Interactions of Streptococcal Glucosyltransferase with Amylase

Glucosyltransferase (Gtf) enzymes of Streptococcus mutans have been identified in salivary pellicles but the binding site for Gtf is unknown. We have shown that Gtfs may interact with salivary amylase. Objective: The purpose of this study was to assess the binding of Gtfs B, C, and D, of S. mutans to salivary molecules (amylase, cystatin-1, histatin-5, and lysozyme), in particular, amylase, using an ELISA assay. Method: Binding of Gtf was measured by Gtf-specific antibody. Further, to map the binding regions in GtfB and amylase, we used tryptic digested GtfB and mutants of non-glycosylated amylase. The following six mutants were studied: Trp58->Leu, Trp59->Leu, a double mutant Trp58->Leu:Trp59->Leu, tyr151->Met, Phe256->Trp and D306 a deletion mutant lacking a mobile loop ³⁰⁶GAGGA³¹⁰. Results: Results showed that GtfB bound to recombinant, non-glycosylated amylase (rHSAmy) but did not bind to native glycosylated amylase. Further, interaction of tryptic digested GtfB peptides revealed that GtfB binding with rHSAmy may be mediated by sequences contained within a 145 kDa region of GtfB. The mutant D306 bound to GtfB whereas the double mutant Trp58->Leu:Trp59->Leu showed only a very weak binding. These results suggest the aromatic residues at positions 58 and 59 of amylase may be involved in GtfB binding but not the mobile loop containing residues GAGGA. Conclusion: These studies extend our earlier observations that Gtf interacts with amylase, and are the first studies to indicate specific interactions between localized regions of Gtf and amylase. Supported by USPHS R37 DEO7907 and R01 DE12585.