【作者】 李磊；

【导师】 王鹏；

【作者基本信息】 山东大学 ， 微生物学， 2010， 博士

【摘要】 碳水化合物是生物体中最为重要的生物分子之一,它们不仅是生物体不可或缺的结构组成部分,更在许多关键生物过程中发挥着不可替代的作用。碳水化合物的存在形式多种多样,如寡糖、多糖以及糖缀合物(包括糖蛋白、糖脂、脂多糖等)。糖型的改变与特定病理状态,如癌症和炎症密切相关,显示出其在临床诊断中的应用潜力以及作为药物开发靶点的可能性。糖及糖缀合物由糖基转移酶(GT)催化合成,反应中GT的底物特异性直接决定了所形成的糖链结构。与自然界中复杂多样的糖构型相对应,GT是一个大的酶家族。据统计,在业已测序的500多个物种的基因组中,1-2%的基因为GT,然而目前鉴定的GT却仅占其中的一小部分。GT催化一个糖基供体上的糖残基转移到一个受体上,即所谓的转糖基反应(Transglycosylation)。绝大多数的GT以糖核苷酸为糖基供体,如UDP-Gal、GDP-Fuc等,这样的GT称为Leloir型GT。另外磷酸脂连接的糖也可作为糖基供体,如dolichol连接的甘露糖、葡萄糖或寡糖作为糖蛋白合成过程中的糖基供体,这样的GT称为non-Leloir型GT。GT的受体可以是单糖、寡糖、多糖、多肽、蛋白、脂、有机小分子甚至DNA。生物活性寡糖的大量制备是现代医学中糖类应用和研究的前提条件。其合成主要通过酶法进行,而酶法合成主要利用Leloir型糖基转移酶。该酶法合成中仍存在两个问题：1)可用于寡糖大量合成的糖基转移酶数量有限；2)糖核苷酸供体昂贵且难以大量获得。这两个问题的有效解决将直接促进寡糖在医药领域的研究和应用。细菌中含有丰富和结构多样的多糖,其中主要的类型是共价结合于细胞表面的O-多糖(O-抗原)。O-多糖由多拷贝(可以多达100个)寡糖重复单元(O-Repeating Unit)构成,它们与核心寡糖和类脂A共价结合在细胞表面形成脂多糖。O-PS在不同细菌间存在巨大的多样性,例如在大肠杆菌(Escherichia coli)中发现了181种不同结构的O-PS,而沙门氏菌(Samonella)中多达90种。根据O-PS的结构不同,细菌菌株被分成不同的O-血清型(O-serotype),例如E. coli 086和0157。越来越多的证据表明O-PS在关联细菌与宿主环境、保护细菌不受免疫清除中扮演着重要角色,且参与细菌感染。因此,对O-多糖生物合成途径的研究有助于以其为靶点的医药试剂的开发。糖基化是真核生物中最为常见的翻译后修饰过程,利用现有的基因组／蛋白库分析推测,自然界中所有的蛋白中50%以上为糖蛋白。糖基化在生物系统中通过多种方式调控其功能,大量研究表明糖在许多细胞识别过程中起着重要作用,如肿瘤转移、细胞粘附、病原体入侵和免疫反应等。另外,更多研究表明,糖基化能够影响蛋白质的生物合成、折叠、抗原性、免疫原性及其在血浆中的半衰期,这些特性应用在增强蛋白药物药效、延长其在血浆中的半衰期上已经获得了成功。因此对糖基化和其相关酶的研究鉴定有助于医药领域糖蛋白药物的开发。本论文目标之一就是解决以上几个糖生物学相关问题。我们从大肠杆菌O-多糖生物合成基因簇鉴定了一个UDP-GlcNAc/Glc 4-Epimerase,对它的理化研究揭示了其底物适应性、动力学等性质,为其应用于寡糖合成提供了重要依据。另外我们结合糖芯片技术和蛋白体外表达技术建立了高通量鉴定糖基转移酶的方法,为今后GT的鉴定提供了一个快速有效的平台。我们表达了O-多糖生物合成基因簇中的所有关键基因,对其生物合成途径进行了体外重建,为该途径详细机理研究提供了一个完整的模型。最后,我们建立了过量表达和纯化细菌N-糖基化中心酶PglB的技术,为其生化和晶体学研究提供了条件。UDP-Gal和UDP-GalNAc为重要的转糖基反应供体,在寡糖(如ABO-antigen、Globo-H)合成中必不可少。然而它们制备困难、价格昂贵,一个有效的解决方法为反应中加入UDP-GlcNAc/Glc 4-Epimerase,和数倍价格以下的UDP-Glc或UDP-GlcNAc。E. coli O86.B7 O-多糖合成基因簇的gne被认为翻译一个UDP-GlcNAc 4-Epimerase。我们对其进行了克隆、过量表达和生化鉴定。毛细管电泳分析表明Gne同时催化UDP-Glc/UDP-Gal和UDP-GlcNAc/UDP-GalNAc之间的转化。Gne对UPD-Glc、UDP-Gal、UDP-GlcNAc和UDP-GalNAc的Km值分别是370、295、323和373μM,与其它来源的此类酶相比较,发现Gne为双功能酶。另外Gne对UDP-GalNAc和UDP-Gal的kcat/Km值是其对UDP-GlcNAc和UDP-Glc值的2至4倍,说明Gne催化UDP-GalNAc和UDP-Gal的活性稍高。将306位丝氨酸突变为苯丙氨酸(增大空间位阻)后Gne丧失了转化乙酰化底物的活性,而保留转化非乙酰化底物的活性,说明306位丝氨酸在决定Gne底物特异性上有重要作用。Gne是细菌O-多糖合成基因簇中发现的第一个双功能的差相异构酶,将该酶应用于寡糖合成将大大降低成本。糖基转移酶是生物界中一大类酶,目前已测序的基因组中发现近40000个GT基因。然而GT鉴定不仅困难而且繁琐,影响了其在寡糖合成中的应用。我们初步建立了一种高通量糖基转移酶活性鉴定的方法。该方法主要分为三个步骤：1)糖基转移酶克隆：本研究选择了来源于细菌的100个GT进行鉴定。为了避免针对每个酶设计酶切位点克隆,我们选择了更高效、快速的LIC克隆方法,将所有97个成功扩增的基因片段克隆入了LIC质粒pMCSG7。2)蛋白体外表达：我们选择了Expressway Cell-Free E. coli Protein Expression System对所有97个目的蛋白进行表达。表达在4小时内完成,western blot检测到77个蛋白表达。3) MALDI-MS Label Free检测：我们将25种单糖、寡糖固定与金分子层形成糖芯片,然后选择7种糖核苷酸对每个酶进行活性测定,总反应数为34300。反应后清洗去除杂质后直接进行MALDI-TOF/MS检测,无需标记。使用该方法我们成功鉴定获得了9个新的GT。细菌多糖是细菌细胞表面的重要组成部分,在诸如宿主-细胞相互作用中起到关键作用。其合成途径研究主要使用体内基因操作,因此其过程中诸多问题仍然未能解决。我们化学合成了O-多糖生物途径起始底物GalNAc-PP-Und,并使用过量表达的相关糖基转移酶顺序合成了RU (Repeating Unit)-PP-Und。同时我们采用伴侣蛋白共表达的方法表达了O-PS聚合酶Wzy,并纯化得到具有活性的蛋白。使用放射性同位素检测的方法,我们证实Wzy在体外催化了O-PS的合成。随后我们纯化了来自086不同型菌种的O-PS链长控制蛋白Wzz,将其与Wzy共同反应,体外合成了与天然LPS具有相同表型的O-PS。本研究使用化学-酶法体外重建了大肠杆菌O-多糖的wzy-依赖型途径,为今后研究该途径详细机理提供了一个完整的模型。一直以来,人们普遍认为N-糖基化仅存在于真核生物细胞中,直到本世纪初,发现人类肠道致病菌空肠弯曲杆菌中存在一个类似真核生物的N-糖基化途径,这也是目前细菌中发现的唯一N-糖基化途径。膜蛋白PglB是该途径中的关键酶,与真核生物中寡糖基转移酶的STT3蛋白同源,作用是转移寡糖到蛋白的特定氨基酸上。本论文第五章建立了大量表达纯化PglB蛋白的方法,并对其整体拓扑学结构进行了生物信息学预测与实验证明。我们将PglB表达于E. coli C43(DE3)中,表达量可达1 mg/L培养物。使用化学合成的GalNAc-PP-Und为底物供体,体外酶活检测证明该过量表达的酶为正确折叠的活性蛋白。该反应证明PglB是寡糖基转移反应中唯一作用的酶,并且进一步证实了PglB对糖基供体特异性不强。另外,使用经典的PhoA/LacZ融合的方法,我们证明了PglB含有11个跨膜结构域,且C端为一个大的周质空间结构域。这与真核生物STT3蛋白相同。总之,本论文以糖基转移酶等的生化研究为出发点,建立了O-多糖生物合成途径体外模型,提高了人们对细菌多糖生物合成的认识；发展了高通量糖基转移酶活性鉴定的方法,为GT鉴定提供了快速高效的平台；得到了大量高纯度的寡糖基转移酶,为N-糖基化机理研究提供了前体。更多还原

【Abstract】 Carbohydrates (also known as Glycans) constitute one of the major classes of bio-macromolecules in living organisms, they not only are essential components of cells but also play important roles in a variety of vital biological processes. Carbohydrates are of diverse structural complexities, they exist as oligosaccharides, polysaccharides and glycoconjugates (including glycoprotein, glycolipid, lipopolysaccharides). Specific changes in glycan profiles have been associated with certain disease states such as cancer and inflammation, illustrating the potential of using glycans in clinical diagnosis and perhaps as targets to develop therapeutics.The biosynthesis of glycans and glycoconjugates is catalyzed by glycosyltransferases (GT), which directly determined the structure of the glycans. Corresponding to the diverse structural complexities of glycans, GT constitute one of the largest enzyme families in nature. Statistically, around 1-2% of the genes in the sequenced genomes are putative GT, whereas only a small portion of which were characterized. GT catalyze the transfer of glycan moieties from glycan donor to acceptor to form a specific glycosidic bond. Most of the GT utilize sugar nucleotides (such as UDP-Gal, GDP-Fuc) as donors, which are called Leloir type GT. Others could utilize lipid (such as dolichol) linked sugar as donor, and called non-Leloir type GT. The acceptor of GT could be monosaccharides, oligosaccharides, polysaccharides, polypeptides, proteins, lipids, small molecules and even DNA.The application of glycans in modern medical science relies on large scale synthesis of such molecules. Enzymatic synthesis is the major approach for the synthesis, and Leloir type GT are mainly used. However, there are two unsolved problems:1) Few GT can be used for large scale glycan synthesis; 2) Sugar nucleotides are both expensive and hard to obtain.O-Polysaccharides (OPS) is one of the major bacterial polysaccharides, constituted by O-repeating units (up to 100 copies). O-PS covalently linked to core oligosaccharide and lipid-A to form complete LPS. O-PS exhibit vast structural complexities, for example, there are 181 O-PS with distinct structures in E. coli and 90 in Samonella. Based on the structural complexities, bacteria are divided into different O-serotypes, such as E. coli 086 and 0157. Increasing evidences show that O-PS play essential roles in bacteria-host interactions, and involved in bacterial infection. Thus, investigations on O-PS biosynthesis pathway will facilitate the development of pharmaceutics targeting on such bacteria.Glycosylation is one of the most common posttranslational modifications of proteins in eukaryotes. A recent analysis on the genome/protein databases revealed that more than half of all proteins in nature would be glycoproteins. Glycosylation modules protein’s structure and function in biological systems in many ways. For example, the oligosaccharide components in glycoproteins are implicated to play roles in many intercellular recognition processes such as cell adhesion, tumor metastasis, pathogen infections, and immune responses. On the other hand, glycosylation can influence protein’s biosynthesis, folding, antigenicity, immunogenicity, serum half-life. Such properties have been utilized in development of glycoprotein pharmaceutics. Thus, investigations on details of protein glycosylation will facilitate the development of glycoprotein pharmaceutics.We targeted on these glycobiology problems in this dissertation. Firstly, We characterized a bifunctional UDP-GlcNAc/Glc 4-Epimerase from E. coli O-PS biosynthesis gene cluster, provided biochemical details that facilitate the application in oligosaccharide synthesis. Secondly, we developed a high-throughput method for indentifying GT activities, provided a platform for rapid and efficient GT identification. Thirdly, we in vitro reconstituted the O-PS biosynthesis pathway, provided a model for detailed mechanism investigation toward this pathway. Lastly, we have overexpressed the first bacterial oligosaccharyltransferase, Pg1B, which makes the investigation related to N-glycosylation more easier.UDP-Gal and UDP-GalNAc are important sugar donors in the synthesis of a variety of human related antigens. However, they are hard to prepare and significantly expensive, one way to solve this problem is the addition of UDP-GlcNAc/Glc 4-epimerase into the reactions. A gne gene in E. coli O86:B7 O-PS gene cluster were thought to encode such an enzyme. To confirm this annotation, overexpression, purification and biochemical characterization of Gne was performed. By using capillary electrophoresis, we showed that Gne can catalyze the interconversion of both UDP-GlcNAc/GalNAc and UDP-Glc/Gal. The Km values of Gne for UDP-Glc, UDP-Gal, UDP-GlcNAc and UDP-GalNAc are 370, 295,323, and 373μM, respectively. The comparison of the kinetic parameters of Gne from E. coli 086:B7 to those of other characterized UDP-GlcNAc/Glc 4-Epimerases indicated that it is a bifunctional UDP-GlcNAc/Glc 4-Epimerase. Moreover, the calculated kcat/Km values for UDP-GalNAc and UDP-Gal are approximately two to four times higher than those for UDP-GlcNAc and UDP-Glc, suggesting that Gne is slightly more efficient for the epimerization of UDP-GalNAc and UDP-Gal. One mutation (S306Y) retained the activity for non-acetylated substrate, but totally abolished the activity for N-acetylated substrate, indicating that residue S306 plays an important role in the determination of substrate specificity.There are nearly 40,000 putative GT genes among the sequenced genomes. However, due to the difficulty in characterizing such enzymes, only a very small portion of them were used in oligosaccharide synthesis. Herein, we developed a high-throughput GT indentification method, including mainly three steps:1) Rapid GT cloning:LIC were used to clone 97 GT. LIC does not need to design specific restriction sites for each GT, it uses less DNA and less time-consuming; 2) In vitro protein expression:Expressway Cell-Free E. coli Protein Expression System were used to express 97 GT, the expression was finished within 4 hours, and 77 of them were detected in western blot; 3) MALDI-MS Label Free Detection:we prepared a glyco-array with 25 acceptors and 7 donors. All the enzymes were analyzed with each donor and acceptor, the number of total reactions was 34300. The bio-chips were rinsed and applied to MALDI-TOF/MS directly. We found 9 new GT using this method.Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria-host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach that enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared GalNAc-PP-Und, the E. coli 086 oligosaccharide repeating unit was assembled by means of sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy using a chaperone coexpression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of more substrates revealed a defined mode of recognition for Wzy toward the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well defined system for further underpinning molecular details of this biosynthetic pathway. Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, Pg1B, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous Pg1B proteins. Pg1B was overexpressed in E. coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified Pg1B was verified using a chemically synthesized sugar donor:GalNAc-PP-Und and a synthesized peptide acceptor. The result confirms that Pg1B is solely responsible for the oligosaccharyltransferase activity and complements the finding that Pg1B exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of Pg1B using the PhoA/LacZ fusion method. The topological model shows that Pg1B possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common Ncyt-CPeri topology with 11 transmembrane segments for the STT3 family proteins.In summary, through detailed investigation on GT, we established an O-PS in vitro biosynthesis model, enriched the basic knowledge of bacterial polysaccharide biosynthesis; developed and high-throughput enzyme assay method, provided a platform for rapid GT identification; in addition, we obtained large quantities of Pg1B protein, facilitated further investigation on the mechanisms of N-glycosylation.更多还原