Studies of Naturally Occurring Structurally Modified Carbohydrates

天然存在的结构修饰碳水化合物的研究

• 批准号: 7811303
• 负责人: Xi Chen
• 金额: $ 47.76万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7811303
• 关键词: Acute; Adhesions; Affect; Affinity; Avian Influenza A Virus; Binding; Biological; Biological Assay; Birds; Carbohydrates; Cell Communication; Cell membrane; Cell surface; Cells; Cessation of life; Cleaved cell; Detection; Diagnosis; Diagnostic; Domestic Fowls; Enzymes; Exoglycosidases; Funding; Future; Glycoconjugates; Glycoproteins; Grant; Hemagglutinin; Human; Human Influenza A Virus; Human Virus; Industry; Infection; Influenza A virus; Label; Lead; Length; Ligands; Link; Measurement; Measures; Mediating; Membrane Glycoproteins; Methods; Microscopy; Movement; Mucous body substance; Neuraminidase; Physics; Play; Polysaccharides; Process; Protein-Carbohydrate Interaction; Proteins; Recombinants; Recovery; Research; Respiratory System; Respiratory tract structure; Role; Schools; Screening procedure; Sialic Acids; Specificity; Structure; Substrate Specificity; Surface; System; Testing; United States National Institutes of Health; Veterinary Medicine; Viral; Viral Hemagglutinins; Viral Physiology; Virion; Virus; Virus Diseases; anti-influenza drug; base; carbohydrate structure; combinatorial; design; influenza virus strain; influenzavirus; inhibitor/antagonist; multidisciplinary; neutravidin; pandemic disease; parent grant; particle; pathogen; preference; public health relevance; receptor; respiratory; response; surface coating; tool

DESCRIPTION (provided by applicant): This is a competitive revision application submitted in response to NOT-OD-09-058 entitled "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications". The parent grant is R01GM076360 entitled "Studies of naturally occurring structurally modified carbohydrates". Sialylated carbohydrate structures presented on the cell surface play diverse and important biological roles in cell-cell interaction processes. Our parental grant is focusing on the studies of carbohydrate-protein interactions using naturally occurring sialosides synthesized in our lab. In our preliminary studies, we have established efficient approaches for studying two types of sialic acid (Sia)-recognizing protein systems: bacterial sialidases and mammalian Siglecs. For sialidase (sialic acid-releasing exoglycosidase) studies, an efficient microtiter-plate-based high-throughput sialidase substrate specificity assay has been developed using para-nitrophenyl sialyldisaccharides obtained by an effective one-pot three-enzyme chemoenzymatic synthetic approach as substrates. For mammalian Siglecs studies, we have developed a combinatorial chemoenzymatic approach to obtain biotinylated sialosides which can be combined with microtiter-plate-based high-throughput format to find the preferred ligands for Siglecs without the tedious product purification process. Excited by the results obtained, we plan to take the advantages of the sialoside products produced as well as the chemoenzymatic synthetic strategies and the assay methods developed in the parent grant to expand the carbohydrate-protein interaction study to the detection of the preferred glycan ligands and potent glycan-based inhibitors for hemagglutinins on the surface of influenza A virus. In addition, substrate specificity of neuraminidases on the surface of different influenza A virus strains will be characterized and compared to that of human neuraminidases. We hypothesize that 1) the influenza virus NA substrate specificity is different among different viral strains, and is different from human and bacterial sialidases; 2) a correlation exists between the viral HA ligand specificity and the viral NA substrate specificity. To test these hypotheses, two specific aims are to 1) determine the substrate specificity of recombinant human sialidases and neuraminidases on the surface of purified avian and human influenza A virus particles and 2) determine the ligand specificity and identify potential inhibitors of hemagglutinins on influenza A virus particles. We believe the completion of the proposed study will lead to 1) a better understanding of the relationship of ligand specificity of hemaglutinin and substrate specificity of neuraminidase of influenza viruses; 2) finding a set of compounds that can be used as diagnostic tools for influenza virus infection; and 3) identifying potential inhibitors against viral hemagglutinin binding to host. PUBLIC HEALTH RELEVANCE: Influenza A virus is an acute viral disease of the respiratory tract that affects millions of people each year. The virus has two types of surface glycoproteins: hemagglutinin and neuraminidase. Both of these glycoproteins recognize sialic acid-containing structures on the host cell surface and play important roles in influenza virus infections. Hemagglutinin binds to the sialic acid- containing glycans on host respiratory cell surface and initiate infection. Neuraminidase is believed to cleave the sialic acid from the glycans on the surface of infected cells to release the newly formed viral particles to allow the spreading of virus. The studies proposed here can help to understand the relationship of the ligand specificity of hemagglutinin and the substrate specificity of neuraminidases and their involvement in infection. Molecules that can be used for diagnosis of influenza A virus infection and potential inhibitors that can against the binding and the infection of influenza virus can also be identified.

描述(由申请人提供)：这是一份针对NOT-OD-09-058号文件提交的竞争性修订申请，标题为“NIH宣布恢复法资金可用于竞争性修订申请”。母基金为R01GM076360，题为“天然结构修饰碳水化合物的研究”。细胞表面的唾液酸化碳水化合物结构在细胞-细胞相互作用过程中发挥着多种重要的生物学作用。我们的父母资助重点是使用我们实验室合成的天然唾液酸苷来研究碳水化合物和蛋白质之间的相互作用。在我们的初步研究中，我们已经建立了两种有效的方法来研究两种类型的唾液酸(SIA)识别蛋白系统：细菌唾液酸酶和哺乳动物Siglecs。针对唾液酸外糖苷酶的研究，建立了一种高效的微滴板高通量唾液酸酶底物专一性检测方法，该方法以一锅三酶化学法合成的对硝基苯基唾液酸二糖为底物。对于哺乳动物Siglecs的研究，我们开发了一种组合化学酶方法来获得生物素化的唾液酸苷，该方法可以与基于微滴板的高通量格式相结合，从而找到Siglecs的首选配体，而不需要繁琐的产物纯化过程。受到这些结果的鼓舞，我们计划利用唾液酸苷产品的优势，以及在母公司赠款中开发的化学酶合成策略和检测方法，将碳水化合物-蛋白质相互作用研究扩展到检测甲型流感病毒表面首选的葡聚糖配体和有效的基于葡聚糖的凝集素抑制剂。此外，还将表征不同甲型流感病毒株表面神经氨酸酶的底物特异性，并将其与人类神经氨酸酶进行比较。我们假设1)流感病毒NA底物的特异性在不同的病毒株之间是不同的，也不同于人和细菌唾液酸酶；2)病毒HA配体特异性和病毒NA底物特异性之间存在相关性。为了验证这些假设，有两个特定的目的：1)确定重组人唾液酸酶和神经氨酸酶在纯化的禽流感病毒颗粒和人流感病毒颗粒表面的底物专一性；2)确定血凝素对甲型流感病毒颗粒的配基专一性和鉴定潜在的抑制物。我们相信，这项拟议研究的完成将有助于1)更好地了解血凝素的配体特异性与流感病毒神经氨酸酶的底物特异性的关系；2)找到一套可用作流感病毒感染诊断工具的化合物；以及3)寻找潜在的抑制病毒血凝素与宿主结合的药物。 公共卫生相关性：甲型流感病毒是一种急性呼吸道病毒性疾病，每年影响数百万人。该病毒有两种表面糖蛋白：血凝素和神经氨酸酶。这两种糖蛋白都识别宿主细胞表面含唾液酸的结构，并在流感病毒感染中发挥重要作用。血凝素与宿主呼吸道细胞表面含唾液酸的糖链结合，引发感染。神经氨酸酶被认为可以从受感染细胞表面的糖链上裂解唾液酸，释放新形成的病毒颗粒，从而允许病毒的传播。这些研究有助于了解血凝素的配基特异性和神经氨酸酶的底物特异性之间的关系及其在感染中的作用。还可以确定可用于甲型流感病毒感染诊断的分子，以及可以对抗流感病毒结合和感染的潜在抑制剂。