Peptide Mapping of a Novel Discontinuous Epitope of the Major Surface Adhesin from Streptococcus mutans

Craig J Van Dolleweerd,Charles G Kelly,Daniel Chargelegue,Julian K-C Ma

doi:10.1074/jbc.m400820200

Craig J Van Dolleweerd, Charles G Kelly + Show 2 more

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https://doi.org/10.1074/jbc.m400820200

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Abstract

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.

Highlights

Tein streptococcal antigen I/II (SA I/II) of Streptococcus mutans, one of the major consisting of three tandem repeats of a 39-aa sequence, tocausative agents of dental caries
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
When only one fragment was incubated with Guy’s 13, antibody binding to solid phase SA I/II was unaffected compared with the control wells which received only Guy’s 13

Summary

Introduction

Tein SA I/II of Streptococcus mutans, one of the major consisting of three tandem repeats of a 39-aa sequence, tocausative agents of dental caries. 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. 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]. 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]. It was shown that Guy’s 13 recognizes two non-overlapping recombividual antigenic determinants and can be formed by nant polypeptides that show no sequence homology.

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