Effects Of N-acetylglucosaminyltransferase V On Cell Signal Transduction And Gene Regulation

N-Acetylglucosaminyltransferase V (GnT-V) is an important N-glycan processing enzyme located in Golgi bodies. Using UDP-GlcNAc as the donor substrate, GnT-V catalyzes the transfer of the GlcNAc group to the mannose of core a 1,6 arm of N-sugar chain to form a (31,6 GlcNAc linkage. It distributes mainly in intestine, brain and lung. Human GnT-V contains 741 amino acids with six potential sites for N-glycosylation and bears high homology to GnT-V of rat. Its gene is located on chromosome 2q21 containing 17 exons. GnT-V protein is encoded by exons 2-17 as open reading frame. GnT-V was reported to overexpress in many malignant tumors, and the increase of its product - β1,6 branching is a characteristic of cancer progress and metastasis potential. GnT-V has been purified and cloned, but many unknown questions about its function remain to be resolved. In the presented study, using H7721 human hepatocarcinoma cell line transfected with sense or antisense cDNA of GnT-V, the effects of GnT-V on signal transduction and its mechanism as well as the alteration of gene expression were investigated. We expected to elucidate whether and how the transfected glycosyltransferase modulate the cell signal transduction and gene expression? Are the changes in signal transduction and gene expression related to the structural alteration of the sugar chain on some surface receptors resulting from the transfection with sense or antisense GnT-V? The elucidation of these problems will be of benefit to the understanding of GnT-V function, and will provide valuable information concerning molecular mechanism of tumor metastasis. Four respects are included in this investigation.Part I Effects of GnT-V on cell proliferation, cell sensitivity to EGF and the EGFreceptor of H7721 cell lineUsing MTT method, it was found that the proliferation of cells transfected with sense GnT-V cDNA was facilitated, and both of the total 3H-TdR incorporation and the specific incorporation per cell were also increased. Oppositely, these parameters were reduced in cells transfected with antisense cDNA of GnT-V. These results suggested that cell proliferation and DNA synthesis were modulated by GnT-V. The sensitivity of the growth rate to EGF (epidermal growth factor) stimulation was higher in GnTV-S/H7721 cells thanthat in Mock cells, while it was less in GnTV-AS/H7721 cells than in Mock cells. Therefore, the structural modification of N-glycan and the functional changes of EGF receptor (EGFR) in different transfected cells were investigated. It was found that the β1,6 GlcNAc branch on the N-glycans of immuno-precipitated EGFR on GnTV-S/H7721 cells was increased while it was reduced in GnTV-AS/H7721 cell despite the unaltered expression of EGFR protein. Correspondingly, the EGF binding affinity of surface EGFR and the tyrosine auto-phosphorylation of immuno-precipitated EGFR were also enhanced in GnTV-S/H7721 cells and reduced in GnTV-AS/H7721 cells. These results indicate that the alteration of cell proliferation and DNA synthesis caused by different GnT-V cDNA transfection may at least partly result from the modification of N-glycan structure and function of EGFR. It seems that the increased β1,6 GlcNAc branch on the N-glycans of EGFR may benefit to its binding with EGF and the resulting tyrosine auto- phosphorylation, while the decrease of this branch may prevent these processes.Part II Effects of GnT-V on the phosphorylation and activity of EGFR signalingmolecules in H7721 cellsModification of the N-glycan structure and function of EGFR by the transfection of different GnT-V cDNAs may lead to the alterations of the phosphorylation and activity in the downstream molecules of signal transduction. There are two EGFR signaling pathways, one is the classical Ras/Raf/MEK/MAPK pathway and the other is PI-3-K/PKB pathway. In this part, the phosphorylation and/or activity of some key molecules in these two signaling pathways in differently transfected H7721 cells were investigated. By means of specific antibodies combined with Weste...