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: 6296256
• 负责人: MICHAEL J LEVINE
• 金额: $ 14.59万
• 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/6296256
• 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.

唾液成分在细菌定植和繁殖过程中起着重要作用