均一透明质酸的酶法合成策略及生物学功能评价

发布时间：2017-12-28 21:24

本文关键词：均一透明质酸的酶法合成策略及生物学功能评价　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：糖胺聚糖(glycosaminoglycans,GAGs)是哺乳动物中普遍存在的天然杂多糖,结构上由二糖重复单元连接而成,以蛋白聚糖或游离形式存在。糖胺聚糖的重要生理功能及其与重大疾病的密切关系近年来受到国内外科学家的广泛关注。透明质酸(hyaluronan,HA)是结构最简单的糖胺聚糖,由二糖重复单位[-D-glucuronic acid-β 1,3-N-acetyl-D-glucosamine-β 1,4-]连接而成,不同于其他糖胺聚糖,透明质酸没有硫酸化修饰且不与蛋白共价结合,以游离形式存在。HA广泛存在于动物组织及某些微生物中,由于其良好的保水性、粘弹性和生物相容性,已经广泛应用于保健品、化妆品及医药等领域。近年来研究发现,HA除了作为细胞外基质的结构组成外,还参与了细胞识别、信号转导等重要生理过程,在肿瘤的发生、发展及炎症反应过程中发挥了多重生物效应。代谢异常是肿瘤的重要特征,肿瘤微环境中,HA合成和降解的动态平衡发生异常,糖链长度变化是一个重要体现,也是肿瘤恶性程度的评价指标之一。肿瘤细胞表面存在大量HA特异性受体和结合蛋白,HA通过与受体或结合蛋白的相互作用,激活细胞内多条信号通路,参与肿瘤发生发展和炎症反应等生物效应过程。尤为值得关注的是,有别于其它生物效应分子的合成后修饰过程,HA的生物活性受其糖链长度的调控,不同糖链长短的HA表现出截然不同的生物效应。受生物体内多糖合成过程的影响,商品化HA具有高度不均一性(分子量分布范围较宽且呈弥散性分布),严重阻碍了HA生物学功能的正确表述。目前,商品化HA的生产方法主要有两种。一是动物组织提取,这种技术产量较低,且存在动物原料受限和动物病毒残存问题。二是微生物发酵,用这种方法生产HA成本低、规模大且不受原料的限制,但存在细菌内毒素、核酸、蛋白质及重金属污染的风险。上述方法在商品化HA的制备方面得到了较大发展,但在均一化HA的精细化制备方面还极具挑战性。本论文围绕均一 HA的制备这一科学问题,对巴斯德氏菌来源的透明质酸合酶(PmHAS)的催化反应机理进行了深入研究,采用逐步酶法合成HA寡糖片段和一步法同步化合成均一 HA,发展了均一 HA的两步偶联酶法合成策略。论文第二章节通过深入研究PmHAS的催化反应机理,发现PmHAS催化新生HA糖链合成过程中存在起始限速步骤,造成了产物的高度不均一性。通过对PmHAS的底物选择性分析,发现透明质酸三糖是PmHAS催化合成均一 HA所必需的最小受体底物;进一步研究发现,催化反应体系中受体与供体的摩尔浓度比例是影响HA糖链长度的关键因素,通过对反应体系中受体/供体摩尔浓度进行化学计量控制,实现了均一HA的糖链长度可控酶法合成。随后进一步拓展了均一HA酶法合成策略在化学结构明确、HA糖链长度可控的HA缀合物制备领域的应用。制备了生物素标记的透明质酸(HA-biotin conjugates)以及透明质酸-药物缀合物(HA-oroxylin conjugates)。HA-biotin conjugates 能够作为分子探针,对外源 HA的细胞内代谢过程进行可视化检测;HA-oroxylin conjugates作为模式药物,为HA修饰抗肿瘤靶向药物的研发奠定了基础。论文第三章开展了透明质酸-硫酸软骨素(HA-CS)嵌合型糖胺聚糖的合成。由于HA与CS结构的相似性及PmHAS灵活的底物适应性,通过逐步反应的方法合成了 CS2HA2-N3、HA2CS2HA2-N3、CS2HA4-N3 和 CS4HA4-N3 四种 HA-CS嵌合型糖胺聚糖,用于研究微观结构改变对HA生物功能的影响。该方法也可应用于其他不同嵌合型糖胺聚糖的合成中,为嵌合型糖胺聚糖的生物功能研究奠定了基础。论文第四章节中,结合本实验室在糖核苷酸酶法合成方面的工作基础,建立了一种以单糖为起始的多酶偶联的HA合成策略。以廉价单糖GlcNAc和GlcA为起始底物,将糖核苷酸的酶法合成与透明质酸的聚合反应相偶联,一锅多酶催化高效合成HA。并利用此方法成功合成了 HA衍生物,用于交联HA的制备和应用研究,具有十分广阔的应用前景。
[Abstract]:Glycosaminoglycans (glycosaminoglycans, GAGs) are ubiquitous natural Heteropoly saccharides in mammals. They are made up of two sugar repeating units, and exist in proteoglycan or free form. The important physiological functions of glycosaminoglycan and its close relationship with major diseases have been widely concerned by scientists at home and abroad in recent years. Hyaluronic acid (hyaluronan, HA) is the most simple glycosaminoglycan structure, composed of two repeat units [-D-glucuronic acid- beta 1,3-N-acetyl-D-glucosamine- beta 1,4-] connection, different from other glycosaminoglycans, hyaluronic acid and no sulfated modification does not bind covalent protein, is present in a free form. HA is widely distributed in animal tissues and some microorganisms. Because of its good water retention, viscoelasticity and biocompatibility, it has been widely applied in health products, cosmetics and medicine. In recent years, studies have found that HA, as a structural component of extracellular matrix, is also involved in cell recognition, signal transduction and other important physiological processes. It plays multiple biological effects in the process of tumor occurrence, development and inflammatory response. Metabolic abnormality is an important feature of cancer. In the microenvironment of cancer, the dynamic balance of HA synthesis and degradation is abnormal. The change of sugar chain length is an important manifestation, and it is also an evaluation index of malignancy of tumor. A large number of HA specific receptors and binding proteins exist on the surface of tumor cells. HA activates many signaling pathways in cells through interaction with receptors or binding proteins, and participates in biological processes such as oncogenesis, development and inflammatory response. What is particularly noteworthy is that it is different from other biological effect molecules in the process of post modification. The biological activity of HA is regulated by the length of its sugar chain. HA with different sugar chain length shows different biological effects. Influenced by the process of polysaccharide synthesis, commercialized HA is highly heterogeneous, with a wide distribution of molecular weight and diffuse distribution, which seriously hampers the correct expression of HA's biological functions. At present, there are two main methods of production of commercialized HA. The first is the extraction of animal tissue, which is low in production, and there is a problem of animal raw material limitation and animal virus surviving. The two is microbial fermentation. In this way, the production of HA is low cost, large scale and not limited by raw materials, but it has the risk of bacterial endotoxin, nucleic acid, protein and heavy metal pollution. The above method has been greatly developed in the preparation of commercial HA, but it is still very challenging in the fine preparation of homogenized HA. This paper focuses on the preparation of homogeneous HA this scientific problem, hyaluronic acid synthase on Pasteur's source of bacteria (PmHAS) catalytic reaction mechanism were studied, using stepwise enzymatic synthesis of HA oligosaccharides and one-step synchronous combined Cheng Junyi HA, the two step development strategy coupled enzymatic synthesis of uniform HA. In the second chapter, through in-depth study of the catalytic mechanism of PmHAS, it is found that PmHAS catalyzes the initial rate limiting steps in the process of HA synthesis, resulting in highly heterogeneous products. Based on the PmHAS substrate selectivity analysis found that hyaluronic acid three sugar is required for PmHAS catalytic synthesis of homogeneous HA minimal receptor substrate; further study found that the ratio of molar concentration of receptor and donor catalytic reaction system is a key factor affecting HA sugar chain length, by measuring the chemical reaction system of donor / acceptor the molar concentration, the synthesis of uniform HA sugar chain controlled enzymatic method. Subsequently, the application of the homogeneous HA enzyme synthesis strategy in the preparation of HA conjugates with clear chemical structure and controlled HA chain length was further expanded. The biotin labeled hyaluronic acid (HA-biotin conjugates) and hyaluronic acid drug conjugates (HA-oroxylin conjugates) were prepared. HA-biotin conjugates can be used as a molecular probe to visualize the metabolic process of exogenous HA. HA-oroxylin conjugates as a model drug lays the foundation for the development of HA targeted anticancer drug. In the third chapter, the synthesis of hyaluronic acid - chondroitin sulfate (HA-CS) chimeric glycosaminoglycan was developed. Due to the similarity of HA and CS structure and PmHAS flexible substrate adaptability, four HA-CS chimeric glycosaminoglycans, CS2HA2-N3, HA2CS2HA2-N3, CS2HA4-N3 and CS4HA4-N3, were synthesized by stepwise reaction method, which was used to study the effect of microstructure change on HA biological function. This method can also be used in the synthesis of other different chimeric glycosaminoglycan, which lays a foundation for the study of the biological function of chimeric glycosaminoglycan. In the fourth part of the paper, combined with the work of our lab in the field of nucleotides synthesis, a HA synthesis strategy based on monosaccharide was established. Using the cheap monosaccharide GlcNAc and GlcA as the starting substrate, the enzymatic synthesis of sugar nucleotides was coupled with the polymerization of hyaluronic acid, and the one pot multi enzyme catalyzed and highly efficient synthesis of HA. The HA derivatives have been successfully synthesized by this method, and are used for the preparation and application of crosslinked HA, which has a very broad application prospect.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ281

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