SALIVARY AMYLASE--ROLE IN DENTAL CARIES PATHOGENESIS

唾液淀粉酶是唾液蛋白多功能的一个很好的例子。淀粉酶的多功能性质包括：1)淀粉水解；2)与羟基磷灰石(釉质)结合；以及3)在溶液中与细菌(如绿色链球菌)结合，并在结合到羟基磷灰石时结合。对于唾液淀粉酶，其在溶液中与细菌的结合可能导致细菌清除(保护)，而其在釉质膜中的存在可能促进牙菌斑的形成(有害)。它在溶液中与绿色链球菌的结合以及在结合到羟基磷灰石表面时都依赖于其天然构象的维持。这项建议的目的是从其在口腔生理学中的作用(S)来阐明淀粉酶的结构-功能关系。在分子水平上表征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病过程中是至关重要的。在这一资助期间，我们建议产生针对唾液淀粉酶三种功能的具有生化和生理缺陷的不同突变体。突变体将使用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分析来筛选突变体的生物学活性。这些突变体的结构将用蛋白质结晶学来确定，以了解突变对功能的影响。这些第一代突变体将提供关于一个功能的增强或减弱如何影响另外两个功能的线索。我们将从活性显着改变的突变体的体外生物测定和结构分析中获得这些线索。根据这些结果，将构建更多代表第二代和第三代的突变体。这样的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，从而降低患龋齿的可能性。

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.