Cell-Cell Interaction-dependent Regulation of N-Acetylglucosaminyltransferase III and the Bisected N-Glycans in GE11 Epithelial Cells

Abstract

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. In this study, the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans) were investigated in GE11 epithelial cells. N-glycans were purified from whole cell lysates by hydrazinolysis and then detected by high performance liquid chromatography and mass spectrometry. Interestingly, the population of the bisecting GlcNAc-containing N-glycans, the formation of which is catalyzed by N-acetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense conditions compared with those cultured under sparse conditions. The expression levels and activities of GnT-III but not other glycosyltransferases, such as GnT-V and ␣1,6-fucosyltransferase, were also consistently increased in these cells. However, this was not observed in mouse embryonic fibroblasts or MDA-MB231 cells, in which E-cadherin is deficient. In contrast, perturbation of E-cadherin-mediated adhesion by treatment with EDTA or a neutralizing anti-E-cadherin antibody abolished the up-regulation of expression of GnT-III. Furthermore, we observed the significant increase in GnT-III activity under dense growth conditions after restoration of the expression of E-cadherin in MDA-MB231 cells. Our data together indicate that a E-cadherindependent pathway plays a critical role in regulation of GnT-III expression. Given the importance of GnT-III and the dynamic regulation of cell-cell interaction during tissue development and homeostasis, the changes in GnT-III expression presumably contribute to intracellular signaling transduction during such processes.It has been well known that sugar chains have various effects on the functional aspects of glycoproteins and play important roles in cell differentiation, adhesion, and proliferation (1, 2). The sugar chains of glycoproteins are produced via catalysis by various glycosyltransferases. As a result, a specific structure is determined by the expression pattern of these enzymes. The expression of glycosyltransferases is regulated not only by physiological conditions, such as developmental stages, but by pathological conditions, such as rheumatoid arthritis and tumorigenesis (3, 4). The increasing body of evidence suggests that the structures of the glycan components, such as sialyl Lewis antigens (5, 6), mucin-type O-glycans (7), or N-glycans (3, 8), which are controlled by glycosyltransferases, greatly contribute to cell adhesion, cell invasion, and cancer metastasis. A malignant phenotype has been reported to be highly associated with N-linked oligosaccharides (N-glycan) containing ␤1,6-branching, which is catalyzed by N-acetylglucosaminyltransferase V (GnT-V) 4 by transfer of GlcNAc from UDP-GlcNAc to a ␤1,6 mannose in N-glycans (8). It has also been reported that GnT-V activities and ␤1,6-branched N-glycan levels are increased in highly metastatic tumor cell lines (9, 10). Conversely, cancer metastasis is greatly suppressed in GnT-V knock-out mice (11). These re...

Inverse Correlation between the Extent of N-Glycan Branching and Intercellular Adhesion in Epithelia

“…Consistent with these observations, the relative molecular weight of N-glycans linked to the N-cadherin decreases as nonconfluent RPE cells become confluent (21). Also, during development of cell-cell adhesion of epithelial GE11 cells, GnT 2 -III, the enzyme that stops branching of N-glycans is up-regulated (22).…”

Potential roles of N-glycosylation in cell adhesion

“…GnT-III activity was increased under dense culture conditions compared with sparse culture conditions [56]. A significant up-regulation of GnT-III expression was observed only in epithelial cells that express E-cadherin, but not in E-cadherin-deficient cells.…”

Branched N-glycans and their implications for cell adhesion, signaling and clinical applications for cancer biomarkers and in therapeutics

“…They also found that TIMP-1 was aberrantly glycosylated by GnT-V showed a weaker inhibition with respect to both matrix metalloproteinase (MMP)-2 and MMP-9, and this aberrancy was closely associated with cancer cell invasion and metastasis in vivo as well as in vitro (72). As described above, GnT-V and GnT-III have adverse effects in cancer invasion and metastasis (5,6,(23)(24)(25)(26).…”

“…The other major glycoprotein on the cell surface is integirin. In fact, the overexpression of GnT-III resulted in a reduction in β1, 6 GlcNAc branching structures, along with an increase in the bisected N-glycans on integrins (6,25,26). This resulted in the inhibition of integrin-mediated cell spreading and migration, and the level of cellular phosphorylation.…”