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Study on the Cellular ER Stress Induced by Blocking Expression of N-acetylglucosaminyltransferase V

Author: FangHuan
Tutor: ShenZongHou;OuZhouLuo
School: Fudan University
Course: Biochemistry and Molecular Biology
Keywords: N-acetylglucosaminyltransferase V N-glycan glycosyprotein ER stress unfolded protein response cDNA array antisense oligonucleotides lectin chaperone BIP
CLC: Q343
Type: PhD thesis
Year: 2004
Downloads: 132
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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.

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