肝素NDST4催化模式的研究及糖基转移酶活性快速分析方法的建立

发布时间：2018-04-13 05:36

本文选题：硫酸乙酰肝素 + 化学酶法合成　；参考：《山东大学》2017年硕士论文

【摘要】：肝素(heparin,HP)类药物作为抗凝血和血栓治疗药物应用于临床已经有50余年的历史,市场需求不断增长。目前,HP类药物原料的生产依然依靠动物组织提取,来源有限而且质量不稳定。建立安全高效的体外合成HP类化合物的方法代替现有原料生产方式,一直是研究人员努力的方向。经过10余年的努力,酶法合成具有生物活性的HP寡糖的研究取得了突破性进展,但现有技术体系中存在着各种瓶颈问题,制约着HP类药物的规模化制备。比如,N位点硫酸化葡糖胺(N-sulfated glucosamine,GlcNS)直接定位合成困难和催化骨架合成的糖基转移酶(glycosyltransferases,GTs)活性快速分析方法缺失是现有酶法合成技术体系中亟待解决的两个问题。针对上述两个问题,本论文以提高HP寡糖酶法合成技术体系的效率和规模化应用潜力为目的,完成了两部分工作:通过结构均一确定模式底物,揭示了人源硫酸乙酰肝素(heparan sulfate,HS)N-脱乙酰基酶/N-硫酸基转移酶4 N-Deacetylase/N-sulfotransferase 4,NDST4)的催化模式;建立了偶联酿酒酵母尿苷二磷酸(uridinediphosphate,UDP)水解酶(yeast nucleoside diphosphatase,YND1)的 GT 反应高通量筛选体系。1.硫酸乙酰肝素NDST4催化模式的研究N-脱乙酰基酶/N-硫酸基转移酶(N-deacetylase/N-sulfotransferase,NDST)是HS生物合成途径中糖链骨架合成完成后的第一个硫酸化修饰酶,其催化获得的N位点硫酸化的葡糖胺是后续一系列硫酸基转移酶和葡萄糖醛酸异构酶底物识别的结构基础。HS糖链中硫酸根基团的排列形式决定了其与蛋白因子的结合能力,进而影响含有该糖链的蛋白聚糖的生物活性。因此,系统的研究NDST的底物特异性等催化机制不但对揭示HS生物合成控制机理具有重要的理论价值,而且相关结论可以直接指导HP寡糖的体外酶法合成。人体细胞内存在4种具有不同的底物特异性NDST同工酶,共同调节HS的N位点硫酸化修饰。伴随着结构均一确定的HS寡糖酶法合成体系的建立,NDST1的催化模式已被充分阐明。但NDST其他3种同工酶的催化模式还未被充分研究。论文利用由昆虫杆状病毒系统(baculovirus expression system)表达的重组NDST4与一系列化学结构确定的模式底物,在体外模拟HS生物合成过程中N位点硫酸化修饰过程,通过解析反应产物的结构,揭示了 NDST4的催化机制。论文研究结果表明:(1)单独的重组NDST4不具有N-脱乙酰基酶活性;(2)NDST4具有较强的N-硫酸基转移酶活性;(3)与NDST1随机结合底物糖链中的GlcNAc位点,定向催化的模式不同,NDST4的N-硫酸基转移酶活性没有表现出方向性。基于以上结论,该同工酶在HS糖链的体内生物合成中不是调控糖链高硫酸化区域(NS domain)和低硫酸化区域(NAc domain)相间排列的关键酶;应用方面,NDST4具备成为转化葡糖胺(glucosamine,GlcN)为N位点硫酸化葡糖胺(N-sulfated glucosamine,GlcNS)工具酶的潜力。2.偶联酿酒酵母UDP水解酶的Leloir-GT活性高通量筛选方法的建立GT是生物体内负责催化糖苷键合成酶类,它不但是糖生物学研究重点之一,而且是糖生物工程重要的工具酶分子之一,广泛用于体外糖链及糖缀复合物的酶法制备。绝大多数GT需要以核苷活化的单糖为给体,被称为Leloir-GT。但严格的底物特异性限制了天然GT酶分子合成能力,产生了"糖链及其复合物的应用价值"与"酶法合成能力缺陷"的矛盾。以酶法合成HP寡糖技术体系为例,骨架合成工具酶分子巴斯德氏菌(Pasteurella multocida)肝素前体聚合酶2(pmHS2)严格的单糖供体选择性是制约现有体系效率的瓶颈之一。利用蛋白质工程改造该GT的底物选择性是突破艾杜糖醛酸(iduronic acid,IdoA)转化这一瓶颈问题的有效手段。定向进化的成功在很大程度上依赖于针对GT活性的高通量筛选方法的建立。当前大多数Leloir-GTs活性测定方法基于质谱和色谱分离方法等复杂仪器或方法,不满足pmHS2分子定向进化的要求。针对以上问题,论文在可溶性重组表达酿酒酵母(Saccharomyces cerevisiae)高尔基体膜蛋白UDP水解酶(YND1)的基础上,建立了一种Leloir-GT活性高通量筛选方法。我们通过将YND1对UDP的水解反应、Leloir-GT反应和P032-的磷钼蓝显色反应偶联起来建立了一个简便、灵敏、快速的高通量筛选体系,并用GAG骨架合成中最重要的3种GTs共同验证了该体系的有效性和普适性。具体研究内容包括:(1)通过将YND1(GeneID:856722)第1504-1554位的疏水性跨膜区用3组甘氨酸-丝氨酸重复碱基(502F-518H/GSGSGS)进行替换的方法,实现了酿酒酵母跨膜蛋白YND1以可溶且有活性的状态在大肠杆菌中表达;(2)重组YND1的酶学性质研究表明其最佳反应条件与大多数Leloir-GT的理想反应条件一致,表明重组YND1催化的核苷酸水解反应可以实现与GT催化的糖基转移反应相偶联;(3)将GT反应与重组表达的YND1偶联,优化了反应与分析条件,建立了一种建立了一种快速、简便、准确性高的GT活性检测方法;以糖胺聚糖骨架合成中重要的糖基转移酶KfiA、pmHS2和KfoC为Leloir-GTs的模式工具酶分子,确定了该方法的有效性;(4)借助YND1偶联GT反应消除反应副产物UDP潜在抑制作用,可以明显促进GT催化反应,有效提高一定反应时间内目标糖链的得率。
[Abstract]:Heparin (heparin, HP) as the anticoagulant drugs and drug treatment of thrombosis in clinical application has more than 50 years of history, the growing market demand. At present, HP drug raw material production still rely on Extraction from animal tissues, but the quality is not stable. The limited source method for in vitro synthesis of HP compounds to establish safety high efficiency instead of the existing mode of production of raw materials, has always been the direction. After 10 years of efforts, the research of HP oligosaccharides with biological activity of enzyme synthesis method has made a breakthrough, but there are various existing bottlenecks in the technical system, restricting the scale of HP drugs. For example, the N site of sulfation glucosamine (N-sulfated glucosamine, GlcNS) direct synthesis of glycosyl synthesis and catalytic skeleton difficult transferase (glycosyltransferases, GTs) activity analysis method is the lack of the technology of enzymatic synthesis Two problems to be solved in the system. To solve the above two problems, this thesis is to improve the efficiency and the size of the potential application of synthesis technology system HP oligosaccharide enzyme method for the purpose, has completed two tasks: to determine the pattern substrate by uniform structure, reveals the human heparan sulfate (heparan sulfate, HS N-) deacetylase /N- sulfotransferase 4 N-Deacetylase/N-sulfotransferase 4, NDST4) model was established by catalytic coupling; Saccharomyces cerevisiae uridine two phosphoric acid (uridinediphosphate, UDP) (yeast nucleoside diphosphatase, YND1 hydrolase) of N- GT high throughput screening system for.1. heparan sulfate NDST4 catalytic model deacetylase /N- sulfate transferase (N-deacetylase/N-sulfotransferase, NDST) is the first HS sulfuric acid biosynthetic pathway carbohydrate skeleton synthesis after the completion of the modified enzyme, the catalytic site of the N Glucosamine sulfate is determined with the binding capacity of protein factors follow a series of sulfate radical transfer sulfate groups structure based.HS sugar chain and glucuronic acid isomerase enzyme substrate recognition in the arrangement form, thereby affecting the sugar chains of proteoglycans containing biological activity. Therefore, the substrate specificity of the catalytic mechanism the research of NDST system not only to reveal the HS biosynthesis mechanism has important theoretical value, but also can directly guide the related conclusion synthesis of HP oligosaccharides by enzymatic method in vitro. The cells of the body memory has a substrate specificity of different isoforms of NDST in 4, N locus sulfation regulated HS. With the establishment of HS oligosaccharides by enzymatic method the synthetic system uniform structure to identify the catalytic mode of NDST1 has been fully elucidated. But the catalytic model NDST other 3 isozymes have not been adequately studied. By using the insect rod Rotavirus (baculovirus expression system) system model to determine the expression of the recombinant NDST4 substrate and a series of chemical structure, in vitro HS biosynthesis N locus in the sulfation process, based on structure analysis of the reaction products, reveals the catalytic mechanism of NDST4. The research results show that: (1) recombinant NDST4 alone does not have N- deacetylase activity; (2) NDST4 N- has strong sulfate transferase activity; (3) and NDST1 random GlcNAc binding sites of substrate sugar chain, directional catalytic mode, showing no direction of the enzymatic activity of N- sulfate NDST4 transfer. Based on the above conclusions, the isozyme in biosynthesis HS sugar chain is not in the regulation of sugar chain of high sulfated region (NS domain) and low (NAc domain) regional acidification key enzymes are arranged alternately; the application, NDST4 has become the transformation of glucosamine (glucosamine, GlcN N) sites of sulfated glucosamine (N-sulfated glucosamine, GlcNS) activity of high-throughput screening method of Leloir-GT enzyme potential.2. coupling of Saccharomyces cerevisiae UDP hydrolase of GT is the organism responsible for catalyzing the glycosidic bond synthetase, which is not only the glycobiology research priorities, and is one of the important tool enzyme molecular biological sugar engineering, is widely used in the enzymatic chain and glucose in vitro with complex preparation. Most GT need to give monosaccharide with nucleoside activation, known as Leloir-GT. but strict substrate specificity limits of natural GT enzyme molecule synthesis ability, resulting in a "contradiction value" and its complexes with sugar chain "synthesis ability of enzymatic defects. In enzymatic synthesis of HP oligosaccharides technology system as an example, Pasteur's molecular skeleton synthesis tool enzyme bacteria (Pasteurella multocida) heparin precursor (pmHS2) polymerase 2 monosaccharide donor strictly. The selectivity is one of the bottleneck of the existing system. The efficiency of the transformation of the GT using protein engineering substrate selectivity is breakthrough iduronic acid (iduronic acid, IdoA) effective means of transforming the bottleneck. Directed evolution success largely depends on setting up the high-throughput screening method for GT activity. Most of the current Leloir-GTs activity the determination method of mass spectrometry and chromatographic separation method based on complex instruments or methods, does not meet the pmHS2 requirements of directed molecular evolution. In view of the above problems, the soluble recombinant expressing yeast (Saccharomyces cerevisiae) Golgi membrane protein UDP hydrolase (YND1) on the basis of established a high-throughput screening method. The activity of Leloir-GT by us the hydrolysis of YND1 to UDP, Leloir-GT and P032- reaction of molybdenum blue color reaction coupling together to establish a simple, sensitive and fast High throughput screening system with speed, and GAG skeleton 3 most GTs synthesis in common to verify the validity of the system and universality. The research contents include: (1) by YND1 (GeneID:856722) hydrophobic transmembrane region in 1504-1554 place with 3 sets of glycine serine repeat base (502F-518H/GSGSGS) replacement method, realized the expression of yeast transmembrane protein YND1 in soluble and active form in Escherichia coli; (2) study on the enzymatic properties of recombinant YND1 indicated that the optimal reaction conditions with most Leloir-GT ideal reaction conditions, showed that the nucleotide hydrolysis reaction catalyzed by recombinant YND1 can realize sugar the base with the GT catalytic transfer reaction phase coupling; (3) the YND1 coupling reaction of GT with recombinant expression, and analysis of reaction conditions were optimized, the establishment of a set up a fast, simple, GT activity detection method with high accuracy to; KfiA enzyme glycosyl transfer important glycosaminoglycan skeleton synthesis, pmHS2 and KfoC as the model tool Leloir-GTs enzyme molecules, determine the effectiveness of the proposed method; (4) using YND1 GT coupling reaction elimination reaction byproducts of UDP potential inhibition, GT can significantly promote the catalytic reaction, improve reaction time of sugar chain the yield.

【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】：R915

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