Volume 16, No 1, Jan 2006

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 16 Issue 1, January 2006: 82-92

ORIGINAL ARTICLES

Blocking of N-acetylglucosaminyltransferase V induces cellular endoplasmic reticulum stress in human hepatocarcinoma 7721 cells

Huan Fang^{1, *}, Wei Huang^{2, *}, Ying Ying Xu^{1, *}, Zong Hou Shen¹, Chao Qun Wu², Shou Yi Qiao², Yan Xu², Long Yu², Hui Li Chen¹

¹State Key Laboratory of Genetic Engineering, Department of Biochemistry, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China;

²Department of Genetics, School of Life Science, Fudan University, 220 Handan Road, Shanghai 200433, China Correspondence: Zong Hou Shen, Chao Qun Wu(zhshen@shmu.edu.cn cqwu@fudan.edu.cn)

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/7721) was constructed from 7721 hepatocarcinoma cells in previous study. In this study, cDNA array gene expression profiles were compared between GnT-V-AS/7721 and parental 7721 cells. The data indicated that GnT-V-AS/7721 showed a characteristic expression pattern consistent with the ER stress. The molecular mechanism of the ER stress was explored in GnT-V-AS/7721 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/eIF2α pathway, the activation of ER eIF2α kinase PERK was observed. To confirm the results from GnT-V-AS/7721 cells, the key molecules in the UPR were examined again in 7721 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 7721 cells. Rate of ³H-Man incorporation corrected with rate of ³H-Leu incorporation in GnT-V-AS/7721 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/7721. 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.

Cell Research (2006) 16:82-92. doi:10.1038/sj.cr.7310011; published online 16 January 2006

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