lysates from E. coli expressing recombinant A- or P-region polypeptides. In A lysates from E. coli expressing recombinant polypeptides Pro0-IV or 110 or PBS (inhibitor-1) were mixed with Guy's 13 and with lysates from E. coli expressing the recombinant polypeptides indicated (inhibitor-2). The mixtures of Guy's 13 + inhibitors were added to SA I/II-coated ELISA plates, and following overnight incubation, non-bound components were removed by extensive washing. The amount of Guy's 13 binding to the solid phase SA I/II was quantitated using a HRP-conjugated secondary antibody. The no inhibitor control represents the level of binding of Guy's 13 IgG to SA I/II in the absence of any lysate (i.e. PBS only). Results are presented as the mean (±S.D.) A450 for triplicate wells. In B lysates from E. coli expressing either fragment Pro0-IV or fragment 110 (inhibitor-1) were mixed with lysate from E. coli expressing A-region recombinant polypeptides 121, 128, or 129 (inhibitor-2) and with Guy's 13 IgG. Results are presented as the mean (±S.D.) percent inhibition relative to the no inhibitor control for triplicate wells.

 
Recombinant polypeptide 110 (spanning aa 984–1539 of SA I/II), which was shown previously to have no Guy's 13-binding activity in immunoblotting or in ELISAs (14), was used as a negative control. The lack of inhibition observed following mixing of the 110 lysate with either Pro0-IV lysate or with each of the A-region lysates (Fig. 2A) confirmed the requirement for the simultaneous presence of both A- and P-region binding sites.

These results show that all of the recombinant fragments of the A-region were able to inhibit Guy's 13 IgG binding to solid phase SA I/II when mixed with Pro0-IV recombinant polypeptide, although the extent of inhibition varied depending on the recombinant polypeptide used. None of the recombinant fragments were able to inhibit Guy's 13 binding on their own. In view of the repeat nature of the A-region it is, perhaps, not surprising that all of the A-region recombinant polypeptides tested gave some levels of inhibition. The recombinant polypeptide 121 (aa 121–218 of SA I/II) inhibited Guy's 13 binding by >90% when mixed with Pro0-IV polypeptide.

Two further non-overlapping clones of A-region fragment 121 were generated: fragment 128 (aa 121–169 of SA I/II) and fragment 129 (aa 170–218) (see Fig. 1). In combination with Pro0-IV fragment, A-region fragment 129 is an effective inhibitor of Guy's 13 binding to solid phase SA I/II (see Fig. 2B), giving levels of inhibition (80%) nearly equivalent to 121 (88.5%), while fragment 128 did not inhibit Guy's 13 IgG binding above controls. The A-region binding site therefore lies within aa 170–218 of SA I/II. An A-region binding site can therefore be localized to this fragment and the ELISA results indicate that homologous A-region binding sites may be present in at least one of the remaining A-region tandem repeats.

Mapping of the Binding Sites Using Synthetic Peptides—The P-region binding site had previously been mapped to a recombinant polypeptide, representing aa 816–983 of SA I/II (14). To further characterize the binding of Guy's 13 to SA I/II, 19 overlapping 20-mer peptides spanning the P-region were synthesized (Table I). A further peptide, p1037, which lies C-terminal to the P-region, was included as a negative control. Each of the peptides was tested for their ability to inhibit Guy's 13 IgG binding to solid phase SA I/II in combination with A-region fragment 121. Three independent experiments were performed, and the results were plotted as the mean percentage inhibition (±S.D.) relative to a no inhibitor control, which represents Guy's 13 binding to solid phase SA I/II in the absence of any inhibitor lysate or peptide (Fig. 3). As a positive control, a mixture of 121 and Pro0-IV lysates produced high levels of inhibition (89%). No inhibition (<5%) was observed with any fragment or peptide in combination with control recombinant polypeptide 110 (gray bars). Most of the 121 lysate + peptide combinations also did not give any inhibition (<5%), comparable with the no inhibitor control and with the samples utilizing the negative control 110 lysate + peptide combinations. The exceptions were peptides p886, p926, p955, and p965, which produced varying levels of inhibition at 27%, 52%, 87%, and 20%, respectively.


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  		TABLE I
Nomenclature and sequence of peptides spanning amino acids 803-1004 of the P-region of SA I/II from S. mutans NG5 Peptide p1037 was included as a negative control. Amino acid numbering is from SA I/II of S. mutans strain NG5.

 



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  		FIG. 3.
Inhibition of Guy's 13 binding to solid phase SA I/II using overlapping 20-mer peptides spanning the P-region of SA I/II. Peptides were mixed with inhibitor-1 (either 121 or 110 lysate) and Guy's 13 IgG. The mixtures were added to SA I/II-coated ELISA plates, and following overnight incubation, non-bound components were removed by extensive washing. The amount of Guy's 13 binding to the solid phase SA I/II was quantitated using a HRP-conjugated secondary antibody and is presented as the percent inhibition relative to a no inhibitor control, which represents wells where both inhibitor-1 and -2 are PBS. Results are presented as the mean (±S.D.) percentage inhibition from three independent experiments.

 
Peptides p926 and p955 (mixed with 121 lysate) inhibited the binding of Guy's 13 to solid phase SA I/II by more than 50%. Sequence homology between these two peptides is the likely reason for this. Indeed, all four inhibitory peptides (p886, p926, p955, and p965) share a common sequence motif, discussed later.

Dose-dependent inhibition of Guy's 13 binding to immobilized SA I/II has been demonstrated before for both the A and P sites (14). In the presence of saturating amounts of the 121 lysate, dose-dependent inhibition by peptide p955 was demonstrated. Maximal inhibition was achieved with 40 µM p955, with an IC50 (50% inhibitory concentration) of 3.3 µM (data not shown).

Six 20-mer peptides spanning aa 161–230 of SA I/II (each peptide overlapping by 10 residues) were synthesized to map more finely the A-region binding site. However, none of these peptides significantly inhibited the binding of Guy's 13 to solid phase SA I/II when mixed with lysate from E. coli expressing recombinant fragment Pro0-IV (results not shown).

Homologues of Peptide p955—The peptide p955 sequence is located within tandem repeat IV of the proline-rich region of SA I/II. The repeat nature of the P-region suggested that p955-homologous peptides from the other P-region tandem repeats might also inhibit Guy's 13 binding. Table II shows the sequence of p955 aligned with the three other peptide homologues from P-region tandem repeats I, II, and III, which were synthesized and tested for their ability to inhibit Guy's 13 IgG binding to solid phase SA I/II by preincubation with the 121 fragment (or with the 110 fragment as a negative control).


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  		TABLE II
Synthetic peptides from the P-region of SA I/II from S. mutans NG5 homologous to peptide p955 The location of the peptides within each of the P-region repeats is shown. Amino acid numbering is from SA I/II of S. mutans strain NG5.

 
The results (Fig. 4A) showed that, in addition to p955, peptides p892 and p931 (from P-region repeats II and III, respectively) resulted in 88% inhibition of binding of Guy's 13 to solid phase SA I/II, whereas p853 (from P-region repeat I) was not an effective inhibitor.



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  		FIG. 4.
Inhibition of Guy's 13 binding to solid phase SA I/II using peptides. Peptides were mixed with inhibitor-1 (either 121 or 110 lysate) and with Guy's 13 IgG and the amount of inhibition measured as described in the legend to Fig. 3. Results from three independent experiments are plotted as the mean (±S.D.) percentage inhibition relative to a no inhibitor control. A, inhibition of Guy's 13 binding to solid phase SA I/II using peptide homologues of p955. B, inhibition of Guy's 13 binding to solid phase SA I/II using alanine-substituted peptide analogues of p955.

 
Alanine Scanning Mutagenesis of Peptide p955—ASM of peptide p955 was performed. Thirteen peptide analogues (A3 to A15) were synthesized, each containing a single alanine substitution at positions 3 to 15 of peptide p955.

Each of the alanine-substituted peptides was tested with the 121 fragment (or with the 110 fragment as a negative control) for inhibition of Guy's 13 IgG binding to solid phase SA I/II (see Fig. 4B). As a positive control, Guy's 13 IgG preincubated with fragment 121 and either Pro0-IV or p955 resulted in high level (90%) inhibition.

The results enabled the alanine substitutions to be classified into three groups: (i) those alanine substitutions that had no effect on the inhibition by p955 of Guy's 13 binding to SA I/II (positions 4, 7, 9, 10, 13, and 14), (ii) those substitutions that completely abolished the inhibitory effects of p955 (positions 5 and 8), and (iii) those alanine substitutions that resulted in partial abolition of the inhibitory effects of p955 (positions 3, 6, 11, 12, and 15).

N- and C-terminal Deletions of Peptide p955—Ten peptides were synthesized that exhibited progressive deletions of amino acid residues from the N terminus of peptide p955. A further 10 peptides were synthesized with progressive deletions of amino acid residues from the C terminus of peptide p955.

The results of competition ELISAs using the N-terminal deleted peptides showed that deletion of the N-terminal residue (Asn) of p955 had no effect on the inhibition of Guy's 13 binding to solid phase SA I/II. Deletion of the first 2 residues (Asn-Lys), however, reduced by 38% the inhibition exhibited by p955, while deletion of the first 3 residues completely abolished the inhibitory effect of p955 (data not shown).

The results of the competition ELISAs using the C-terminal deleted peptides showed that deletion of the last 5 residues of p955 (Thr-Asp-Pro-Val-Tyr) had very little effect on the ability of the peptide to inhibit Guy's 13 binding to solid phase SA I/II. Deletion of the last 6–8 residues resulted in progressively lower levels of inhibition, culminating in total loss of inhibition when the last 9 residues were removed (data not shown).

Binding of Guy's 13 IgG to Solid Phase A-region or P-region Binding Site in the Presence of Peptides or Lysates—To confirm the results of the competition ELISAs, the ability of peptides or lysates to promote binding of Guy's 13 to solid phase-associated binding sites was investigated.

The results show that when A-region fragment 121 was adsorbed to the solid phase of an ELISA plate, Pro0-IV lysate and peptide p955 both promoted the binding of Guy's 13 (Fig. 5A). No effect was seen with a control monoclonal antibody (MOPC 31C) (gray bars). The results using P-region tandem repeat homologues of p955 (see Table II) show that p892 and p931 also promote binding of Guy's 13 to 121 while p853 does not. Lysates 121, 129, and 110 did not promote binding of Guy's 13 to immobilized 121. These findings are consistent with the results of the competition ELISAs (see Figs. 2 and 4A) and confirm the simultaneous requirement for both the A- and P-region binding sites.



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  		FIG. 5.
Binding of Guy's 13 IgG to solid phase-associated recombinant fragments in the presence of peptides. Lysate from E. coli expressing recombinant polypeptide 121 was coated onto ELISA plates. Following blocking, a mixture of 0.2 µg/ml Guy's 13 IgG (or MOPC 31C) and either peptide or lysate (the fluid phase sample) was added to the wells. Following overnight incubation and extensive washing, antibody that remained bound was detected by the addition of a HRP-conjugated anti-mouse IgG secondary antibody. A shows the results using P-region homologues of peptide p955 as the fluid phase component. B shows the results using the alanine-substituted peptide analogues of p955 in the fluid phase. Results are presented as the mean (±S.D.) A450 of triplicate wells.

 
The results using the alanine-substituted peptide analogues of p955 in this direct binding ELISA (see Fig. 5B) also mirrored the results obtained using these peptides in the competition ELISAs. Thus, peptides (such as A7, A10, and A13), which are strong promoters of Guy's 13 binding to solid phase 121 fragment, are also strong inhibitors (in combination with fragment 121) of Guy's 13 binding to immobilized SA I/II (see Fig. 4B).

As a control, Pro0-IV lysate (encoding the P-region binding site) coated onto the solid phase did not bind Guy's 13 in the presence of fluid phase p955 but did bind Guy's 13 in the presence of fluid phase 121 and 129 lysates, which both encode A-region binding sites (data not shown).

Negative control 110 fragment coated onto the solid phase did not bind Guy's 13 IgG in the presence of p955 or any of the A- or P-region lysates (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In an attempt to delineate the Guy's 13 conformational epitope, we made use of earlier findings that the Guy's 13 epitope was formed from non-contiguous sequences that, when added together, were able to reconstitute the Guy's 13 epitope. The epitope is unusual because it can be reassembled in vitro irrespective of whether the two regions lie in cis or in trans. This allowed each binding site to be studied in turn and enabled each to be more finely mapped by cloning and expression of successively smaller recombinant fragments and, ultimately, with synthetic peptides.

The A site was mapped to a recombinant polypeptide (fragment 129) spanning aa 170–218 of the A-region of SA I/II. This sequence is identical to aa 170–218 of PAc from S. mutans serotype c strain MT8148 (21). The data indicated that at least one further binding site was present in the A-region, consistent with the repeat nature of this region. Attempts to map the binding site in the 129 fragment using overlapping 20-mer peptides were unsuccessful.

Collectively, alignment of the 20-mer synthetic peptides p886, p926, p955, and p965, the results of ASM, the use of N- and C-terminally truncated peptides, and a comparison of the peptide homologues allowed a consensus P-region binding site to be defined by a 14-amino acid sequence: XPX(E/D)PXYXXX-PXPP. This sequence can be found at aa 893–906, 932–945, and 956–969 of SA I/II and implies the presence of at least three P-region binding sites for Guy's 13. ASM of p955 showed that residues Pro957, Pro960, Tyr962, Pro966, Thr967, Pro968, and the acidic residue Glu959 were most important for peptide binding.

Although the P site is shown to reside on a peptide, the data from the ASM show that this partial epitope is not defined simply by a linear sequence of residues (22, 23). Thus many of the residues in this partial epitope are most likely not involved in making direct contact with the complementarity determining region residues of the antibody. Similarly, most of the residues of the A site are also unlikely to be involved in interaction with the paratope. This indicates that each binding site still retains some secondary structure, consisting of independent contact residues interspersed among non-contact residues. Non-contact residues may be required for either the formation of a stable secondary structure or for interactions between the A- and P-region binding sites, which then allows the assembled epitope to be recognized by Guy's 13.

Other workers (24–26) have described the mapping of discontinuous epitopes using synthetic peptides. Those reports have all described antibodies that are able to bind independently to at least two, non-overlapping peptides, indicating that the epitopes are discontinuous and that there is little interaction between the epitope fragments (binding sites) in the assembled epitope. In contrast, Spendlove et al. (27) described an antibody that, while able to bind independently to three peptides, showed increased binding when the peptides were mixed together. This synergistic binding is indicative of cooperativity between each of the binding sites within the assembled epitope.

The discontinuous Guy's 13 epitope represents an extreme form of cooperativity. Under the denaturing and reducing conditions of immunoblotting Guy's 13 was shown to interact with SA I/II through two binding sites in an independent fashion (14). While this is not likely to be representative of the conditions under which Guy's 13 would be expected to encounter cell-surface SA I/II, it nevertheless proved invaluable in establishing the discontinuous nature of the epitope. In fluid phase immunoassays, cooperativity between the binding sites was essential for Guy's 13 binding to take place. Taken together the results indicate that the two binding sites interact with each other to form the assembled epitope and that Guy's 13 interacts with both binding sites within the assembled epitope.

Structural analysis of proline-rich regions in other proteins (28, 29), where proline is present as every third residue, suggests that these regions adopt a conformation with 3 residues per turn, known as the polyproline II (PP II) helix, with a 3.1-Å rise/residue (30). By analogy it is predicted that the P-region of SA I/II, which also contains frequently repeating (PXX)n motifs, may also adopt a PP II helix-like conformation (31).

Feng et al. (32) have proposed a model for the PP II helix of Src homology 3 ligands. This model, adapted for peptide p955 and depicted in Fig. 6 is consistent with the ASM results. The critical, as defined by ASM, acidic (Glu959) and tyrosine (Tyr962) residues lie in close proximity on one edge of the proposed PP II helix model. The residues that make a smaller entropic contribution lie in the lower plane, while residues that form the upper edge appear to make little contribution to peptide p955 function.



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  		FIG. 6.
Proposed conformation of peptide p955. The core region (residues 3–15) of p955 is depicted. Gray colored residues had a major effect on p955 function when mutated to alanine. Striped residues only had a moderate effect on p955 function. Unshaded residues are those that, when mutated to Ala, had no effect on p955 function. Amino acid differences between p955 and the other functional peptide homologues (i.e. p892 and p931) are indicated by the arrows.

 
The results of this study have demonstrated that A-region sequences of SA I/II are able to interact with P-region sequences. Thus, we hypothesize that the PP II-like helix of the proline-rich region interacts with the alanine-rich region, which itself is predicted to adopt an {alpha}-helical structure (5, 33–35). The interaction of PP II-like helices with {alpha}-helices has been described before for turkey pancreatic polypeptide, a 36-amino acid hormone (28, 36), where residues 2 to 8 form a PP II-like helix that is closely packed against an {alpha}-helix formed from residues 14–32. Homologous polypeptides from the pancreas of a wide range of species have been isolated and sequenced, and despite sequence variation, secondary structure analyses (37) and immunochemical studies (38) indicate that the tertiary structure involves the interaction of the PP II- and {alpha}-helices. This idea is now proposed here as the structural basis for the recognition of SA I/II by Guy's 13. Thus, hydrophobic interactions between the proline side chains of the PP II-helical P-region and side chains from the {alpha}-helical A-region permit the assembly of the Guy's 13 epitope. Thus, although the Guy's 13 epitope is formed from two discontinuous binding sites within SA I/II, each site has a conformational requirement determined by its secondary structure.

Recently, the crystal structure of a recombinant protein spanning the variable (V) region of antigen I/II (SR) from S. mutans serotype f strain OMZ 175 has been determined (39). While this recombinant protein does not encompass the A- and P-region binding sites defined in our studies, it does include some flanking alanine-rich and proline-rich sequences. The structure shows that the alanine-rich sequences form an {alpha}-helix that, indeed, lies in close proximity to the proline-rich sequences. The structure also suggests that the A- and P-regions are orientated in an antiparallel configuration. Juxtaposition of the A- and P-regions is also suggested by our present report as well as our earlier work (14). In this regard it is interesting to note that one of the A-region 82 aa repeats, with a predicted {alpha}-helical structure, has approximately the same length (123 Å)as one of the P-region 39 aa repeats, with a proposed PP II-helical structure (121 Å), suggesting that an interaction between the two regions might require the longitudinal association of a PP II helix with an {alpha}-helix. If this is true, it may explain why an A-region binding site could not be localized to a 20-mer peptide. The minimal P-region binding site was defined by the 14-residue sequence XPX(E/D)PXYXXXPXPP, which if it adopts a PP II helix conformation, would have a length of 43.4 Å. This distance can only be spanned by an {alpha}-helical peptide of >=29 residues.

The binding of antibodies, such as Guy's 13, to S. mutans cell surface SA I/II might block adhesion epitopes by exerting their effect locally (by steric hindrance) or at a distance (through induced conformational changes in the SA I/II molecule). Such antibodies, when used as a passive mucosal vaccine, could affect the adhesin function of the protein, leading to prevention of colonization of the oral cavity by the bacterium. These studies, which begin to map the interaction of Guy's 13 with SA I/II at a molecular level, represent an initial step in understanding these mechanisms.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 44-207-188-43-57; Fax: 44-207-188-43-75; E-mail: craig.van_dolleweerd{at}kcl.ac.uk.

1 The abbreviations used are: SA I/II, streptococcal antigen I/II; aa, amino acid residues; ASM, alanine scanning mutagenesis; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; PP II, polyproline II. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Karen Homer for assistance with mass spectrometric analysis of the peptides.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Loesche, W. J. (1986) Microbiol. Rev. 50, 353-380[Free Full Text]
  2. Russell, M. W., and Lehner, T. (1978) Arch. Oral Biol. 23, 7-15[CrossRef][Medline] [Order article via Infotrieve]
  3. Ogier, J. A., Wachsmann, D., Schöller, M., Lepoivre, Y., and Klein, J. P. (1990) Arch. Oral Biol. 35, (suppl.) 25S-31S
  4. Okahashi, N., Sasakawa, C., Yoshikawa, M., Hamada, S., and Koga, T. (1989) Mol. Microbiol. 3, 221-228[CrossRef][Medline] [Order article via Infotrieve]
  5. Kelly, C., Evans, P., Bergmeier, L., Lee, S. F., Progulske-Fox, A., Harris, A. C., Aitken, A., Bleiweis, A. S., and Lehner, T. (1989) FEBS Lett. 258, 127-132[CrossRef][Medline] [Order article via Infotrieve]
  6. Jenkinson, H. F., and Demuth, D. R. (1997) Mol. Microbiol. 23, 183-190[CrossRef][Medline] [Order article via Infotrieve]
  7. Smith, R., and Lehner, T. (1989) Oral Microbiol. Immunol. 4, 153-158[Medline] [Order article via Infotrieve]
  8. Lehner, T., Caldwell, J., and Smith, R. (1985) Infect. Immun. 50, 796-799[Abstract/Free Full Text]
  9. Ma, J. K. C., Hunjan, M., Smith, R., and Lehner, T. (1989) Clin. Exp. Immunol. 77, 331-337[Medline] [Order article via Infotrieve]
  10. Ma, J. K. C., Hunjan, M., Smith, R., Kelly, C., and Lehner, T. (1990) Infect. Immun. 58, 3407-3414[Abstract/Free Full Text]
  11. Ma, J. K. C., Hiatt, A., Hein, M., Vine, N. D., Wang, F., Stabila, P., van Dolleweerd, C., Mostov, K., and Lehner, T. (1995) Science 268, 716-719[Abstract/Free Full Text]
  12. Kruger, C., Hu, Y., Pan, Q., Marcotte, H., Hultberg, A., Delwar, D., van Dalen, P. J., Pouwels, P. H., Leer, R. J., Kelly, C. G., van Dollenweerd, C., Ma, J. K., and Hammarstrom, L. (2002) Nat. Biotechnol. 20, 702-706[CrossRef][Medline] [Order article via Infotrieve]
  13. Ma, J. K. C., Hikmat, B. Y., Wycoff, K., Vine, N. D., Chargelegue, D., Yu, L., Hein, M. B., and Lehner, T. (1998) Nat. Med. 4, 601-606[CrossRef][Medline] [Order article via Infotrieve]
  14. van Dolleweerd, C. J., Chargelegue, D., and Ma, J. K.-C. (2003) Infect. Immun. 71, 754-765[Abstract/Free Full Text]
  15. Russell, M. W., Bergmeier, L. A., Zanders, E. D., and Lehner, T. (1980) Infect. Immun. 28, 486-493[Abstract/Free Full Text]
  16. Bollet, C., Gevaudan, M. J., de Lamballerie, X., Zandotti, C., and de Micco, P. (1991) Nucleic Acids Res. 19, 1955[Free Full Text]
  17. Schier, R., Balint, R. F., McCall, A., Apell, G., Larrick, J. W., and Marks, J. D. (1996) Gene (Amst.) 169, 147-155[CrossRef][Medline] [Order article via Infotrieve]
  18. Wellings, D. A., and Atherton, E. (1997) Methods Enzymol. 289, 44-67[CrossRef][Medline] [Order article via Infotrieve]
  19. Dörner, B., Ostresh, J. M., Houghten, R. A., Frank, R., Tiepold, A., Fox, J. E., Bray, A. M., Ede, N. J., James, I. W., and Wickham, G. (2000) in Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Chan, W. C., and White, P. D., eds) University Press, Oxford
  20. Kelly, C. G., Younson, J. S., Hikmat, B. Y., Todryk, S. M., Czisch, M., Haris, P. I., Flindall, I. R., Newby, C., Mallet, A. I., Ma, J. K.-C., and Lehner, T. (1999) Nat. Biotechnol. 17, 42-47[Medline] [Order article via Infotrieve]
  21. Okahashi, N., Sasakawa, C., Yoshikawa, M., Hamada, S., and Koga, T. (1989) Mol. Microbiol. 3, 673-678[CrossRef][Medline] [Order article via Infotrieve]
  22. Geysen, H. M., Barteling, S. J., and Meloen, R. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 178-182[Abstract/Free Full Text]
  23. Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) J. Immunol. Methods 102, 259-274[CrossRef][Medline] [Order article via Infotrieve]
  24. Korth, C., Stierli, B., Streit, P., Moser, M., Schaller, O., Fischer, R., Schulz-Schaeffer, W., Kretzschmar, H., Raeber, A., Braun, U., Ehrensperger, F., Hornemann, S., Glockshuber, R., Riek, R., Billeter, M., Wuthrich, K., and Oesch, B. (1997) Nature 390, 74-77[CrossRef][Medline] [Order article via Infotrieve]
  25. Reineke, U., Sabat, R., Misselwitz, R., Welfle, H., Volk, H. D., and Schneider-Mergener, J. (1999) Nat. Biotechnol. 17, 271-275[CrossRef][Medline] [Order article via Infotrieve]
  26. Prodinger, W. M., Schwendinger, M. G., Schoch, J., Kochle, M., Larcher, C., and Dierich, M. P. (1998) J. Immunol. 161, 4604-4610[Abstract/Free Full Text]
  27. Spendlove, L., Li, L., Potter, V., Christiansen, D., Loveland, B. E., and Durrant, L. G. (2000) Eur. J. Immunol. 30, 2944-2953[CrossRef][Medline] [Order article via Infotrieve]
  28. Blundell, T. L., Pitts, J. E., Tickle, I. J., Wood, S. P., and Wu, C.-W. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4175-4179[Abstract/Free Full Text]
  29. Darbon, H., Bernassau, J.-M., Deleuze, C., Chenu, J., Roussel, A., and Cambillau, C. (1992) Eur. J. Biochem. 209, 765-771[Medline] [Order article via Infotrieve]
  30. Adzhubei, A. A., and Sternberg, M. J. E. (1993) J. Mol. Biol. 229, 472-493[CrossRef][Medline] [Order article via Infotrieve]
  31. Kelly, C., Evans, P., Ma, J. K. C., Bergmeier, L. A., Taylor, W., Brady, L. J., Lee, S. F., Bleiweis, A. S., and Lehner, T. (1990) Arch. Oral Biol. 35, (suppl.) 33S-38S
  32. Feng, S., Chen, J. K., Yu, H., Simon, J. A., and Schreiber, S. L. (1994) Science 266, 1241-1247[Abstract/Free Full Text]
  33. LaPolla, R. J., Haron, J. A., Kelly, C. G., Taylor, W. R., Bohart, C., Hendricks, M., Pyati, J. P., Graff, R. T., Ma, J. K. C., and Lehner, T. (1991) Infect. Immun. 59, 2677-2685[Abstract/Free Full Text]
  34. Demuth, D. R., Golub, E. E., and Malamud, D. (1990) J. Biol. Chem. 265, 7120-7126[Abstract/Free Full Text]
  35. Demuth, D. R., and Irvine, D. C. (2002) Infect. Immun. 70, 6389-6398[Abstract/Free Full Text]
  36. Glover, I., Haneef, I., Pitts, J., Wood, S., Moss, D., Tickle, I., and Blundell, T. (1983) Biopolymers 22, 293-304[CrossRef][Medline] [Order article via Infotrieve]
  37. Glover, I. D., Barlow, D. J., Pitts, J. E., Wood, S. P., Tickle, I. J., Blundell, T. L., Tatemoto, K., Kimmel, J. R., Wollmer, A., Strassburger, W., and Zhang, Y.-S. (1984) Eur. J. Biochem. 142, 379-385[Medline] [Order article via Infotrieve]
  38. Taylor, T. C., Thompson, D. O., Ebner, K. E., Kimmel, J. R., and Rawitch, A. B. (1988) Mol. Immunol. 25, 961-973[Medline] [Order article via Infotrieve]
  39. Troffer-Charlier, N., Ogier, J., Moras, D., and Cavarelli, J. (2002) J. Mol. Biol. 318, 179-188[CrossRef][Medline] [Order article via Infotrieve]

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