【作者】 李姣；

【导师】 申宗侯； 王晓明；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 N-乙酰氨基葡萄糖基转移酶V(GnT-V)位于真核细胞的高尔基体中，它是N-连接糖蛋白糖链加工合成过程中的关键酶。GnT-V催化的反应是将供体UDP-GlcNAc中的GlcNAc转移至受体糖链中的α-1，6甘露糖苷臂，进而形成含有β-1，6分支结构(GlcNAcβ1，6 Manα1，6)的三或四天线的N-糖链。以往的研究显示，GnT-V与正常细胞的恶性转化和肿瘤细胞的转移密切相关。本课题组前期的研究表明，使用反义寡核苷酸和RNA沉默技术抑制GnT-V的表达(GnT-V的表达下降约56％)，能够诱导人肝癌细胞株SMMC-7721发生内质网应激反应(Endoplasmic Reticulum Stress，ER stress)。在转染反义GnT-V的7721细胞(7721／AS)中，分子伴侣Bip在mRNA和蛋白水平均出现增高，同时出现XBP1mRNA的剪接。总蛋白的糖染色显示，7721／AS细胞糖蛋白的三、四天线的N-糖链明显减少。但对于ER stress出现的具体分子机制还不甚了解，可能与某些糖蛋白的糖链变化有关。本研究在前期研究的基础上，进一步探讨了GnT-V低表达导致7721细胞出现ER stress的具体分子机制；继而从另一侧面初步探讨了GnT-V过表达时对ER stress的影响。本研究分成四部分，以具有N-连接糖链的葡萄糖转运体1(GLUT1)为主要研究对象。首先我们观察了7721，7721／AS和7721／mock三种细胞中GLUT的表达类型，结果表明GLUT1成为三种细胞的主要葡萄糖转运体类型。接下来我们观察了7721／AS细胞的GLUT1糖链结构是否发生变化，结果显示GLUT1的分子量减小，且三、四天线的N-糖链成分减少。然后我们又发现7721／AS细胞中GLUT1转运葡萄糖的功能也显著降低。进一步的研究显示，GLUT1在7721／AS细胞中的分布并未受到影响。为了确认葡萄糖饥饿可以引起细胞发生ER stress，我们对7721细胞进行了葡萄糖饥饿的研究，结果显示，在葡萄糖饥饿的情况下，7721细胞发生了明显的ER stress。最后，受文献的启发，我们初步观察了GnT-V表达水平与内质网应激的关系：我们以自身存在ER stress的人肝癌细胞株—HuH7为对象，发现与其它自身无ER stress的肝癌细胞相比，HuH7的GnT-V表达水平较低，向HuH7细胞中转染GnT-V，发现HuH7的应激水平减弱。80综上所述，GLUT1结构和功能的异常导致7721／AS细胞出现葡萄糖缺乏，进而引起细胞发生ER stress。GnT-V低表达正是通过改变GLUT1的糖链结构而导致7721细胞出现应激反应。相反，GnT-V过表达减弱HuH7细胞中ER stress的现象更加从另一侧面证实了GnT-V表达水平与ER stress的相关性。更多还原

【Abstract】 N-acetylglucosaminyltransferaseV (GnT-V, EC 2. 4. 1. 155), which localizes in Golgi apparatus, catalyzes the transfer of GlcNAc residue from UDP-GlcNAc to anα-1, 6 mannoside arm in the acceptor glycans to synthesize the GlcNAc-β1, 6-Man branching (GlcNAcβ1, 6 Manα1, 6), then forms tri-or tetra-antennary N-linked oligosaccharide chains. GnT-V is a key enzyme in the processing of N-glycans during synthesis of glycoproteins. It is well known that GnT-V is associated with tumorigensis of normal cells and metastasis of tumor cells. In our laboratory, using antisense oligonucleotide and RNAi techniques, it was found that down-regulating GnT-V could activate endoplasmic reticulum stress ( ER stress ) responses in 7721 cells, a human hepatocarcinoma cell line ( the expression of GnT-V was decreased about 56% ). There was an up-regulation of Bip at mRNA and protein levels, and appearance of the spliced form of XBP1mRNA in antisense GnT-V transfectant. In this study, we further investigated the molecular mechanism of ER stress induced by down-regulating GnT-V.We selected glucose transporter (GLUT) as the object of study, since GLUT was a glycoprotein containing an N-linked glycan chain and responsible for basic supply of cells with glucose. Glucose starvation has been shown to induce ER stress in tumor ells. Therefore we hypothesized that GnT-V change glycosylation of GLUT and further affect the function of GLUT. Furthermore, inspired by a literature, we observed the effect of GnT-V on ER stress existed in HuH7 hepatocarcinoma cell line. The results obtained from this study are as follows: (1) GLUT1 has become the predominant GLUT isoform of 7721 cells. (2) The content oftri-or tetra-antennary sugar chain of GLUT1 decreased in antisense GnT-V transfectant. (3) Glucose transport activity of GLUT1 in antisense GnT-V transfectant is at least 2-fold lower than that in mock tansfectant. (4) Modulation of GLUT1 glycosylation does not interfere with the distribution of GLUT1 protein within cells. (5) Glucose deprivation can activate ER stress responses in 7721 cells. (6) GnT-V overexpression attenuates the ER stress in HuH7 cells. In summary, the glucose transport activity and glycosylation of GLUT1 decreased in antisense GnT-V transfectant, which may be one possible mechanism of ER stress induced by down-regulating GnT-V, and GnT-V may contribute to the regulation of glucose uptake by modifying glycosylation of GLUT1 in some tumor cells.更多还原