The Effects Of Core Fucosylation On The Subcellular Distribution Of β-catenin In Human Lung Cancer Cells

Core fucosylation catalyzed by alpha-1,6 fucosyltransferase (alpha-1,6 FucT),is regarded as an important manner of post-transcriptional modification and functional regulation of glycoproteins.Recent developments in glycobiology have revealed that the sugar chains of glycoproteins play important roles in cellular recognition,which is essential for the maintenance of ordered social behaviour of differentiated cells.It has also been confirmed that the alterations of glycoprotein sugar chains are prompted by malignant transformation,tumor invasion,and metastasis.Our previous study indicated that the core fucosylation also has significant effects on the adhesion function of E-cadherin molecules,and thus the core fucosylated E-cadherin may effects on the subcellular distribution ofβ-catenin in human lung cancer cells.As a key molecule of Wnt signal pathway,β-catenin is also required for cell-cell adhesion.Endogenousβ-catenin is found in the adherins conjuction,which includes E-cadherin,α-,β-catenin involved in formulation of cell-skeleon.Considerableβ-catenin is also found in the cytoplasm,which is involved in the activities of Wnt pathway. Galectin-3,a novel binding Partner forβ-catenin,likeβ-catenin shuttles between the nucleus and the cytosol,and is also involved in the regulation of Wnt/β-catenin signaling pathway.Galectin-3 has been implicated in adhesion-mediated processes,cell cycle regulation, apoptosis resistance,tumorigenesis and metastasis.However,there is no report about whether galectin-3 is the substrate of core fucosyltransferase,whether galectin-3 affects the expression or subcellular distribution ofβ-catenin.This thus is worth of deep study as it can help clearly elucidate the role of core fucosylation in carcinogenesis and progression. In this study,we employed two lung cell lines 95C and 95D,which are giant-cell lung cancer cells origining from the same patient with low (C) and high(D) metastatic potentials.We constructed two pairs of cell models by overexpressing core fucosyltransferase in 95C cells and Fut8-targeted RNAi in 95D cells.There are two parts in my experiment. In the first part,we have proved that:Ⅰ) E-cadherin,notβ-catenin, is a substract of core fucosyltransferase identified by LCA blot.Ⅱ) In 95C cells overexpressing core fucosyltransferase,tyrosine residues and tyrosine 654 are less phosphorylated onβ-catenin.On the Contrast, in 95D cells with Fut8-targeted RNAi transfection,total tyrosine phosphorylation and Y654 phosphorylation are significantly enhanced.Ⅲ) in 95C cells overexpressing core fucosyltransferase,nuclearβ-catenin level was enhanced,Concomitantly,95D cells underexpressing core fucosyltransferase express a lessβ-catenin in the nucleus.Theβ-catenin expression level in whole cell lysates didn't change in the two models. Literatures have demonstrated thatβ-catenin with higher tyrosine phosphorylation level has less binding ability with E-cadherin.Therefore, the changes of tyrosine phosphorylation observed in our study could partly interpret the changes of nuclearβ-catenin caused by core fucosylation.In the fist part of our experiment,we have proved that the core fucosylated E-cadherin could up-regulate the tyrosine phosphorylation level of membrane-boundβ-catenin,and thus cause less accumulation ofβ-catenin in the nuclus.Then,we have next interesting question that whether there is some unkown substate of core fucosylation in the cytoplasm,which also affects the subcellular redistribution ofβ-catenin. Galectin-3 is a novel binding partner forβ-catenin.Due to its small size; it is plausible that galactin-3 shuttles freely between the cytosol and the nucleus.Galectin-3 could be involved in the nuclear retention ofβ-catenin,leading to the activation of the Wnt targeted genes.Moreover, there is a competition between galectin-3 andα-catenin in bindingβ-catenin.The result in this part shows thatⅠ) galectin-3 is a novel substract of core Fucosyltransferase identified by LCA blot.Ⅱ) galectin-3 with higher core fucosylation to posses high binding ability withβ-catenin in 95C cells with overexpressing core fucosyltransferase, vice versa in 95D cells underexpressing core fucosyltransferase.Ⅲ) The core fucosyltransferase regulates galectin-3 expression in protein lever, but not in mRNA level.We presume that the modification of core fucosylation induced the conformation change of galectin-3,which effects on the binding ability withβ-catenin.It is intriguing that core fucosyltransferase regulates galectin-3 expression in protein level but not in mRNA level.This is coincidental with a report that the N-glycosylated MUC1-C suppresses expression of microRNA miR-322 and thereby upregulates galectin-3 published in Molecular Cell by Selvi Ramasamy.So we presume that the glycosylation change caused by core fucosyltransferase might effects the expression of miR-322 by a posttranscriptional mechanism,thus,regulates Galectin-3 expression in protein level indirectly.This posttranscriptional mechanism needs further investigation.In conclusion,core-fucosylated E-cadherin up-regalates the tyrosine phosphorylation level of membrane-boundβ-catenin,and thus causes less accumulation ofβ-catenin in the nuclus.The modification of core fucosylation might also induce the conformation change of galectin-3, and effects on the binding ability with cytoplasmicβ-catenin,such the polynary regulatory mechanism reveals complicated functions of core fucosylation in human lung cancer cells.