Introduction of Bisecting GlcNAc into Integrin α5β1 Reduces Ligand Binding and Down-regulates Cell Adhesion and Cell Migration

Abstract

The enzyme ␤1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin ␣ 5 ␤ 1 , and the focal adhesion kinase phosphorylation. E 4 -PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin ␣ 5 subunit as well as ␣ 2 and ␣ 3 subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin ␣ 5 ␤ 1 to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the ␣ 5 subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.Nearly all secreted and cell surface proteins are glycosylated. The attached sugar chains have many biological functions, for example, cell-cell communication, signal transduction, protein folding, and stability (1). Oligosaccharides are synthesized through the action of glycosyltransferase, which catalyzes the formation of glycosidic bonds. ␤1,4-N-Acetylglucosaminyltransferase (GnT-III) 1 catalyzes the transfer of GlcNAc residues in a ␤1,4-linkage to the ␤1,4-mannose residue in the core region of N-glycans, producing a bisecting GlcNAc (Fig. 1). We previously purified GnT-III from rat kidney and cloned the cDNA from both the rat and human (2, 3). In a quest for the biological significance of GnT-III, we found that GnT-III suppresses the dimerization of TrkA, the receptor for nerve growth factor, and EGF receptor phosphorylation (4, 5). It is interesting to note that the metastatic capabilities of B16 mouse melanoma cell are down-regulated by the introduction of the GnT-III gene (6). This anti-metastatic effect has been partly ascribed to the effect of GnT-III on an increase in E-cadherinmediated homotypic adhesion and a suppression of the phosphorylation of the E-cadherin-␤-catenin complex on the cell-cell adhesion (7,8).Cell-extracellular matrix (ECM) interactions play essential roles during the acquisition of migration and invasive behavior of the cells. The integrin family consists of ␣ and ␤ heterodimeric transmembrane receptors for ECM and connects many biological functions, such as development, control of cell proliferation, protection against apoptosis, and malignant transformation (9). The fibronectin (FN) ...

B4GALNT3 Expression Predicts a Favorable Prognosis and Suppresses Cell Migration and Invasion via β1 Integrin Signaling in Neuroblastoma

“…For example, GnT-V, GnT-III, ST6GalNAc I, and ST6Gal-I directly modify carbohydrate structures on ␤ 1 integrin and affect integrin activity. [33][34][35][36] These changes in N-glycosylation 33,37 or O-glycosylation 36 of ␤ 1 integrin lead to altered cell morphologic features and behavior. B4GALNT3 modifies N-and O-glycans decorated with GlcNAc in vitro.…”

Deficiency in surface expression of E‐selectin ligand promotes lung colonization in a mouse model of breast cancer

“…53 The glycosylation profile of integrins has been implicated in the modulation of their function. 54,55 Therefore, sLe x expression may modify the adhesion abilities of either E-cadherin or integrins involved in metastasis. Further studies are needed to investigate these possibilities.…”

β1,4-N-Acetylgalactosaminyltransferase III Enhances Malignant Phenotypes of Colon Cancer Cells

“…Recently, changes in expression levels of glycosyltransferases have been found to alter integrin signaling and thereby regulate cell behavior. For example, GnT-III overexpression inhibits cell spreading and migration on fibronectin and decreases FAK phosphorylation (9). ST6Gal-I overexpression results in increased association of h 1 integrin and talin, indicating that integrin signaling is activated (8).…”

“…ST6Gal-I has been shown to sialylate h 1 integrins and up-regulate colon cancer cell migration (8). GnT-III introduces bisecting GlcNAc into integrin a 5 h 1 and downregulates cell adhesion and migration (9). Aberrant Nglycosylation of h 1 integrin induced by GnT-V causes reduced a 5 h 1 integrin clustering and stimulates cell migration (10).…”