Structure/function studies of lactoferrin as a protease

乳铁蛋白作为蛋白酶的结构/功能研究

• 批准号: 6765440
• 负责人: Andrew George Plaut
• 金额: $ 23.57万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6765440
• 关键词: Actinobacillus actinomycetemcomitans; active sites; bacterial proteins; bioassay; chimeric proteins; clinical research; endopeptidases; enzyme mechanism; enzyme substrate; gram negative bacteria; human milk; human subject; lactoferrin; oligosaccharides; protein structure function; protein transport; proteolysis; technology /technique development

DESCRIPTION: We have recently discovered that human lactoferrin, a member of the lactotransferrin family of iron binding proteins, has proteolytic activity. Lactoferrin is one of the most abundant proteins in milk and most other human secretions, and is likely to contribute to the innate immune defense mechanisms on mucosal surfaces. It was unexpected that such a widely studied protein is a protease, but this has now been confirmed using recombinant lactoferrin expressed in baby hamster kidney cells, Aspergillus awamori, and baculovirus expression systems. Bovine milk lactoferrin has similar activity. We have shown that human lactoferrin is a serine-type protease, and the enzyme active site is a serine-lysine dyad located in the amino-terminal, globular N-lobe of the protein. In addition, we have identified the peptide bonds that lactoferrin cleaves in the Haemophilus influenzae Hap adhesin and IgA protease precursor, two virulence proteins that are secreted to the outer membrane by the autotransporter pathway. Lactoferrin also cleaves proteins from the outer membranes of the dental pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, and from Shigella enteric pathogens. Among the known substrates are adhesins that mediate bacterial attachment to host epithelial cells, and lactoferrin cleavage reduces bacterial adhesiveness, in vitro. In our proposed studies we will develop quantitative assays for lactoferrin enzyme activity. These will be based on fusion proteins that are designed to incorporate lactoferrin-susceptible polypeptides whose cleavage releases an easily measurable reporter. Short substrates will be introduced into immobilized cellulase of Cellulomonas fimi, allowing estimation of lactoferrin activity by measuring released cellulase activity. Longer substrates will be expressed in E. coli as fusion proteins that have bacterial alkaline phosphatase as the reporter. This strategy will then be used to identify the peptide bonds cleaved by lactoferrin in Aae, an autotransporter adhesin protein of the periodontal pathogen Actinobacillus actinomycetemcomitans. Finally, we will explore the role of lactoferrin oligosaccharides in modifying proteolytic activity. This will be by site-specific mutations of one or more asparagine glycan attachment sites in recombinant lactoferrin expressed in insect cells. These basic studies will address the long-term goal of producing proteolytically active, recombinant lactoferrin for clinical control of bacterial attachment, colonization, and infection of oral and other mucosal surfaces.

描述：我们最近发现人乳铁蛋白（铁结合蛋白乳铁蛋白家族的成员）具有蛋白水解活性。乳铁蛋白是牛奶和大多数其他人类分泌物中最丰富的蛋白质之一，可能有助于粘膜表面的先天免疫防御机制。令人惊讶的是，如此广泛研究的蛋白质是一种蛋白酶，但现在已经使用在幼仓鼠肾细胞、泡盛曲霉和杆状病毒表达系统中表达的重组乳铁蛋白证实了这一点。牛乳乳铁蛋白具有类似的活性。我们已经证明，人乳铁蛋白是一种丝氨酸型蛋白酶，酶活性位点是位于蛋白质氨基末端、球状N叶的丝氨酸-赖氨酸二联体。此外，我们还鉴定了乳铁蛋白在流感嗜血杆菌 Hap 粘附素和 IgA 蛋白酶前体中裂解的肽键，这两种毒力蛋白通过自转运蛋白途径分泌到外膜。乳铁蛋白还可以裂解牙科病原体伴放线放线杆菌和牙龈卟啉单胞菌以及肠道病原体志贺氏菌外膜的蛋白质。已知的底物包括介导细菌附着于宿主上皮细胞的粘附素，而乳铁蛋白的裂解可降低体外细菌的粘附性。在我们提出的研究中，我们将开发乳铁蛋白酶活性的定量测定。这些将基于融合蛋白，该融合蛋白被设计为掺入乳铁蛋白敏感的多肽，其裂解释放出易于测量的报告分子。将短底物引入纤维单胞菌的固定化纤维素酶中，从而可以通过测量释放的纤维素酶活性来估计乳铁蛋白活性。较长的底物将在大肠杆菌中表达为融合蛋白，以细菌碱性磷酸酶作为报告基因。然后，该策略将用于鉴定 Aae 中乳铁蛋白裂解的肽键，Aae 是牙周病原体放线杆菌伴放线杆菌的一种自转运粘附素蛋白。最后，我们将探讨乳铁蛋白寡糖在改变蛋白水解活性中的作用。这将通过昆虫细胞中表达的重组乳铁蛋白中的一个或多个天冬酰胺聚糖附着位点的位点特异性突变实现。这些基础研究将解决生产具有蛋白水解活性的重组乳铁蛋白的长期目标，用于临床控制口腔和其他粘膜表面的细菌附着、定植和感染。