Salivary amylase provides an excellent example of the multifunctionality exhibited by salivary proteins. The multifunctional nature of amylase includes: 1) starch hydrolysis; 2) binding to hydroxyapatite (enamel); and 3) binding to bacteria (e.g. viridans streptococci) in solution and when bound to hydroxyapatite. For salivary amylase, its binding to bacteria in solution may result in bacterial clearance (protective) while its presence in the enamel pellicle may facilitate dental plaque formation (harmful). Its binding to viridans streptococci both in solution as well as when bound to the hydroxyapatite surface is dependent upon the maintenance of its native conformation. The goals of this proposal are to elucidate the structure-function relationships of amylase in the context of its role(s) in oral physiology. Characterization of these relationships at the molecular level will improve the understanding of basic mechanisms responsible for the early colonization of streptococci in the oral cavity. The underlying hypothesis of this proposal is that the multifunctionality of this enzyme can play a significant role in dental caries development. In particular, we feel that the structural domains of salivary amylase are critical in the caries process. In this grant period, we propose to generate distinct mutants with biochemical and physiological defects targeted against each of the three functions of salivary amylase. The mutants will be generated using a facile baculovirus expression system and the biological activities of the mutants will be screened with specific assays for bacterial binding, starch hydrolysis or hydroxyapatite binding. The structure of these mutants will be determined using protein crystallography for understanding the effect of mutation on the function. These first generation mutants will provide clues regarding how augmenting or weakening of one function affects the other two. We will obtain these clues from the in vitro biological assays and structure analysis of mutants which exhibit significantly altered activity. Based upon these results, additional mutants representing second and third generations will be constructed. Such mutants will permit the design of strategies to manipulate human salivary amylase-bacterial interactions that favor the host and thus reduce the potential for caries.
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