【作者】 杨晓云；

【导师】 耿美玉；

【作者基本信息】 中国海洋大学 ， 药物化学， 2007， 博士

【摘要】 N-糖链在细胞通讯、生长、分化中起重要的作用,文献报道有证据表明N-糖链在神经发育中也起重要作用。目前糖组学(Glycomics)是后后基因组的研究重点。糖基转移酶是广泛存在的一大类酶,参与了聚糖、糖苷和复合糖类中糖部分的生物合成,具有高度的底物专一性。目前国际上关于糖基转移酶的研究较为初步,与其他类型的酶类相比,如参与代谢的酶类等,在酶的表达,受哪些因素调控等方面研究不够透彻,而且其涉及的生物学效应则更为广泛,复杂。糖基转移酶生物功能的完全阐释将揭示庞大的相关生物信息。N-乙酰氨基葡萄糖基转移酶V(N-acetylglucosaminyltransferase V,GnT-V/Mgat5,EC 2.4.1.155)作为重要的高尔基体糖基转移酶,是糖蛋白N-糖链加工酶之一,具有决定N-糖链类型及复杂型糖链结构的重要作用,催化GlcNAc基团转移至N-糖链核心α1,6臂的α-甘露糖的反应,形成N-糖链的β1,6分支结构,能形成2、3、4天线的N-糖链产物,在蛋白质翻译后修饰中起重要作用,而蛋白糖基化则是生物活性蛋白发挥其生物功能的必要条件之一,其中包括了具有重要功能的生长因子的受体膜蛋白的修饰。N-乙酰氨基葡萄糖基转移酶V是重要的高尔基体糖基转移酶,参与了糖蛋白N-糖链的合成,具有对糖蛋白翻译后糖基化共翻译修饰的重要功能。而且内质网中的蛋白质糖基化共翻译修饰过程能促使蛋白进行正确折叠,同时蛋白质糖基化共翻译修饰也是真核细胞存活必需的。研究发现N-乙酰氨基葡萄糖基转移酶V的缺失能引起受体N-糖链异常,糖基化异常能影响某些类型的细胞表面的受体功能,导致受体信号转导的改变。因此本文构建GnT-V高表达细胞株研究糖蛋白糖链变化对受体功能的影响。神经生长因子NGF是最早发现的神经生长因子,作为重要的神经营养因子,能调节神经的生长、发育、分化,通过高亲和性的受体TrKA调节营养神经元,不仅在成神经细胞瘤中,NGF受体TrkA表达升高与良好的预后相关,而且在神经退行性病变中,如帕金森和阿尔茨海默氏症(AD),NGF也是治疗药物研究的热点。NGF通过高亲和性的受体引起轴突生长细胞分化,而低亲和性受体引起凋亡,微环境中NGF过低则不能引起轴突生长。PC12细胞是大鼠肾嗜镉瘤细胞,具有神经细胞的特点,是研究NGF神经轴突生长良好的细胞模型,文献报道,在正常状态下50-100 ng/mL左右的NGF刺激几天后能诱导PC12细胞轴突生长。因此选取该细胞作为研究细胞模型,观察低浓度NGF刺激下(降低4-8倍)GnT-V高表达后对细胞的影响,而此低浓度下,NGF单独不足以诱导轴突生长。人神经生长因子受体(Trk A)含有4个潜在的N-糖链糖基化位点,该位点在神经营养因子受体Trk家族中高度保守,提示糖基化可能有重要的作用,另外还有9个不太保守的N-糖链糖基化位点。研究发现,成熟的Trk A受体在保守和可变的N-糖链糖基化位点均有糖基化。这些位点的糖基化有两种不同的功能,一是Trk A受体糖基化是阻止配体不依赖的激活所必需的,去糖基化的Trk A受体核心蛋白在没有配体激活时也是磷酸化的并且显示组成性激酶活性作用于信号分子Shc和PLC-γ;二是Trk A受体糖基化是受体定位于细胞膜表面所必需的,只有在细胞膜表面的受体能引发Ras/Raf/MAP激酶通路。用激光共聚焦显微镜发现没有糖基化的活性Trk受体陷入在细胞内,不能定位细胞膜,而且没有糖基化的活性Trk A受体不能激活Ras-MAP、MEK和ErK激酶通路,因此,没有糖基化的Trk A受体核心蛋白不能诱导PC12细胞神经元的分化。而且文献报道去糖基化的Trk不能应答其配体NGF,即衣霉素预处理稳定转染的高表达Trk的PC12细胞后,抑制Trk糖基化,改变了Trk蛋白上GM1(ganglioside,神经节苷脂,酸性糖鞘脂)结合位点,失去了与GM1形成复合物的能力。而且文献报道细胞膜N-糖链在神经胚胎发育中有重要作用,值得注意的是GnT-V在神经元细胞分化时期此酶的表达升高2-3倍,而GnT-V高表达引起的糖基化修饰的Trk A受体核心蛋白对其功能的影响和对PC12细胞神经元的分化影响未见报道。目前N-糖链与肿瘤侵袭转移的关系近年来备受关注,尤其是与GnT-V相关的β1,6分支结构的研究。研究表明在恶性肿瘤中,N-乙酰氨基葡萄糖转移酶V活性增高,其催化产物β1,6分支也增加,肿瘤细胞表面β1,6分支存在能促进其入侵基膜,β1,6分支已成为乳腺癌和结肠癌肿瘤恶化的标志。细胞膜N-糖链在个体发育和肿瘤生成中有显著变化提示N-糖链在细胞分化和细胞增殖转移中有重要作用。而GnT-V在神经中的生物学作用还未见报道。在神经发育中,复合型N-糖链尤其是神经组织胚胎发育必须的。Taguchi et al.报道指出肿瘤的侵袭与神经轴突的应答过程相似的概念,提示在肿瘤转移中起作用的糖基转移酶GnT-V很可能在神经中也起重要作用。神经轴突的生长与肿瘤的迁移有很多相似之处,包括细胞骨架的变化和参与的信号调节通路。至今关于GnT-V在神经中的作用尚无文献报道,我们主要在此方面进行阐释。综上所述,本文选NGF为靶点,研究GnT-V转染PC12细胞后GnT-V高表达对NGF及受体诱导的轴突生长通路的变化。考虑到目前GnT-V在神经中的生物学作用未见报道,因此本文首次在此方面进行初步的探索研究,首先成功构建转染稳定表达GnT-V的PC12细胞和mock组细胞,进行了糖链和蛋白鉴定。研究NGF诱导轴突生长的影响,结果表明,GnT-V高表达,能修饰神经生长因子受体,引起NGF低浓度下神经轴突的生长,转染细胞轴突生长明显增强,提示GnT-V表达增加对神经元分化具有调节作用。研究了GnT-V对PC12细胞的生物效应,这种促进PC12细胞突起生长的作用,是促分化作用。为了阐释N-糖链的变化,将NGF受体TrKA免疫沉淀后,用凝集素PHA-L分析GnT-V酶作用产物β1,6分支结构糖链。同时免疫沉淀TrKA受体,测定NGF受体的信号通路的激活,研究了转染稳定表达GnT-V的细胞,mock组细胞TrKA受体磷酸化随时间的变化,TrKA受体用抗TrKA抗体免疫沉淀垂钓分析磷酸化水平。结果表明,转染稳定表达GnT-V的细胞比mock组细胞磷酸化水平明显升高。GnT-V转染PC12细胞对NGF刺激轴突信号转导通路的敏感性增强。基于前期研究发现低浓度的NGF能引起PC12细胞轴突生长的现象,进一步研究GnT-V对TrKA受体作用的机理。研究发现GnT-V高表达,免疫沉淀神经生长因子细胞膜受体TrKA糖链β1,6分支结构增加,说明此酶GnT-V能直接糖基化修饰神经生长因子高亲和性的受体TrKA,同时发现糖基化修饰的高亲和性的受体TrKA的磷酸化增强。为进一步解释其机制,我们研究了不同时间点TrKA受体的内吞,NGF-β孵育不同的时间点,测定转染稳定表达GnT-V的细胞,mock组细胞NGF-β与受体TrKA结合内吞的情况。结果发现转染稳定表达GnT-V的PC12细胞与mock组相比,TrKA受体内吞延迟,提示GnT-V高表达引起的N-糖链变化,使得NGF受体TrKA内吞延迟,进而增强了受体信号转导通路,导致神经轴突的生长。这些结果表明,GnT-V高表达,修饰神经生长因子受体,增强其功能,能引起低浓度NGF刺激下神经轴突的生长, GnT-V在神经中可能通过修饰糖蛋白受体调节NGF诱导的信号通路,可能在与神经生长因子受体相关的神经退行性病变中起作用。本文研究发现N-乙酰氨基葡萄糖基转移酶V糖基化修饰神经生长因子受体TrKA,能调节TrKA受体的功能,激活TrKA受体介导的轴突生长的信号通路。此论文的研究意义在于首次阐释了N-乙酰氨基葡萄糖基转移酶V对神经生长因子受体的糖基化修饰作用对TrKA受体功能的影响,其研究结果为研究N-糖链在神经中的作用提供了深层次的理论依据,拓展了对N-乙酰氨基葡萄糖基转移酶V在神经中作用的认识。发现N-乙酰氨基葡萄糖基转移酶V具有促神经分化作用,这种对神经生长因子受体TrKA的调节作用,可能与神经生长因子相关的神经退行性病症的药物设计提供了新的靶点,拓展了糖基转移酶研究的领域。更多还原

【Abstract】 N-glycan plays an important role in cell communication, differentiation, and survival. Several lines of evidence have suggested that N-glycan involves nerve development, but its precise mechanism is yet to be elucidated. Glycomics is the most important part of post post-geneomics. The processing of N-glycans in the Golgi apparatus is the most important step in the biosynthesis of the complex type of Asn-linked glycoproteins. It is known that at least six N-acetylglucosaminyltransferases (GnTs), designated as GnT I-VI, are involved in the biosynthesis of a core structure of the complex type of N-glycans with specific enzyme substrate. The glycosytltranferases are a large family, which is the part of glycomics, and elucidation of the mechanism of them will be explained great informations to cells and proteins.N-Acetylglucosaminyltransferases V (GnT-V/Mgat5) plays a pivotal role in the processing of N-linked glycoproteins in the Golgi apparatus. N-acetylglucosaminyltransferase V (GnT-V /Mgat5, EC 2.4.1.155) is one of the pivotal glycosyltransferases of Golgi enzyme which participates in the branching of N-glycans (N-glycans), and produces an unique sugar chain structure, aβ1,6 branching GlcNAc structure (GlcNAcβ1,6Manα1,6-) in C2C2,6 tri- or C2,4C2,6 tetra-antennary N-glycan products (C2C2, C2,4C2, C2C2,6 and C2,4C2,6 express the linkages of the bi-, tri- and tetra-antennae to the carbon atom positions ofα1,3 andα1,6 mannosides in the N-glycan core). This enzyme can motify many active glycoprotein for the posttranslational modifications, which is essential for their function, and the most important proteins are the receptors of the growth factor family. Co-translational modification of proteins in the endoplasmic reticulum by N-glycosylation facilitates their folding and is essential in single-cell eukaryotes. Aberrant glycosylation can affect the functions of several types of cell surface receptors by altering the signaling pathways mediated by these receptors.The modulation of receptor transcript and protein levels by abnormal N-glycosylation resulting from deletion of GnT-V appears to be a novel observation.NGF was the first identified member of a family of neurotrophic factors that function both in vitro and in vivo to promote neuronal survival and differentiation. The biological effects of NGF are mediated by high-affinity binding to cell-surface glycoprotein receptors called TrKA (for tyrosine kinase receptor) encoded by the trk protooncogene [now designated Ntrk (NTRK) for neurotrophic tyrosine kinase receptor in the human (mouse) genome data base]. NGF induces differentiation of the rat pheochromocytoma cell line PC12 into cells resembling sympathetic neurons, thus providing a well-characterized model for the investigation of the mechanism of action of NGF. Following the application of NGF to PC12 cells, long-term transcription medicated events occur. These include the extension of neurites and the acquisition of a differentiated phenotype that is characterized by the development of electrical excitability and the biosynthesis of neurotransmitters.The human nerve growth factor receptor (TrkA) contains four potential N-glycosylation sites that are highly conserved within the Trk family of neurotrophin receptors, and nine additional sites that are less well conserved. Using a microscale deglycosylation assay, it is showed that both conserved and variable N-glycosylation sites are used during maturation of TrKA. Glycosylation at these sites serves two distinct functions. First, glycosylation is necessary to prevent ligand-independent activation of TrKA. Unglycosylated TrKA core protein is phosphorylated even in the absence of ligand stimulation and displays constitutive kinase activity as well as constitutive interaction with the signaling molecules Shc and PLC-γ. Second, glycosylation is required to localize TrKA to the cell surface, where it can trigger the Ras/Raf/MAP kinase cascade. Using confocal microscopy, it is showed that unglycosylated active TrK receptors are trapped intracellularly. Furthermore, the unglycosylated active TrKA receptors are unable to activate kinase in the Ras-MAP kinase pathway, MEK and ErK.Consistent with these biochemical observations, unglycosylated TrKA core protein does not promote neuronal differentiation in TrK PC12 cells even at high levels of constitutive catalytic activity. The highly conserved nature of these N-glycosylation sites across a broad spectrum of species suggests these sites have an important function. TrKA contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain, and it is thought that activation of this kinase represents the initial step in the intracellular signal transduction pathway of NGF. TrKA has 13 glycolated sites, when the addition of tunicamycin, a potent inhibitor of N-glycosylation into culture medium, TrKA cannot phosphated. These data suggested that glycosylated TrKA is required for the receptors function. However, it is no reported that GnT-V modifies TrKA receptor glycosylation and effect the receptors function to regulate the neuronal differentiation, such as PC12 cell differentiation. Further research is needed to delineate the precise mechanism of modification of TrKA glycoprotein. Most importantly, GnT-V activity increased to approximately two- to three-fold the initial level with increasing mRNA level during nerve cellular differentiation. We demonstrate in the present study that GnT-V role on TrKA enhancement of NGF induction of neurite outgrowth. These effects are due to enhancement of the TrKA-associated tyrosine kinase activity elicited by NGF.There is a growing body of in vivo and in vitro evidence indicating that GnT-V are highly involved in cancer progression and metastasis andβ1, 6 GlcNAc branching is a key structure associated with cancer metastasis. The marked changes in the sugar chain structures of cell surface membrane occurring during ontogenesis and oncogenesis suggest that they play pivotal roles in cell differentiation and proliferation. The resultant pathology showed that complex type N-glycans are required for normal embryonic development, especially of neural tissues. Similarities between tumor invasion and physiologic responses, such as neurite outgrowth, have been noted (Taguchi et al., Nature 2000).The axon outgrowth and cancer metastasis are similarities in many ways, including cytoskeleton changes and signal pathways. However, it is unclear that the effect of GnT-V in nerve. Therefore, we study on the role of GnT-V regulation NGF receptor TrKA glycoprotein by over expression. In the present study, we have successfully contraction of GnT-V gene-transfected PC12 Cells and mock cells. And the level of GnT-V expression was higher in GnT-V gene-transfectioned PC12 cells than mock and PC12 cells. We have investigated the biological effect of GnT-V gene transfection into PC12 cells and found that they were sensitively responsive to NGF as indicated by the axon outgrowth. Those findings suggest that the modification of glycan structure on cell surface receptors may alter their function. Our previous works show that over expression of GnT-V gene-transfected PC12 Cells strongly enhances axon outgrowth elicited by a low dose of NGF. The results show that GnT-V strongly promotion differentiation of the PC12 cell. To investigate the change of sugar component of glycoproteins from cell lysates of GnT-V gene-transfectioned PC12 cells, mock and PC12 cells, lectin blot analyses were performed using L-PHA and E-PHA. To explore this interaction, the effect of GnT-V on receptor tyrosine kinase activity was examined by immunoprecipitates TrKA. Here, we attempts have been made to elucidate the mechanism of GnT-V function in nerve.We report here that over expression N-acetylglucosaminyltransferase V directly modifies TrKA protein, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances axon outgrowth in rat pheochromocytoma cells (PC12) elicited by a low dose of NGF that alone is insufficient to induce neuronal differetiation. Furthermore, modification of TrKA glycoprotein could directly enhance NGF-activated autophosphorylation of immunoprecipitated TrKA in vitro. Receptor density at the cell surface is influenced by rates of de novo production, endocytosis, recycling, and degradation. Lattice-dependent regulation of receptors occurs primarily at the cell surface, is dependent on Golgi enzyme activities and the number of N-glycans per receptor, and opposes receptor loss to endocytosis. We examine the possibility that GnT-V modified N-glycans on cytokine NGF receptor TrKA oppose constitutive endocytosis by retaining surface receptors where membrane remodeling is active. Thus, we have investigated the interaction of N-glycan of NGF receptor TrKA and endocytosis. To further elucidate the mechanism of GnT-V, we study the different time point of endocytosis of TrKA receptor, and the result show that TrKA of GnT-V gene-transfected PC12 Cells delayed their removal by constitutive endocytosis than mock cells, suggesting high expression of GnT-V may alter the N-glycan of NGF receptors, effect their endocytosis and let to the NGF receptor TrKA stay longer at the cell membrane, inceasing the effect of NGF and enhancing the down stream signal pathway.In this paper, we firstly investigate the biological effect of GnT-V on the NGF receptor TrKA, and the results show that glycosylation of TrKA by GnT-V have regulated the function of TrKA receptor. It is new idea for GnT-V function on axon outgrowth, which is helpful for further reseach of GnT-V function. Thus, these results strongly suggest that N-acetylglucosaminyltransferase V as a specific endogenous role of NGF receptor function, and these enhanced effects appear to be due, at least in part, to promotion differentiation and protection of neuronal damage. This work is a step toward intriguing innovative therapeutic strategies for trials currently in consider with glycosyltransferase in neurodegenerative disorders. It has been shown that GnT-V function as a neurotrophic factor. Still, the present observations suggest intriguing innovative therapeutic strategies for trials currently in progress with neurotrophicis in neurodegenerative disorders.更多还原