Transcirptional And Functional Study Of Glycosphingolipid Gg4and Its Synthase β3Ga1T4

Glycosphingolipids (GSLs) which composed of carbohydrate chain, long fatty acid andsphingosine are important components in cellular structure. GSLs widely participate inprocesses like cell growth, proliferation, differentiation, apoptosis, cell-cell adhesion, signaltransduction et al. Epithelial-mesenchymal transition (EMT) which can be induced by TGF-βis an important process in cancer development. Our previous findings suggested that theexpression of Gg4(gangliotetraosylceramide, asialo-GM1) and β3GalT4(UDP-galactose:β-N-acetyl-galacosamine β-1,3galactosyltransferase, the synthase of Gg4) in NMuMG(mouse mammary gland cells) decreased significantly in TGF-β induced EMT process; Gg4may consolidate the interaction between E-cadherin and β-catenin; exogenous Gg4canobviously inhibit the EMT process at a concentration of50μmol L-1. However, the regulatorymechanism of Gg4and β3GalT4in EMT is still unclear, and the molecular mechanism andspecific function of Gg4of participating in EMT remind to be clarified.In this study, electrophoretic mobility shift assay (EMSA) and chromatinimmunoprecipitation assay (ChIP) were performed in vitro and in vivo respectively, toconfirm the regulation of Smad3/4on β3GalT4. Next, the Gg4overexpressed and silencedNMuMG cell lines were constructed to prove the effects of Gg4on cell morphology, motilityand proliferation. Thirdly, the attemptations of expressing β3GalT4in Pihcai pastorsi andEscherichia coli by genetic engineering technology were conducted to obtain bioactiveprotein and produce its antibody. Main results are as follows:1. The regulatory mechanism of Smad3/4to β3GalT4in TGF-β signaling pathway.Smad3was phosphorylated and phosphorylated Smads entered into cell nucleus with the helpof Smad4to regulate the expression of β3GalT4were confirmed by EMSA and ChIP assay.The expression of β3GalT4in cancer tissue samples were declined determined by real-timePCR.2. Effect of overexpress and silence Gg4on the motility of NMuMG cell. The Gg4overexpressed and silenced NMuMG cell lines were constructed by genetic engineeringtechnology. Overexpression of Gg4inhibited the cell motility and vice versa, confirmed bycolloidal gold assay.3. Expression of mouse and human β3GalT4in P. pastorsi and E. coli. Firstly, a plasmidwith a six His tag named pPIC9K-β3GalT4(Mouse) was constructed, and its expression wasinduced by0.5%,1%and1.5%methanol in P. pastorsi GS115strain. Next, another threeplasmids named pET-β3GalT4(Mouse), pAC-β3GalT4(Mouse) and pET-β3GalT4(Human)were also constructed and induced by IPTG in E. coli. However, the expression of β3GalT4inP. pastorsi and E. coli were not successful. After the analysis of β3GalT4gene, the rarecodons in β3GalT4coding sequence of mouse (32) and human (33) might be the main reasonfor the failed expression of β3GalT4in P. pastorsi and E. coli.