Blocking of N-acetylglucosaminyltransferase V induces cellular endoplasmic reticulum stress in human hepatocarcinoma 7,721 cells.
N-acetylglucosaminyltransferase V (GnT-V) is an important tumorigenesis and metastasis-associated enzyme. To study its biofunction, the GnT-V stably suppressed cell line (GnT-V-AS/7,721) was constructed from 7,721 hepatocarcinoma cells in previous study. In this study, cDNA array gene expression profiles were compared between GnT-V-AS/7,721 and parental 7,721 cells. The data indicated that GnT-V-AS/7,721 showed a characteristic expression pattern consistent with the ER stress. The molecular mechanism of the ER stress was explored in GnT-V-AS/7,721 by the analysis on key molecules in both two unfolded protein response (UPR) pathways. For ATF6 and Ire1/XBP-1 pathway, it was evidenced by the up-regulation of BIP at mRNA and protein level, and the appearance of the spliced form of XBP-1. As for PERK/eIF2alpha pathway, the activation of ER eIF2alpha kinase PERK was observed. To confirm the results from GnT-V-AS/7,721 cells, the key molecules in the UPR were examined again in 7,721 cells interfered with the GnT-V by the specific RNAi treatment. The results were similar with those from GnT-V-AS/7721, indicating that blocking of GnT-V can specifically activate ER stress in 7,721 cells. Rate of (3)H-Man incorporation corrected with rate of (3)H-Leu incorporation in GnT-V-AS/7,721 was down-regulated greatly compared with the control, which demonstrated the deficient function of the enzyme synthesizing N-glycans after GnT-V blocking. Moreover, the faster migrating form of chaperone GRP94 associated with the underglycosylation, and the extensively changed N-glycans structures of intracellular glycoproteins were also detected in GnT-V-AS/7,721. These results supported the mechanism that blocking of GnT-V expression impaired functions of chaperones and N-glycan-synthesizing enzymes, which caused UPR in vivo.
State Key Laboratory of Genetic Engineering, Department of Biochemistry, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China.
Huan
H
Fang
Wei
W
Huang
Ying Ying
YY
Xu
Zong Hou
ZH
Shen
Chao Qun
CQ
Wu
Shou Yi
SY
Qiao
Yan
Y
Xu
Long
L
Yu
Hui Li
HL
Chen
D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000502:physiology*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035925:Iron-Regulatory Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism* / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry; D017510:Protein Folding; D034622:RNA Interference; D000071701:Regulatory Factor X Transcription Factors; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D000071717:X-Box Binding Protein 1; D019892:eIF-2 Kinase / Q000378:metabolism
D004268:DNA-Binding Proteins; D035925:Iron-Regulatory Proteins; D008562:Membrane Glycoproteins; D017351:N-Acetylglucosaminyltransferases; D011134:Polysaccharides; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; D019892:eIF-2 Kinase; C000605543:XBP1 protein, human; C053387:endoplasmin