Studies of Naturally Occurring Structurally Modified Carbohydrates

描述（由申请人提供）：这是一份竞争性修订申请，是响应no - od -09-058提交的，标题为“NIH宣布竞争性修订申请的恢复法案资金可用性”。母基金编号为R01GM076360，题目为“天然存在的结构修饰碳水化合物的研究”。细胞表面的唾液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。我们的父母资助主要用于研究碳水化合物与蛋白质的相互作用，使用我们实验室合成的天然存在的唾液皂苷。在我们的初步研究中，我们已经建立了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。对于唾液酸释放外糖苷酶的研究，已经开发了一种高效的基于微滴板的高通量唾液酸酶底物特异性测定方法，该方法使用通过有效的一锅三酶化学酶合成方法获得的对硝基苯唾液酸二糖作为底物。对于哺乳动物Siglecs的研究，我们已经开发了一种组合化学酶方法来获得生物素化的唾液苷，这种方法可以与基于微滴板的高通量形式相结合，从而找到Siglecs的首选配体，而无需繁琐的产品纯化过程。由于获得的结果令人兴奋，我们计划利用所生产的硅苷产品以及在母体资助中开发的化学酶合成策略和检测方法的优势，将碳水化合物-蛋白质相互作用的研究扩展到检测甲型流感病毒表面的首选聚糖配体和有效的聚糖基血凝素抑制剂。此外，神经氨酸酶在不同甲型流感病毒株表面的底物特异性将被表征，并与人类神经氨酸酶的底物特异性进行比较。我们推测：1)流感病毒NA底物特异性在不同病毒株之间存在差异，并且与人类和细菌唾液酸酶不同；2)病毒HA配体特异性与病毒NA底物特异性存在相关性。为了验证这些假设，两个特定的目标是：1)确定纯化的禽流感和人甲型流感病毒颗粒表面重组人唾液酸酶和神经氨酸酶的底物特异性；2)确定配体特异性，并确定甲型流感病毒颗粒上血凝素的潜在抑制剂。我们相信本研究的完成将有助于1)更好地理解血凝素配体特异性与流感病毒神经氨酸酶底物特异性的关系；2)发现一组可作为流感病毒感染诊断工具的化合物；3)寻找潜在的抑制病毒血凝素与宿主结合的抑制剂。

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.