【作者】 郭鹏；

【导师】 申宗候； 陈惠黎；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2003， 博士

【摘要】 N-乙酰氨基葡萄糖转移酶V(GnT-V)(EC.2.41.155)是分布在高尔基体中的一种重要的N-糖链加工酶，它以UDP-GlcNAc为供体底物，将GlcNAc基团转移至受体N-糖链核心α1，6臂的甘露糖(Man)上，形成GlcNAcβ1，6Man分支结构，主要分布在小肠，脑和肺等组织。人类GnT-V由741个氨基酸组成，有6个潜在的N-糖基化位点，基因定位于染色体2q21，含有17个外显子，其开放阅读框架由外显子2-17进行编码。GnT-V在很多恶性肿瘤中部有过量表达，其产物复杂型N-糖链β1，6GlcNAc分支的增多可能是肿瘤发展和转移潜能的重要特征。本实验室对GnT-V的研究已有多年，但我们发现GnT-V的功能尚有很多问题未解决。本文以稳定转染GnT-V正义或反义cDNA质粒的H7721人肝癌细胞为材料，从下列四个方面对GnT-V影响信号转导及其机制以及引起基因表达的变化进行了研究，企图阐明糖基转移酶转染是怎样调节细胞信号转导的?而细胞信号转导的改变是否与细胞表面某些受体的糖链结构的改变(由GnT-V的正义或反义cDNA转染引起的)有关?能否影响细胞基因表达?这些问题的阐明不仅有利于进一步认识GnT-V的功能，而且为肿瘤转移的分子机理提供有价值的资料。 第一部分 GnT-V对H7721细胞生长、EGF敏感性和EGF受体的影响 用MTT方法发现转染正义GnT-V cDNA的H7721细胞增殖速度加快，而且无论是~3H-TdR的总参入量或每个细胞的参入量均见增加，说明DNA合成增强，而转染反义GnT-VcDNA的H7721细胞则完全相反，DNA合成和细胞增殖速度均见降低，这提示GnT-V可调节细胞的生长。GnTV-S／H7721细胞的生长对表皮生长因子(EGF)刺激比Mock细胞敏感，而GnTV-AS／H7721细胞对EGF敏感性则低于Mock细胞。因此本文对不同转染细胞EGF受体(EGFR)的糖链结构及功能的改变作进一步研究，结果发现，不同转染细胞中，免疫沉淀EGFR的蛋白量没有改变，但GnTV-S／H7721细胞EGFR上N-糖链中β1，6 GlcNAc分支比Mock细胞增多，而GnTV-AS／H7721细胞相反。随着EGFR上N-糖链的变化，其配体结合与自身酪氨酸磷酸化也相应改变，GnTV-S／H7721细胞对EGF亲和力和自身酪氨酸磷酸化增强，而GnTV-AS／H7721细胞这两方面能力都减弱。由此可见不同GnT-VcDNA转染后引起的DNA合成和细胞增殖速度的变化的原因之一很可能是EGFR糖链结构和功能的改变，而EGFR糖链β1，6GlcNAc分支增加有利于和EGF的结合及随之引起的酪氨酸磷酸化，而EGFR糖链田，6口cNAc分支减少则不利于上述过程。第二部分G nT-V对H7721细胞中EGF受体信号转导分子磷酸化 及活力的影响 EGFR的结构与功能被转染的正义或反义GnT一V cDNA改变后，可能会进一步引起下游信号分子磷酸化或活力的改变。EGFR的信号通路主要有两条，一是Ras-raf-MEK一MAPK经典途径，另一条是新发现的PI一3K一PKB途径。本部分对GnT’V-S/H7721和GnTv一As/H7721细胞这两条通路中一些关键信号分子的磷酸化及/或活力变化进行了研究。用特异性抗体结合Westem blot结果发现，正义或反义GnT一V cDNA的转染并不引起PKB、p44/42MAPK和MEK蛋白质表达的变化，而GnTv一S/H”21细胞PKB T308、5473位点磷酸化和免疫沉淀PKB的酪氨酸磷酸化以及以GSK召a/日磷酸化为指标的PKB的活性都较Mock细胞增加，GnTV一AS/H7721细胞中这些指标的变化则相反。GnTV一S/H7721细胞p42/4 4 MAPK的磷酸化也较Mock细胞增强，而GnTV一AS舰7721细胞则减弱，表明EGFR的这两条信号通路中信号分子的磷酸化或活力都受GnT一V的影响。为了研究这两条信号通路中信号分子磷酸化或活力改变与EGF的关系，我们用EGF短时间刺激血清饥饿的细胞，结果发现，当细胞血清饥饿12小时后，PKB活力和p42/4 4 MAPK磷酸化明显减少至无法检测出，但MEK磷酸化仍可测出。EGF刺激后不同转染细胞中的PKB活性、p42/44 MAPK和MEK磷酸化都明显增加，其中GnTV一S/H 7721细胞PKB比活性、p42/4 4 MAPK和MEK磷酸化程度较Mock细胞增加明显，而GnTV一AS/H7721细胞中上述指标的增加程度则不及Mock细胞。为了研究PKB活性、P42/44 MAPK磷酸化与N一糖链的关系，我们用N一糖链合成抑制剂衣霉素处理细胞后，发现不同转染细胞中PKB活性和p42/.44MAPK磷酸化都普遍降低，而且它们在不同转染细胞间的差别减小:衣霉素处理细胞后再用EGF刺激细胞，不同转染细胞的PKB活性都略为增高，但不及未用衣霉素处理者明显，且在不同转染细胞之间的PKB活性差别几乎消失。由此可见，PKB的基础活性和p42/44 MAPK的基础磷酸化及外源性EGF刺激细胞后所引起的PKB活性诱导性升高也与N一糖链有关。为了研究PKB活性和P42/4 4 MAPK磷酸化与EGFR的关系，采用EGFR抗体处理细胞后，发现不同转染细胞中PKB活性和p42/44 MAPK磷酸化的差别也减小，表明EGFR的N一糖链参与了其下游的信号转导。本部分从正反两个方向均证实由正义或反义Gn丁一v的转染引起的EGFRN一糖链结构改变对E〔净的信号转导起重要的调节作用。第三部分G nT一V对整联蛋白表达、糖链结构的影响及其与PKB磷酸化的关系 整联蛋白是细胞膜上另一个与PKB磷酸化(5473)有关更多还原

【Abstract】 N-Acetylglucosaminyltransferase V (GnT-V) is an important N-glycan processing enzyme located in Golgi bodies. Using UDP-GlcNAc as the donor substrate, GnT-V catalyzes the transfer of the GlcNAc group to the mannose of core a 1,6 arm of N-sugar chain to form a (31,6 GlcNAc linkage. It distributes mainly in intestine, brain and lung. Human GnT-V contains 741 amino acids with six potential sites for N-glycosylation and bears high homology to GnT-V of rat. Its gene is located on chromosome 2q21 containing 17 exons. GnT-V protein is encoded by exons 2-17 as open reading frame. GnT-V was reported to overexpress in many malignant tumors, and the increase of its product - β1,6 branching is a characteristic of cancer progress and metastasis potential. GnT-V has been purified and cloned, but many unknown questions about its function remain to be resolved. In the presented study, using H7721 human hepatocarcinoma cell line transfected with sense or antisense cDNA of GnT-V, the effects of GnT-V on signal transduction and its mechanism as well as the alteration of gene expression were investigated. We expected to elucidate whether and how the transfected glycosyltransferase modulate the cell signal transduction and gene expression? Are the changes in signal transduction and gene expression related to the structural alteration of the sugar chain on some surface receptors resulting from the transfection with sense or antisense GnT-V? The elucidation of these problems will be of benefit to the understanding of GnT-V function, and will provide valuable information concerning molecular mechanism of tumor metastasis. Four respects are included in this investigation.Part I Effects of GnT-V on cell proliferation, cell sensitivity to EGF and the EGFreceptor of H7721 cell lineUsing MTT method, it was found that the proliferation of cells transfected with sense GnT-V cDNA was facilitated, and both of the total 3H-TdR incorporation and the specific incorporation per cell were also increased. Oppositely, these parameters were reduced in cells transfected with antisense cDNA of GnT-V. These results suggested that cell proliferation and DNA synthesis were modulated by GnT-V. The sensitivity of the growth rate to EGF (epidermal growth factor) stimulation was higher in GnTV-S/H7721 cells thanthat in Mock cells, while it was less in GnTV-AS/H7721 cells than in Mock cells. Therefore, the structural modification of N-glycan and the functional changes of EGF receptor (EGFR) in different transfected cells were investigated. It was found that the β1,6 GlcNAc branch on the N-glycans of immuno-precipitated EGFR on GnTV-S/H7721 cells was increased while it was reduced in GnTV-AS/H7721 cell despite the unaltered expression of EGFR protein. Correspondingly, the EGF binding affinity of surface EGFR and the tyrosine auto-phosphorylation of immuno-precipitated EGFR were also enhanced in GnTV-S/H7721 cells and reduced in GnTV-AS/H7721 cells. These results indicate that the alteration of cell proliferation and DNA synthesis caused by different GnT-V cDNA transfection may at least partly result from the modification of N-glycan structure and function of EGFR. It seems that the increased β1,6 GlcNAc branch on the N-glycans of EGFR may benefit to its binding with EGF and the resulting tyrosine auto- phosphorylation, while the decrease of this branch may prevent these processes.Part II Effects of GnT-V on the phosphorylation and activity of EGFR signalingmolecules in H7721 cellsModification of the N-glycan structure and function of EGFR by the transfection of different GnT-V cDNAs may lead to the alterations of the phosphorylation and activity in the downstream molecules of signal transduction. There are two EGFR signaling pathways, one is the classical Ras/Raf/MEK/MAPK pathway and the other is PI-3-K/PKB pathway. In this part, the phosphorylation and/or activity of some key molecules in these two signaling pathways in differently transfected H7721 cells were investigated. By means of specific antibodies combined with Weste更多还原