Immunological characteristics of a synthetic peptide associated with a catalytic domain of mutans streptococcal glucosyltransferase

Abstract

The immunogenicity of a multiple antigenic peptide construct consisting of four copies of the synthetic 21-mer peptide DANFDSIRVDAVDNVDADLLQ was measured. The composition of this peptide was derived from a sequence in the N-terminal region of mutans streptococcal glucosyltransferases (GTFs) containing an aspartic acid implicated in catalysis. The peptide (CAT) construct was synthesized as a tetramer on a lysine backbone and subcutaneously injected into Sprague-Dawley rats for polyclonal antibody formation or intraperitoneally injected into BALB/c mice, and then spleen cell fused with Sp2/OAgl4 murine myeloma cells for monoclonal antibody formation. The resulting rat antisera and mouse monoclonal antibodies reacted with CAT and with native GTF isozymes from Streptococcus sobrinus and Streptococcus mutans (in enzyme-linked immunosorbent assay and Western blot [immunoblot] analyses). Functional inhibition of the water-insoluble glucan synthetic activity of S. sobrinus GTF-I was demonstrated with an immunoglobulin M anti-CAT monoclonal antibody (>80% inhibited) and with rat sera (approximately 17% inhibited). The monoclonal antibody preparation also modestly inhibited the water-soluble glucan synthetic activity of an S. mutans GTF mixture. These results suggest that the CAT peptide contains B-cell epitopes that are similar to those of intact mutans streptococcal GTFs and has the potential to elicit antibody that can inhibit GTF function. Thus, sequences within this peptide construct may have value for inclusion in a synthetic dental caries vaccine.

Protective Immunity againstStreptococcus mutansInfection in Mice after Intranasal Immunization with the Glucan-Binding Region ofS. mutansGlucosyltransferase

“…We have previously subcloned the putative catalytic and glucan-binding regions (CAT and GLU, respectively) of S. mutans GTF-I and demonstrated that antibodies to either CAT (representing amino acid residues 253 to 628) or GLU (representing amino acid residues 1183 to 1473) could inhibit glucan synthesis by GTF, although anti-GLU antibodies were much more effective than anti-CAT antibodies (75% versus 22% inhibition, respectively) (10). These findings support earlier observations that antibodies to peptides corresponding to sequences within the CAT or GLU caused a moderate inhibition of GTF activity (3,5,16,27,28). Furthermore, subcutaneous immunizations in the salivary gland vicinity of rats with synthetic peptides representing components of the glucan-binding or catalytic region of GTF moderately reduced smooth-surface caries (30).…”

Glucansucrases: mechanism of action and structure–function relationships

“…Other mutations affecting Asp residues near Asp‐451 from GTF‐B had no significant effect on enzymatic activity [96]. The functional importance of this region has also been confirmed by the fact that an antibody directed against a peptide of sequence homologous to this region inhibited glucansucrase activity [97–100].…”

Immunoglobulin A reaction to oral streptococci in saliva of subjects with different combinations of caries and levels of mutans streptococci

“…Theoretic-ally, some such reactions could be more advantageous from a protective aspect, as they could interfere with important virulence or adhesion factors of the bacteria. For example, an antigen of a molecular weight 185-190 kDa, designated as antigen I/II (19), B {20), IF (10), PI (5) or PAc (16) has been particularly studied, and glucosyltransferases {GTF) {4, 23,26) are other interesting candidates {1, 8).…”