SALIVARY AMYLASE--ROLE IN DENTAL CARIES PATHOGENESIS

唾液淀粉酶——在龋齿发病机制中的作用

• 批准号: 6516504
• 负责人: NARAYANAN RAMASUBBU
• 金额: $ 18.07万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ DENTAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6516504
• 关键词: Streptococcus mutans; X ray crystallography; amylases; bacteria infection mechanism; bacterial proteins; binding sites; conformation; dental caries; enzyme activity; enzyme complex; enzyme structure; hydrolysis; hydroxyapatites; mutant; oral bacteria; pathologic process; protein binding; saliva; site directed mutagenesis; starch; tooth enamel

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.

唾液淀粉酶提供了唾液蛋白表现出的多功能性的一个很好的例子。 淀粉酶的多功能性质包括：1）淀粉水解； 2）与羟基磷灰石（搪瓷）结合； 3）与溶液中的细菌（例如Viridans链球菌）结合并与羟基磷灰石结合。 对于唾液淀粉酶，其与溶液中细菌的结合可能会导致细菌清除率（保护性），而其在牙釉质中的存在可能会促进牙菌斑的形成（有害）。 它与溶液中的Viridans链球菌的结合以及与羟基磷灰石表面结合时，它取决于其天然构象的维持。该提案的目标是阐明淀粉酶在其在口服生理学中的作用的背景下的结构功能关系。 这些关系在分子水平上的表征将提高对口腔中链球菌早期定植的基本机制的理解。 该提议的基本假设是该酶的多功能性在牙齿的发育中可以发挥重要作用。 特别是，我们认为唾液淀粉酶的结构结构域在龋齿过程中至关重要。 在这个赠款期间，我们建议以靶向唾液淀粉酶的三种功能的生化和生理缺陷产生不同的突变体。 突变体将使用简单的杆状病毒表达系统产生，并将使用特定的细菌结合，淀粉水解或羟基磷灰石结合的特定测定法对突变体的生物学活性进行筛选。 这些突变体的结构将使用蛋白质晶体学确定，以了解突变对功能的影响。 这些第一代突变体将提供有关一个功能的增强或削弱如何影响其他两个功能的线索。 我们将从体外生物学测定法和突变体的结构分析中获得这些线索，这些突变体显示出显着改变活性。 基于这些结果，将构建代表第二代和第三代的其他突变体。 这样的突变体将允许设计策略来操纵人唾液淀粉酶 - 细菌 - 细菌相互作用，从而有利于宿主，从而降低了龋齿的潜力。