【作者】 薛梦阳；

【导师】 王鹏；

【作者基本信息】 山东大学 ， 微生物学， 2014， 博士

【摘要】 生物体内存在两种重要的氨基己糖,分别是N-乙酰氨基葡萄糖(GlcNAc)和N-乙酰氨基半乳糖(GalNAc),在结构和功能方面均发挥重要的作用。它们不仅是细菌细胞壁骨架和糖胺聚糖的基本组分,还存在于具有重要生物功能的糖蛋白或糖脂的糖链部分的核心结构中。合成含有这两种氨基己糖及其结构类似物的糖缀合物,对研究它们参与介导的生物过程及糖类药物开发具有重要的意义。研究糖链的生物学功能,首先需要获得结构明确且均一的糖缀合物。直接从生物来源分离得到的糖或糖缀合物的结构具有微不均一性；利用有机化学法合成糖缀合物的步骤复杂,且收率较低。利用糖基转移酶催化的反应具有高度区位特异性和立体特异性的特点,模拟生物体内糖链的合成步骤,在体外用酶法制备结构均一的糖链成为有机化学合成法的有效替代途径。但利用Leloir型糖基转移酶在体外大量制备生物活性寡糖仍然存在两个问题：1)用于寡糖合成的糖基转移酶的数量和种类有限；2)糖核苷酸供体底物数量有限、难以获得且价格昂贵。这两个制约糖生物学研究发展的问题已经严重阻碍了糖及糖缀合物在医药领域的研究和应用。糖核苷酸在化学结构上是核苷二磷酸或核苷一磷酸与不同单糖的异头碳羟基连接形成的衍生物,是单糖的活化形式,能够被糖基转移酶识别并添加到糖链中。实现糖核苷酸衍生物的体外大量制备,为生物活性寡糖的体外制备提供原材料,对糖生物工程重要产品开发、糖缀合物生理生化功能研究及药物开发具有重大意义。论文的目标之一是氨基糖核苷酸及其衍生物的酶法合成研究。主要是酶法高效合成UDP-GlcNAc/UDP-GalNAc结构类似物。人源的UDP-GalNAc焦磷酸化酶(Homo sapiens UDP-GalNAc pyrophosphorylase, AGX1)对GalNAc-1-P具有极高的催化活性,通过一步反应能催化合成UDP-GalNAc.通过构建重组载体,AGX1在原核表达系统中获得了高纯度的可溶性表达。论文中系统研究了AGX1的生物化学性质,AGX1在含有10mM MgCl2的pH8.0100mM Tris-HCl缓冲液中具有最佳活性,最适反应温度为37℃。AGX1对不同核苷三磷酸的底物适应性研究发现,该酶可以利用UTP/dUTP/dTTP作为底物,而对同样是嘧啶类核苷酸的CTP则完全没有催化能力。结合该酶的晶体结构,推测核苷三磷酸底物中的碱基部分C4-位环外氧原子与AGX1的特定氨基酸位点之间形成氢键相互作用从而能够与酶结合。CTP的C4-位上氨基的存在阻碍了氢键的形成,影响了底物与酶的结合。AGX1对不同糖-1-磷酸的底物适应性研究发现,C2-乙酰氨基在酶与底物识别结合过程中发挥重要作用。不含N-乙酰氨基的Glc-1-P不能被AGX1利用。酶动力学研究发现,当以UTP/dUTP/dTTP为NTP供体时,AGX1利用GalNAc-1-P的能力略高于GlcNAc-1-P。对核苷三磷酸的喜好顺序为：UTP>dUTP>dTTP。通常情况下,真核来源的焦磷酸化酶的底物适应性都比较有限,而AGX1却具有较为广泛的底物适应性,这是一个非常重要的发现,其意义在于可以利用该酶广泛的底物适应性在体外合成多种稀有核苷酸,对于糖基转移酶的生化性质研究和糖核苷酸物质库的丰富具有重要意义。基于AGX1的底物利用广泛性特点,我们研究了利用AGX1体外合成稀有糖核苷酸的技术路线,制备了2种稀有糖核苷酸,dUDP-GalNAc和dTDP-GalNAc,这些产物可以用于相关糖基转移酶的性质研究。空肠弯曲菌(Campylobacter jejuni)是第一个被发现含有蛋白N-糖基化过程的原核生物。论文第三章通过对空肠弯曲菌糖基化途径的分析,找到一种新型的UDP-GlcNAc焦磷酸化酶(Campylobacter jejuni UDP-GlcNAc pyrophosphorylase, CjGlmU)。CjGlmU在原核表达系统中得到了高纯度的可溶性表达。CjGlmU对不同糖-1-P底物适应性的研究发现,CjGlmU对Glc-1-P同样具有较高的反应产率,这表明C2-位N-乙酰基不是酶对底物识别的关键位点。但随着N-乙酰基上修饰基团空间位阻的变大,CjGlmU的催化能力也随之减弱,分析可能是较大的空间位阻影响了酶与底物的相互结合。C4-位羟基的构型是影响酶与底物识别及催化能力的关键因素,同样是以UTP作为供体底物,CjGlmU对GalNAc-1-P的催化效率仅为GlcNAc-1-P的12%左右。CjGlmU对不同核苷三磷酸底物的适应性的研究表明,CjGlmU对嘧啶类核苷酸底物的利用率要远远高于嘌呤类核苷酸底物。与EcGlmU(Escherichia coli K12来源的UDP-GlcNAc焦磷酸化酶)相比,CjGlmU具有更广泛的核苷三磷酸底物适应性。CTP能够很好地被CjGlmU利用,合成CDP-GlcNAc的效率在60%以上。CjGlmU可以催化合成16种稀有糖核苷酸,其中对7种稀有糖核苷酸的产率能够达到60%以上,其他的9种也均在20%左右。基于该酶广泛的底物适应性,我们在体外成功地制备了3种UDP-GlcNAc的衍生物,分别是CDP-GlcNAc, UDP-GlcNBu和UDP-GlcNPr。CjGlmU的研究工作为体外大量制备非天然糖核苷酸奠定了基础,利用该酶的催化反应,能够合成数十种稀有糖核苷酸。通过对产物的分离纯化,极大丰富了糖核苷酸化合物库,为糖基转移酶的活性研究和含非天然糖残基的糖缀合物的合成研究提供了丰富的供体底物。氨基糖核苷酸作为Leloir型糖基转移酶的糖供体,参与了生物体内重要糖缀合物的合成过程。论文的另一部分工作重点就是将合成制备的氨基糖核苷酸及其衍生物用于糖基转移酶合成复杂糖缀合物的研究中。糖基转移酶是体外制备生物活性寡糖的一类重要的工具酶,探索不同种来源的糖基转移酶并研究其生物化学性质,能够极大地促进糖生物学的研究和糖类药物的开发。论文第四章,我们克隆了一种来源于幽门螺杆菌(Helicobacter pylori)的p1,3-GlcNAc糖基转移酶(β1,3-N-acetylglucosaminyltransferase, HpLgtA),该酶以UDP-GlcNAc为糖供体,催化将GlcNAc以β1,3键连接到半乳糖残基上。该酶也是poly-LacNAc合成中的一个关键酶。在大肠杆菌表达系统中,该酶被过量表达为带有N-端MBP标签和C-端His6标签的可溶性蛋白。实验中使用了34种糖核苷酸研究HpLgtA的底物适应性,并对比了已知活性的来源于脑膜炎奈瑟氏球菌(Neisseria meningitidis)的同功能的糖基转移酶(NmLgtA)。HpLgtA与NmLgtA都可以利用其中的8种糖核苷酸作为底物,但利用特异性上存在差异。二者均对N-酰基带有修饰基团的UDP-GlcNAc结构类似物具有催化活性；N-酰基上取代基团空间位阻的不断增大使酶的催化活性逐渐降低。除此之外,HpLgtA还可以利用C6-位带有修饰基团的结构类似物。UDP-Glc和UDP-GalNAc均不是这两种糖基转移酶的合适底物。由此推测,N-酰基的存在及C4-羟基的构型是影响酶与底物相互作用的关键位点。二者氨基酸序列上38%的相似性也可以揭示这一发现。对其中3种底物的酶动力学研究表明,HpLgtA比NmLgtA具有稍高的催化效率,UDP-GlcNAc是二者的最适底物。随着N-酰基上修饰基团的空间位阻逐渐变大,二者的催化效率也逐渐降低。利用该酶较广泛的底物适应性,可以在体外制备含有GlcNAc结构类似物的糖链(poly-LacNAc),并对糖链功能的改变做进一步的研究。透明质酸(Hyaluronan, HA)是最简单的一种糖胺聚糖,以游离形式(不与蛋白共价结合)存在于生物体内。具有良好的水溶性与粘弹性,已经广泛应用在医药,美容等领域。透明质酸糖链的长度的和不同的化学修饰都能影响其生物学功能。论文第五章,利用来源于A型巴斯德氏菌(Pasteurella multocida)的透明质酸合酶(hyaluronan synthase, PmHAS)作为工具,以生物素标记的葡萄糖醛酸(GlcA-Biotin)作为受体底物,采用逐步合成的方法在体外制备了生物素标记的透明质酸二糖,三糖。并进一步选用单糖,二糖和三糖作为前体对PmHAS的聚合能力进行了研究。研究工作发现,PmHAS对单糖(GlcA或GlcNAc)不具有聚合能力。但能以生物素标记的单糖、二糖及三糖作为前体物质,利用UDP-GlcA和UDP-GlcNAc作为糖基供体,通过聚合反应,最终生成HA糖链。而且以三糖为前体时的聚合速率要远远高于二糖和单糖,前体的糖链长度越大,越有利于PmHAS催化的聚合反应的进行。综上所述,本论文建立了一套高效的酶法合成制备UDP-GlcNAc/GalNAc结构类似物的体系。利用真核来源的AGX1和原核来源的CjGlmU分别合成了6种和16种UDP-GlcNAc/GalNAc的结构类似物。同时系统研究了一种新型的幽门螺杆菌来源的p1,3-GlcNAc糖基转移酶的底物适应性。利用A型巴斯德氏菌来源的透明质酸合酶(PmHAS)在体外合成并分离制备了生物素衍生化的透明质酸二糖和三糖,并进一步研究了PmHAS的生物化学性质。更多还原

【Abstract】 Carbohydrate (or glycans), including monosaccharide, oligosaccharide, polysaccharide and glycoconjugates are wildly existing in all living organisms. They are not only essential components of cells but also participate in various important biopathways.There are two common N-acetylhexosamines in living orgamism. They are N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). They are not only essential components of bacterial cell walls and glycosaminoglycans, but also prevalent in the core structures of glycans in glycoproteins and glycolipids. Besides, they play important roles in various metabolic processes. Such as O-GlcNAcylation involved in regulating signaling pathways, and glycolipids which have GalNAc residues involved in cellular interaction, differentiation and other processes. Studying oligosaccharides, polysaccharides and glycoconjugates which containing these two monosaccharides or their derivatives will help us to understand physiological and pathological processes as well as developing pharmaceuticals.In order to research the biological roles of glycans, we need to prepare glycans which have defined and homogeneous structures. The structures of glycans isolated from biological sources have microheterogeneity. Chemical approach for synthesizing glycans has tedious process, strict conditions and often has low yield. Enzymatic synthesis, which mimics the biosynthetic pathway, is considered more attractive with the advantages of strone regio-and stereo-specificity, high conversion percentage, and mild reaction conditions.In the enzymatic synthesis approach, Leloir type glycosyltransferases (GT) are mainly used. But there still are two unsolved problems:1) Few GT which has broad substrate specificity could be used for large scale synthesis of glycans;2) Sugar nucleotides are expensive and hard to obtain.Sugar nucleotides, also known as active sugars, are constructed by linking the anometic carbon of monosaccharides to nucleotide diphosphates or nucleotide monophosphates. They are sugar donors of Leloir type glycosyltransferases, and important precursors in biological synthesis of glycans. Analogs of natural sugar nucleotides could be used to study the structure-function relationship of glycosyltransferases and medicinal chemistry. Due to the easier and fewer steps of de novo pathway of sugar nucleotide synthesis, it has been the common stratege for preparing sugar nucleotides.One aim of this thesis is to enzymatically synthesize UDP-GalNAc/GlcNAc and their derivatives. UDP-GalNAc pyrophosphorylase from Homo sapiens (AGX1) can catalyze the generation of UDP-GalNAc from GalNAc-1-P and UTP in one step. It shows higher enzymatic activity towards GalNAc-1-P. In Chapter2, AGX1was cloned and overexpressed in Escherichia coli BL21(DE3) with a N-terminal His6-tag. After purification by Ni-column, the purity of recombinant AGX1was higher than95%. The enzyme exhibited maximum activity in pH8.5Tris-HCl buffer with the presence of10mM Mg2+at37℃. Then we systematically studied nucleotide substrate specificity of AGX1during its uridyltransfer reaction, AGX1could use dUTP and dTTP as substrates and to generate their corresponding nucleotide sugars. AGX1had a finite NTP substrates tolerance when using GalNAc-1-P and GlcNAc-1-P as its sugar-1-P substrates. From the substrate specificity of AGX1and the crystal structure, we presumed that the exocyclic oxygen04of uracil base was crucial for substrate binding and enzyme catalysis. Small changes of2’-hydroxyl group at ribose (dUTP/dTTP) make no differences in substrate recognition, but lead to slight decrease in the enzyme catalysis. When using purine nucleotide triphoshpate substrates, the enzyme-substrate binding and enzymatic activity was affected due to the large steric hindrance.Camphylobacter jejuni contains a post-translation N-glycosylation system. In Chapter3, we study the substrate specificity of a novel UDP-GlcNAc pyropyosphorylase from C. jejuni (CjGlmU), which has34%amino acid sequence similarity with UDP-GlcNAc pyrophosphoylase from E. coli K12 (EcGlmU). This enzyme show high enzymatic activity after one-step Ni-NTA purification. CjGlmU exhibited broader substrate spcificity than EcGlmU. It could use7kinds of NTPs as substrates, especially for CTP, the conversion percentage could reach62%. This enzyme prefer pyrimidine nucleotide triphosphate than purine nucleotide triphosphate. The sugar-1-P substrate specificity showed that this enzyme could tolerate N-acetyl modified GlcNAc-1-P derivatives. Finally,3kinds of unnatural sugar nucleotides were synthesized. We have set up a relatively easy strategy to prepare unnatural sugar nucleotides.Poly-N-acetyllactosamine (poly-LacNAc) is the most common and important sugar structure of cell-surface of membrane glycoconjugates which play a significant role in cell-cell interactions and cell-matrix adhesion. β-1,3-N-acetylglucosaminyltransferase (LgtA) from Helicobater pylori and Neisseria meningitidis have ever been widely used in the enzymatic and chemoenzymatic synthesis of poly-LacNAc both in vivo and in vitro. We show here the cloning and expressing of HpLgtA and NmLgtA as an N-MBP and C-His6-tagged fusion protein in E. coli. The basic characteristics of both LgtAs were systematically studied. In addition, a library of34UDP-sugars was used to study the donor substrate specificity of HpLgtA as well as NmLgtA. Overall, HpLgtA and NmLgtA have quite different tolerance toward substrate modifications. Both HpLgtA and NmLgtA can use uridine5’-diphosphate-N-acetylglucosamine (UDP-GlcNAc), and some of its C2’-modified derivatives as donor substrates. Besides, HpLgtA could also use several derivatives with C6’-modifications. The kinetics study shows that HpLgtA has a relatively higher enzymatic efficiency than NmLgtA. The donor substrate promiscuity of HpLgtA and NmLgtA will allow efficient chemoenzymatic synthesis of diverse poly-LacNAc derivatives for binding study with galectins and for studies of their possible therapeutic applications.Hyaluronan is one kinds of important glycosaminoglycans, it takes various important parts in several biopathways. In chapter5, we use the hyaluronan synthase from Pasteurella multocida (Type A)(PmHAS) as a tool, use GlcA-Biotin as starting material for synthesizing biotinylated hyaluronan oligosaccharides by stepwise. We successfully synthesized HA disaccharide and trisaccharide. Furthmore, these HA oligosaccharides were used to study the relationship between acceptor length and PmHAS catalytic activity. PmHAS exhibited the highest activity toward trisaccharide, and showed no activit towards no modified monosaccharide:GlcA and GlcNAc. This new discovery will help us to better understand the catalytic mechanism of PmHAS.In summary, this thesis established a strategy of enzymatic synthesis and chromatographical preparation of UDP-GlcNAc/GalNAc derivatives with one enzyme from bacteria and one enzyme from human.5kinds of unnatural sugar nucleotides were prepared in vitro. We also study the sugar nuclotides substrate specificity of a novel glycosyltransferase from H. pylori. Besides, we successfully synthesized biotinylated hyaluronan oligosaccharides by stepwise stratege, also used them to study the relationship between accetpor length and PmHAS catalytic activity.更多还原