【作者】 方欢；

【导师】 申宗候； 欧周罗；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 N-乙酰氨基葡萄糖转移酶V(GnT-V)是存在于高尔基体中的N糖链外链加工酶，催化UDP-GlcNAc中GlcNAc转移到N糖链五糖核心上α甘露糖(αman)形成β1，6键，是合成C2 C2，6型三天线和C2，4C2，6型四天线N糖链所必需的，细胞表面β1，6分支结构的多少主要取决于GnT-V活性及表达的高低。大量研究表明肿瘤细胞侵袭很大程度上是由于细胞表面形成过量的N糖链β1，6分支结构所致，过多的β1，6分支结构导致生成多天线的N糖链结构改变了糖蛋白分子的生物学性状，使肿瘤细胞粘附功能发生异常而易发生向周围组织侵袭。本教研室以往的研究发现用反义核酸技术阻断体外培养H7721人肝癌细胞的GnT-V基因表达，建立了GnT-V基因沉默细胞株GnT-V-AS／H7721，并且观察到由于GnT-V基因沉默而引起的细胞表型改变，例如生长缓慢，对血清饥饿耐受下降，对凋亡诱导剂全反式维甲酸(ATRA)更加敏感等。为探讨其机制用基因芯片技术(DNA microarray)对H7721细胞和GnT-V-AS／H7721细胞基因转录水平表达谱进行比较，发现GnT-V-AS／HH7721细胞基因表达谱具有内质网应激(ER stress)的特征。ER stress是由于内质网内未折叠蛋白堆积到一定程度引起的一种真核细胞应激现象，是近年来新发现的重要的凋亡起始机制之一，目前国外对ER stress的研究报导较多，国内尚未见研究成果报导。本文以H7721细胞为材料从分子水平对GnT-V表达受阻后引起ER stress的现象进行了较为系统的研究并探讨其产生机制。一．GnT-V-AS／H7721和H7721细胞基因转录水平表达情况的总体比较本文运用基因芯片技术对GnT-V-AS／H7721和H7721细胞基因转录水平的表达进行检测和比较，结果表明GnT-V-AS／H7721细胞基因转录水平具有以下特点：(1)介导内质网中蛋白折叠的伴侣蛋白如BIP、蛋白质二硫键异构酶、脯氨酰肽酰异构酶D和A以及一些热休克蛋白表达上调为H7721细胞的3倍以上，其中BIP更上调为8.21倍，(2)蛋白质合成系统的一些基因表达下降，如S5、S10、S14、S19、S21、L8、L10、L18、L27、L29、L37的表达仅为H7721细胞的0.11—0.45，(3)涉及泛肽和蛋白酶体的蛋白降解途径的基因表达上调，如泛肽水解酶，E2-泛肽结合酶，蛋白酶体亚基P40,P55,HC3,HC8,HC9表达为H7721细胞的2.6—6.6倍。通过复习文献，表明这些现象符合近年来才发现的，普遍存在于真核细胞中的ER stress现象，因此推测GnT-V表达受阻可能是GnT-V-AS／H7721细胞产生ER stress的起始原因。二．GnT-V-AS／H7721和H7721细胞ER stress过程中的关键信息分子的检测比较为验证GnT-V-AS／H7721细胞株存在ER stress现象，本文以GnT-V-AS／H7721细胞为实验细胞，H7721细胞和二巯基苏糖醇(DTT)处理的H7721细胞作为阴性和阳性对照，检测ER stress过程中的关键分子的表达。ER stress过程中的关键分子主要有BIP、XBP1和PERK等，其中伴侣蛋白BIP是ER stress信号传导过程中最终上调的靶目标，其表达上调是公认的细胞发生ER stress标志。转录因子XBP1是哺乳动物细胞ER stress信号传导过程中的中枢性调节分子，细胞发生ER stress时其mRNA发生特异性地剪接。PERK是位于内质网的eIF2α蛋白激酶家族成员，细胞发生ER stress时特异性地发生活化，并通过磷酸化翻译起始因子eIF2α而下调细胞蛋白合成水平。对这三种ER stress关键分子的检测发现：和阴性细胞相比较，实验细胞中BIP的表达无论是蛋白水平还是转录水平都有明显的上调，但上调程度都要低于DTT处理的ER stress阳性细胞；XBP-1 mRNA在实验细胞中部分被剪接，在阳性细胞中XBP-1 mRNA完全被剪接，而在阴性细胞中其以非剪接形态存在；此外和DTT处理的ER stress阳性细胞相似，实验细胞中的PERK发生磷酸化，表明ER stress过程中通过磷酸化eIF2α抑制蛋白合成机制的活化，这和芯片所检测到的GnT-V-AS／H7721细胞蛋白合成系统水平下调相一致。这些结果表明GnT-V-AS／H7721细胞中存在有ER stress现象，并且相对于DTT引起的ER stress程度较轻，可能是慢性的ER stress过程。因有研究表明非特异性的双链RNA导入细胞可以激活胞内蛋白激酶PKR而引起eIF2α磷酸化，抑制胞内蛋白质合成系统。GnT-V-AS／H7721中含有整合的反义GnT-V cDNA，并且通过转录产生反义RNA与GnT-V的mRNA结合而起作用，反义GnT-V cDNA的整合也有可能作为非特异因素，为排除整合及双链RNA原因导致的非特异因素干扰，采用了反义寡聚核苷酸(asODN)技术阻断H7721细胞中GnT-V的表达后，结果发现BIP的表达同样发生上调，并且出现少量的剪接XBP-1 mRNA，这表明GnT-V表达受阻可以特异性地引起胞内ER stress。本教研室以往的研究发现GnT-V-AS／H7721细胞相对于H7721细胞对凋亡诱导剂ATRA更加敏感，而ER stress是引起凋亡的重要因素之一，当刺激信号过于剧烈，细胞产生的应答仍不足以克服过强的刺激信号导致的细胞损伤时，位于内质网胞浆侧的caspase12能特异地被激活并参与ER stress引起的凋亡过程，本文发现ATRA处理的GnT-V-AS／H7721细胞中存在caspase12活化，表明GnT-V-AS／H7721细胞对ATRA诱导的凋亡敏感性的增高和胞内的ER stress有关。三．GnT-V-AS／H7721和H7721细胞内质网中N糖链合成及胞内糖蛋白N糖链结构的比较GnT-V表达受阻可以特异性地引起细胞发生ER stress，但GnT-V是高尔基体中的N糖链外链加工酶，并不参与内质网中N糖链的合成及蛋白折叠过程。通过相关文献和NCBI数据库检索发现直接参与内质网中N糖链合成及蛋白折叠的酶和伴侣蛋白大多数是有N糖链的糖蛋白，GnT-V表达受阻后胞内N糖链外链加工方式的改变可能导致它们N糖链结构发生改变，而正确的糖链结构是糖蛋白发挥生物学功能所必需的，因此考虑GnT-V表达受阻后内质网内N糖链合成酶和伴侣蛋白功能的异常是导致细胞ER stress的原因之一。为了研究GnT-V表达受阻对细胞N糖链合成的影响，本文以GnT-V-AS／H7721细胞为实验细胞，H7721细胞和衣霉素(tunicamycin TM)处理H7721细胞作为对照，以~3H-甘露糖掺入法研究三种细胞株糖蛋白N糖链合成的差异，结果发现GnT-V-AS／H7721细胞和阳性对照相似，二者糖蛋白N糖链合成分别下降为H7721细胞的56％和26％，证明GnT-V表达受阻后可以影响内质网中糖蛋白N糖链合成，表明内质网中的N糖链合成酶功能发生下降，导致内质网中N糖链糖蛋白糖基化异常而引起细胞发生ER stress。为了检测了GnT-V表达受阻后胞内糖蛋白N糖链结构的改变，同样以三种细胞株作为实验对象，在去除细胞膜表面糖肽后，用HRP标记的刀豆凝集素(ConA)和蔓陀罗凝集素(DSA)分析胞内糖蛋白的N糖链结构，结果发现GnT-V-AS／H7721细胞相对于H7721细胞对HRP-ConA染色增深，而对HRP-DSA染色变浅，表明GnT-V表达受阻后胞内糖蛋白N糖链β1,6-GlcNAc分支结构含量减少，说明细胞内糖蛋白N糖链结构因GnT-V表达受阻而普遍发生改变，提示内质网中N糖链合成酶及伴侣蛋白的N糖链结构也发生了改变，因此认为GnT-V表达受阻可能影响内质网中N糖链合成酶和伴侣蛋白的N糖链结构，从而影响到它们的功能，引起ER stress。更多还原

【Abstract】 N-acetylglucosaminyltransferase V (GnT-V) was an important N-glycan processing enzyme located in Golgi apparatus. It transfered the UDP-GlcNAc residue to the α mannose of five oligosaccharide core in N-glycan, which formed α β 1,6GlcNAc linkage in the C2C2,6 tri-antennary and C2,4C2,6 tetra-antennary N-glycans respectively.The amount of β 1,6 branching structures on cell surface was associated with the expression level and activity of GnT-V.Previous studies indicated that excessive β 1,6 branching structures on cell surface was essential for the metastasis of malignant cell for the redundant structures increased the antennaries on glycosyproteins on cell surface and change the fuctions of the latters.Our laboratory constrcted the GnT-V-AS/H7721cell line by introduced antisense cDNA of GnT-V into H7721 cells,a human hepatocarcinoma cell line. The growth rate of GnT-V-AS/H7721 was decreased in serum-containing medium,while the cell death was accelerated in serum-free medium. Besides that the cells were also more susceptible to the apoptosis induced by ATRA than the mock-transfected cells.However the mechanism was not resolved.To find the reason, gene expression profile was detected in GnT-V-AS/H7721 and H7721 cells with cDNA array in our laboratory.The characteristics of ER stress was found in GnT-V-AS/H7721 cells.ER stress ,which was induced by the accumulation of unfolded proteins in ER,was an protective stress reaction by eukaryotes and an important inistial mechanism for apoptosis. This paper studied the ER stress phenomenon and mechanism in cells after blocking expression of GnT-V and three parts were included in the investigation.一. Comparison of gene expression profile between H7721 and GnT-V-AS/H7721 cells with cDNA arraycDNA array analysis was used to study the difference in gene expression between H7721 and GnT-V-AS/H7721 cells.Among 5832 genes 788 genes showed different expression in the two kinds of cells,of which 390 genes expressed higher and 398 lower in GnT-V-AS/H7721 cells than that in H7721 cells.The 788 genes differently expressed were divided into 18 groups according to their fuction.The global analysis indicated that four groupes genes in all groupes were most differently expressed in two types of cells.which were chaperones assisting proteins’ folding in ER,heat shock proteins, ingredients associated with proteins’ synthesis and ingredients in the proteins’ degradation pathway of ubiquitin and proteasome.The expression level of heat shock proteins and chaperones such as BIP,PDI(protein disulfide isomerase) and PPI(peptidylprolyl isomerase) in GnT-V-AS/H7721 cells were over 3 times than that in H7721 and particularly BIP was 8.21 times higher,however, the expression level of ingredients associated with proteins’ synthesis were downregulated for the mRNA level of ribosomes such as S5,S10,S14,S19,S21,L8,L10,L18,L27,L29 and L37 were only 0.11-0.45 times than that in H7721.The compositions related to ubiquitin and proteasome including ubiquitin hydrolyzing enzyme,E2 ubiquitin conjugating enzyme and proteasome subunits P40 P55 HC3 HC8, HC9 in GnT-V-AS/H7721 were 2.6-6.6 times higher than that in H7721 cells.To take those together the GnT-V-AS/H7721 cells showed the ER stress phenomenon and suggested that blocking expression of GnT-V in the cells may be the initial reason.二. Determination of important signal transduction molecules during ER stress in GnT-V-AS/H7721 cells For investigating the ER stress phenomenon in GnT-V-AS/H7721 cellsdetected by cDNA array,the key signal molecules were determinated and compared in GnT-V-AS/H7721, H7721 and H7721 treated with DTT(dithiothreitol).The two latters were used as negative and positive control cells respectively in this paper.The results indicated that both transcriptional and translational level of BIP in GnT-V-AS/H7721 were much higher than that in H7721 cells which were resembles to that in H7721 treated with DTT and BIP was regarded as the marker of ER stress. It was also as same as in positive control cells that protein kinase PERK was phosphorylated in GnT-V-AS/H7721. The kinase was the unfolded protein sensor in ER.This was consistent with the downregulation of protein synthesis examinated by cDNA array and indicated the activation of the pathway that phosphorylated eIF2 α inhibited synthesis of proteins.Additionally XBP1 mRNA which functioned as centrally regulating molecule during ER stress was spliced partially,while it was completely spliced in positive control cells and unspliced in H7721.These results gave the proof that blocking expression of GnT-V caused H7721’s ER stress and response was more weak than that caused by DTT. It may be the chronic process.Additionally,previous studies reported that the ds-RNA may activate the kinase PKR which phosphorylated eIF2 α and inhibited synthesis of proteins.In this paper the antisense cDNA of GnT-V was integrated with GnT-V-AS/H7721’s gene and functioned through the binding of antisense RNA and mRNA of GnT-V.Additionally,the integration of GnT-V cDNA in genome also may be a non-specific factor. In order to testify the specificity of ER stress induced by blocking of GnT-V expression in H7721 cells, the asODN of GnT-V was transiently transfected into H7721 cells. The specificity of ER stress was evidenced by the results that transient blocking of GnT-V expression by asODN also upregulated BIP mRNA and partially activated XBP1 mRNA splicing system in H7721 cells. GnT-V-AS/H7721 was previously found to be more sensitive to the treatment of apoptosis agent ATRA than the mock transfected cell,however,the strong ER stress may cause cells’apoptosis.We suspected whether activation of ER stress in GnT-V-AS/H7721 cells may contribute to this characteristic.Caspase-12 which was an ER stress specific apoptosis enzyme was investigated in three types ofcells. In the experiment caspase12 in GnT-V-AS/H7721 treated by ATRA was activated partially which was fully activated in H7721 cells treated with high concentration DTT. This may suggest that GnT-V-AS/7721’s sensitiveness to apoptosis induced by ATRA was associated with its ER stress.三. Comparison of the synthesis and structures of intracellular N-glycans in H7721 and GnT-V-AS/H7721 cellsThe blocking expression of GnT-V can specially induced the ER stress in H7721 cells but GnT-V was in Golgi which did not assist the N-glycans synthesis and protein folding in ER , so the mechanism was complicated.The structures of intracellular N-glycans may changed for their processing mode were changed after blocking GnT-V. Moreover,according to the report the enzymes and chaperones assisting the N-glycans synthesis and proteins folding in ER were glycoproteins containing N-glycans .For consideration that the correct structures of glycans were essential for the function of glycoproteins, we suspected that the abnormal function of the enzymes and chaperones assisting the N-glycans synthesis and proteins folding after blocking of GnT-V’s expression was one of reasons of the ER stress. H7721 and H7721 treated with TM (tunicamycin)were used as negative and positive control respectively in the experiments. To test the effect on the synthesis of N-glycans in ER after blocking expression of GnT-V,the rate of ~3H-man incorporation was investigated in three types of cells.The rate of GnT-V-AS/H7721 was similar with H7721 treated with TM,which were decreased greatly compared with H7721.This suggested the abnormality of function of the N-glycan synthesis enzymes in ER.They may cause the abnormal N-glycosylation for glycoproteins.which induced ER stress in cells. To examine the change of intracellular N-glycans after blocking GnT-V, DSA and ConA labeled by HRP was used to stain the proteins from three types of cells after removing of N-glycans on cellular surface.For the DSAstaining the intracellular glycosyprotein from GnT-V-AS/H7721 showed a deeper stain than the other two, however, the glycoproteins from H7721 was deepest when using ConA staining.This indicated that the β 1,6GlcNAc structures on intracellular glycoproteins were decreased after blocking expression of GnT-V.The result indicated that the structures of N-glycans in intracellular glycosyproteins was changed generally for blocking expression of GnT-V. It suggested that the structures of N-glycans in enzymes and chaperones assisting the N-glycans synthesis and proteins folding were also changed.This presented the concept that the change of their structures of N-glycans after blocking expression of GnT-V distorted the function of the enzymes and chaperones .which caused the ER stress.更多还原