SALIVARY AMYLASE--ROLE IN DENTAL CARIES PATHOGENESIS

唾液淀粉酶提供了一个很好的例子，展示了唾液蛋白的多功能性。淀粉酶的多功能性包括：1)淀粉水解；2)与羟基磷灰石（牙釉质）结合；3)在溶液中与细菌（如翠绿链球菌）结合，并与羟基磷灰石结合。对于唾液淀粉酶，它与溶液中的细菌结合可能导致细菌清除（保护性），而它存在于牙釉质膜中可能促进牙菌斑的形成（有害）。无论是在溶液中还是在与羟基磷灰石表面结合时，它与翠绿链球菌的结合都依赖于其天然构象的维持。本提案的目的是阐明淀粉酶在口腔生理中的作用背景下的结构-功能关系。在分子水平上描述这些关系将提高对链球菌在口腔中早期定植的基本机制的理解。这一提议的基本假设是，这种酶的多功能性可以在龋齿的发展中发挥重要作用。特别是，我们认为唾液淀粉酶的结构域在龋齿过程中是至关重要的。在本研究期间，我们拟针对唾液淀粉酶的三种功能产生具有生化和生理缺陷的不同突变体。该突变体将使用一个简单的杆状病毒表达系统产生，并将通过细菌结合、淀粉水解或羟基磷灰石结合的特定测定来筛选突变体的生物活性。这些突变体的结构将使用蛋白质晶体学来确定，以了解突变对功能的影响。这些第一代突变体将提供关于增强或削弱一种功能如何影响其他两种功能的线索。我们将从体外生物试验和结构分析中获得这些线索，这些突变体表现出明显改变的活性。基于这些结果，将构建代表第二代和第三代的额外突变体。这种突变体将允许设计策略来操纵人类唾液淀粉酶与细菌的相互作用，从而有利于宿主，从而减少龋齿的可能性。

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.