SALIVARY AMYLASE--MICROBIAL INTERACTIONS AND HYDROXYAPATITE BINDING

唾液淀粉酶——微生物相互作用和羟基磷灰石结合

• 批准号: 6238416
• 负责人: MICHAEL J LEVINE
• 金额: $ 3.71万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6238416
• 关键词: Streptococcus; amylases; cell adhesion; chemical binding; chemical models; circular dichroism; complementary DNA; enzyme activity; genetic manipulation; human subject; hydrolysis; hydroxyapatites; microorganism interaction; model design /development; molecular cloning; molecular site; nucleic acid sequence; oral bacteria; point mutation; polymerase chain reaction; protein structure; saliva; site directed mutagenesis

Components of saliva can play an important role in the colonization and metabolism of oral bacteria. One abundant salivary component that has recently been shown to interact with oral viridans streptococci is salivary amylase. In fact, amylase is now thought to possess at least three biological functions: 1) hydrolysis of complex carbohydrates; 2) binding to bacteria; and 3) binding to hydroxyapatite (HAP). The goal of the present application is to understand the structural basis of the three functions of amylase in the oral cavity. This will be accomplished by amplification of human salivary amylase cDNA from total salivary gland mRNA using the polymerase chain reaction (PCR). the amplified amylase cDNA will be cloned and expressed in an E. coli system and the bioactive recombinant human salivary amylase purified and compared to native amylase for enzymatic activity, streptococcal binding, HAP binding and secondary structure by circular dichroism (CD) spectroscopy. This optimized expression system will then be used to express amylases in which point mutations are produced using site-directed mutagenesis of specific residues implicated in enzymatic function. These mutant amylases will be compared to the wild type protein for enzymatic and streptococcal binding activities. A molecular model of salivary amylase based on the published amino acid sequence and available crystal structure data of other amylases will be constructed and used to interpret effects of the site-directed mutations on the biological functions. Finally, the HAP binding domain of salivary amylase will be mapped using selective fragmentation of amylase immobilized on HAP. Once identified, recombinant amylase cDNAs containing mutations in this region will be constructed and expressed as described above. These mutant amylases will then be tested for their ability to promote bacterial adhesion to HAP a well as for enzymatic activity and streptococcal binding. An understanding of amylase function may allow the design of amylase analogs having enhanced enzymatic and bacterial binding activities but diminished ability to bind HAP. Such a construct might promote bacterial clearance from the oral cavity while simultaneously inhibiting bacterial adhesion to teeth. The availability of such an agent may prove useful as a component of biologically compatible and efficacious anti-plaque agents or artificial salivas.

唾液的成分可以在定植中发挥重要作用， 口腔细菌的代谢。 一种丰富的唾液成分， 最近显示与口腔草绿色链球菌相互作用的是唾液 淀粉酶 事实上，淀粉酶现在被认为具有至少三种 生物学功能：1）复杂碳水化合物的水解; 2）结合 3）与羟基磷灰石（HAP）结合。 的目标 本申请是为了了解这三者的结构基础 淀粉酶在口腔中的作用。 这将通过 人唾液腺淀粉酶cDNA的扩增 使用聚合酶链反应（PCR）。 扩增的淀粉酶cDNA将 在E.大肠杆菌系统和生物活性重组体 纯化人唾液淀粉酶并与天然淀粉酶比较， 酶活性、链球菌结合、HAP结合和次级 通过圆二色性（CD）光谱进行结构化。 这种优化的 表达系统将用于表达淀粉酶， 使用特定残基的定点诱变产生突变 与酶功能有关。 这些突变淀粉酶将被比较 用于酶和链球菌结合的野生型蛋白质 活动 唾液淀粉酶的分子模型基于已发表的 其他淀粉酶的氨基酸序列和可用的晶体结构数据 将被构建并用于解释现场导向的效果， 生物功能的突变。 最后，HAP结合结构域的 唾液淀粉酶将使用淀粉酶的选择性片段化来作图 固定在HAP上。 一旦鉴定，含有淀粉酶cDNA的重组体 该区域中的突变将如所述构建和表达 以上 然后将测试这些突变淀粉酶的能力， 促进细菌与HAP的粘附以及酶活性， 链球菌结合。 对淀粉酶功能的了解可能有助于 具有增强的酶和细菌结合的淀粉酶类似物的设计 活动，但结合HAP的能力减弱。 这样的结构可能 促进口腔细菌清除，同时 抑制细菌粘附到牙齿上。 这种药剂的可用性 可以证明可用作生物相容性和有效的 抗牙斑剂或人造唾液。