本文選題：硫酸乙酰肝素 + 化學(xué)酶法合成��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：肝素(heparin,HP)類藥物作為抗凝血和血栓治療藥物應(yīng)用于臨床已經(jīng)有50余年的歷史,市場需求不斷增長。目前,HP類藥物原料的生產(chǎn)依然依靠動物組織提取,來源有限而且質(zhì)量不穩(wěn)定。建立安全高效的體外合成HP類化合物的方法代替現(xiàn)有原料生產(chǎn)方式,一直是研究人員努力的方向。經(jīng)過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進展,但現(xiàn)有技術(shù)體系中存在著各種瓶頸問題,制約著HP類藥物的規(guī)模化制備。比如,N位點硫酸化葡糖胺(N-sulfated glucosamine,GlcNS)直接定位合成困難和催化骨架合成的糖基轉(zhuǎn)移酶(glycosyltransferases,GTs)活性快速分析方法缺失是現(xiàn)有酶法合成技術(shù)體系中亟待解決的兩個問題。針對上述兩個問題,本論文以提高HP寡糖酶法合成技術(shù)體系的效率和規(guī)�；瘧�(yīng)用潛力為目的,完成了兩部分工作:通過結(jié)構(gòu)均一確定模式底物,揭示了人源硫酸乙酰肝素(heparan sulfate,HS)N-脫乙酰基酶/N-硫酸基轉(zhuǎn)移酶4 N-Deacetylase/N-sulfotransferase 4,NDST4)的催化模式;建立了偶聯(lián)釀酒酵母尿苷二磷酸(uridinediphosphate,UDP)水解酶(yeast nucleoside diphosphatase,YND1)的 GT 反應(yīng)高通量篩選體系。1.硫酸乙酰肝素NDST4催化模式的研究N-脫乙酰基酶/N-硫酸基轉(zhuǎn)移酶(N-deacetylase/N-sulfotransferase,NDST)是HS生物合成途徑中糖鏈骨架合成完成后的第一個硫酸化修飾酶,其催化獲得的N位點硫酸化的葡糖胺是后續(xù)一系列硫酸基轉(zhuǎn)移酶和葡萄糖醛酸異構(gòu)酶底物識別的結(jié)構(gòu)基礎(chǔ)。HS糖鏈中硫酸根基團的排列形式?jīng)Q定了其與蛋白因子的結(jié)合能力,進而影響含有該糖鏈的蛋白聚糖的生物活性。因此,系統(tǒng)的研究NDST的底物特異性等催化機制不但對揭示HS生物合成控制機理具有重要的理論價值,而且相關(guān)結(jié)論可以直接指導(dǎo)HP寡糖的體外酶法合成。人體細胞內(nèi)存在4種具有不同的底物特異性NDST同工酶,共同調(diào)節(jié)HS的N位點硫酸化修飾。伴隨著結(jié)構(gòu)均一確定的HS寡糖酶法合成體系的建立,NDST1的催化模式已被充分闡明。但NDST其他3種同工酶的催化模式還未被充分研究。論文利用由昆蟲桿狀病毒系統(tǒng)(baculovirus expression system)表達的重組NDST4與一系列化學(xué)結(jié)構(gòu)確定的模式底物,在體外模擬HS生物合成過程中N位點硫酸化修飾過程,通過解析反應(yīng)產(chǎn)物的結(jié)構(gòu),揭示了 NDST4的催化機制。論文研究結(jié)果表明:(1)單獨的重組NDST4不具有N-脫乙酰基酶活性;(2)NDST4具有較強的N-硫酸基轉(zhuǎn)移酶活性;(3)與NDST1隨機結(jié)合底物糖鏈中的GlcNAc位點,定向催化的模式不同,NDST4的N-硫酸基轉(zhuǎn)移酶活性沒有表現(xiàn)出方向性。基于以上結(jié)論,該同工酶在HS糖鏈的體內(nèi)生物合成中不是調(diào)控糖鏈高硫酸化區(qū)域(NS domain)和低硫酸化區(qū)域(NAc domain)相間排列的關(guān)鍵酶;應(yīng)用方面,NDST4具備成為轉(zhuǎn)化葡糖胺(glucosamine,GlcN)為N位點硫酸化葡糖胺(N-sulfated glucosamine,GlcNS)工具酶的潛力。2.偶聯(lián)釀酒酵母UDP水解酶的Leloir-GT活性高通量篩選方法的建立GT是生物體內(nèi)負責催化糖苷鍵合成酶類,它不但是糖生物學(xué)研究重點之一,而且是糖生物工程重要的工具酶分子之一,廣泛用于體外糖鏈及糖綴復(fù)合物的酶法制備。絕大多數(shù)GT需要以核苷活化的單糖為給體,被稱為Leloir-GT。但嚴格的底物特異性限制了天然GT酶分子合成能力,產(chǎn)生了"糖鏈及其復(fù)合物的應(yīng)用價值"與"酶法合成能力缺陷"的矛盾。以酶法合成HP寡糖技術(shù)體系為例,骨架合成工具酶分子巴斯德氏菌(Pasteurella multocida)肝素前體聚合酶2(pmHS2)嚴格的單糖供體選擇性是制約現(xiàn)有體系效率的瓶頸之一。利用蛋白質(zhì)工程改造該GT的底物選擇性是突破艾杜糖醛酸(iduronic acid,IdoA)轉(zhuǎn)化這一瓶頸問題的有效手段。定向進化的成功在很大程度上依賴于針對GT活性的高通量篩選方法的建立。當前大多數(shù)Leloir-GTs活性測定方法基于質(zhì)譜和色譜分離方法等復(fù)雜儀器或方法,不滿足pmHS2分子定向進化的要求。針對以上問題,論文在可溶性重組表達釀酒酵母(Saccharomyces cerevisiae)高爾基體膜蛋白UDP水解酶(YND1)的基礎(chǔ)上,建立了一種Leloir-GT活性高通量篩選方法。我們通過將YND1對UDP的水解反應(yīng)、Leloir-GT反應(yīng)和P032-的磷鉬藍顯色反應(yīng)偶聯(lián)起來建立了一個簡便、靈敏、快速的高通量篩選體系,并用GAG骨架合成中最重要的3種GTs共同驗證了該體系的有效性和普適性。具體研究內(nèi)容包括:(1)通過將YND1(GeneID:856722)第1504-1554位的疏水性跨膜區(qū)用3組甘氨酸-絲氨酸重復(fù)堿基(502F-518H/GSGSGS)進行替換的方法,實現(xiàn)了釀酒酵母跨膜蛋白YND1以可溶且有活性的狀態(tài)在大腸桿菌中表達;(2)重組YND1的酶學(xué)性質(zhì)研究表明其最佳反應(yīng)條件與大多數(shù)Leloir-GT的理想反應(yīng)條件一致,表明重組YND1催化的核苷酸水解反應(yīng)可以實現(xiàn)與GT催化的糖基轉(zhuǎn)移反應(yīng)相偶聯(lián);(3)將GT反應(yīng)與重組表達的YND1偶聯(lián),優(yōu)化了反應(yīng)與分析條件,建立了一種建立了一種快速、簡便、準確性高的GT活性檢測方法;以糖胺聚糖骨架合成中重要的糖基轉(zhuǎn)移酶KfiA、pmHS2和KfoC為Leloir-GTs的模式工具酶分子,確定了該方法的有效性;(4)借助YND1偶聯(lián)GT反應(yīng)消除反應(yīng)副產(chǎn)物UDP潛在抑制作用,可以明顯促進GT催化反應(yīng),有效提高一定反應(yīng)時間內(nèi)目標糖鏈的得率。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R915

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