Tomoya Isaji

Branched N-glycans regulate the biological functions of integrins and cadherins

Glycosylation is one of the most common post‐translational modifications, and approximately 50% of all proteins are presumed to be glycosylated in eukaryotes. Branched N‐glycans, such as bisecting GlcNAc, β‐1,6‐GlcNAc and core fucose (α‐1,6‐fucose), are enzymatic products of N‐acetylglucosaminyltransferase III, N‐acetylglucosaminyltransferase V and α‐1,6‐fucosyltransferase, respectively. These branched structures are highly associated with various biological functions of cell adhesion molecules, including cell adhesion and cancer metastasis. E‐cadherin and integrins, bearing N‐glycans, are representative adhesion molecules. Typically, both are glycosylated by N‐acetylglucosaminyltransferase III, which inhibits cell migration. In contrast, integrins glycosylated by N‐acetylglucosaminyltransferase V promote cell migration. Core fucosylation is essential for integrin‐mediated cell migration and signal transduction. Collectively, N‐glycans on adhesion molecules, especially those on E‐cadherin and integrins, play key roles in cell–cell and cell–extracellular matrix interactions, thereby affecting cancer metastasis.

N-Acetylglucosaminyltransferase III Antagonizes the Effect of N-Acetylglucosaminyltransferase V on α3β1 Integrin-mediated Cell Migration

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of β1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that α3β1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in β1,6-GlcNAc-branched N-glycans on integrin α3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in β1,6-GlcNAc-branched N-glycans on the α3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the α3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions.

Deletion of Core Fucosylation on α3β1 Integrin Down-regulates Its Functions

The core fucosylation (α1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently α1,6-fucosylation has been further reported to be very crucial by the study of α1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on α3β1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in α3β1 integrin-mediated events has been studied by using Fut8+/+ and Fut8–/– embryonic fibroblasts, respectively. We found that the core fucosylation of α3β1 integrin, the major receptor for laminin 5, was abundant in Fut8+/+ cells but was totally abolished in Fut8–/– cells, which was associated with the deficient migration mediated by α3β1 integrin in Fut8–/– cells. Moreover integrin-mediated cell signaling was reduced in Fut8–/– cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of α3β1 integrin.

Potential roles of N-glycosylation in cell adhesion

The functional units of cell adhesion are typically multiprotein complexes made up of three general classes of proteins; the adhesion receptors, the cell-extracellular matrix (ECM) proteins, and the cytoplasmic plaque/peripheral membrane proteins. The cell adhesion receptors are usually transmembrane glycoproteins (for example E-cadherin and integrin) that mediate binding at the extracellular surface and determine the specificity of cell-cell and cell-ECM recognition. E-cadherin-mediated cell-cell adhesion can be both temporally and spatially regulated during development, and represents a key step in the acquisition of the invasive phenotype for many tumors. On the other hand, integrin-mediated cell-ECM interactions play important roles in cytoskeleton organization and in the transduction of intracellular signals to regulate various processes such as proliferation, differentiation and cell migration. ECM proteins are typically large glycoproteins, including the collagens, fibronectins, laminins, and proteoglycans that assemble into fibrils or other complex macromolecular arrays. The most of these adhesive proteins are glycosylated. Here, we focus mainly on the modification of N-glycans of integrins and laminin-332, and a mutual regulation between cell adhesion and bisected N-glycan expression, to address the important roles of N-glycans in cell adhesion.

N-Glycosylation of the β-Propeller Domain of the Integrin α5 Subunit Is Essential for α5β1 Heterodimerization, Expression on the Cell Surface, and Its Biological Function

The N-glycosylation of integrin α5β1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on theα5 integrin. Removal of the putative N-glycosylation sites on the β-propeller, Thigh, Calf-1, or Calf-2 domains of the α5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1–5) on the β-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the α5 subunit carrying only three potential N-glycosylation sites (3–5 sites) on theβ-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Δ3–5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the β-propeller domain of the α5 subunit is essential for heterodimerization and biological functions of α5β1 integrin and might also be useful for studies of the molecular structure.

N-Glycosylation of the I-like Domain of β1 Integrin Is Essential for β1 Integrin Expression and Biological Function: IDENTIFICATION OF THE MINIMAL N-GLYCOSYLATION REQUIREMENT FOR α5β1*

N-Glycosylation of integrin alpha5beta1 plays a crucial role in cell spreading, cell migration, ligand binding, and dimer formation, but the detailed mechanisms by which N-glycosylation mediates these functions remain unclear. In a previous study, we showed that three potential N-glycosylation sites (alpha5S3-5) on the beta-propeller of the alpha5 subunit are essential to the functional expression of the subunit. In particular, site 5 (alpha5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin beta1 subunit was investigated using sequential site-directed mutagenesis to remove the combined putative N-glycosylation sites. Removal of the N-glycosylation sites on the I-like domain of the beta1 subunit (i.e. the Delta4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the beta1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either alpha5S3-5 or alpha5S5 mutant of the alpha5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the beta1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the alpha5S3-5/beta1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the beta1 subunit, it might also be useful for the study of molecular structures.N-Glycosylation of integrin α5β1 plays a crucial role in cell spreading, cell migration, ligand binding, and dimer formation, but the detailed mechanisms by which N-glycosylation mediates these functions remain unclear. In a previous study, we showed that three potential N-glycosylation sites (α5S3–5) on the β-propeller of the α5 subunit are essential to the functional expression of the subunit. In particular, site 5 (α5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin β1 subunit was investigated using sequential site-directed mutagenesis to remove the combined putative N-glycosylation sites. Removal of the N-glycosylation sites on the I-like domain of the β1 subunit (i.e. the Δ4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the β1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5 mutant of the α5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the β1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the α5S3-5/β1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the β1 subunit, it might also be useful for the study of molecular structures.

Roles of N-Acetylglucosaminyltransferase III in Epithelial-to-Mesenchymal Transition Induced by Transforming Growth Factor β1 (TGF-β1) in Epithelial Cell Lines

Background: The inhibitory effects of GnT-III on cancer metastasis remain unclear. Results: GnT-III influenced EMT-like changes through not only prolongation of E-cadherin turnover but also suppression of β-catenin·p-Smad complex formation. Conclusion: GnT-III plays important roles in TGF-β-induced EMT-like changes. Significance: The expression of E-cadherin is regulated not only by transcriptional factors but also by post-transcriptional modifications. The epithelial-to-mesenchymal transition (EMT) plays crucial roles in embryonic development, wound healing, tissue repair, and cancer progression. Results of this study show how transforming growth factor β1 (TGF-β1) down-regulates expression of N-acetylglucosaminyltransferase III (GnT-III) during EMT-like changes. Treatment with TGF-β1 resulted in a decrease in E-cadherin expression and GnT-III expression, as well as its product, the bisected N-glycans, which was confirmed by erythro-agglutinating phytohemagglutinin lectin blot and HPLC analysis in human MCF-10A and mouse GE11 cells. In contrast with GnT-III, the expression of N-acetylglucosaminyltransferase V was slightly enhanced by TGF-β1 treatment. Changes in the N-glycan patterns on α3β1 integrin, one of the target proteins for GnT-III, were also confirmed by lectin blot analysis. To understand the roles of GnT-III expression in EMT-like changes, the MCF-10A cell was stably transfected with GnT-III. It is of particular interest that overexpression of GnT-III influenced EMT-like changes induced by TGF-β1, which was confirmed by cell morphological changes of phase contrast, immunochemical staining patterns of E-cadherin, and actin. In addition, GnT-III modified E-cadherin, which served to prolong E-cadherin turnover on the cell surface examined by biotinylation and pulse-chase experiments. GnT-III expression consistently inhibited β-catenin translocation from cell-cell contact into the cytoplasm and nucleus. Furthermore, the transwell assay showed that GnT-III expression suppressed TGF-β1-induced cell motility. Taken together, these observations are the first to clearly demonstrate that GnT-III affects cell properties, which in turn influence EMT-like changes, and to explain a molecular mechanism for the inhibitory effects of GnT-III on cancer metastasis.

A Mutual Regulation between Cell−Cell Adhesion and N-Glycosylation: Implication of the Bisecting GlcNAc for Biological Functions

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. E-cadherin-mediated cell-cell adhesion is believed to be both temporally and spatially regulated during development, and represents a key step in the acquisition of the invasive phenotype for many tumors. Here, we focus mainly on a mutual regulation between E-cadherin-mediated cell-cell adhesion and N-acetylglucosaminyltransferase III (GnT-III) expression, and discuss its implications for biological functions.

Cell-Cell Interaction-dependent Regulation of N-Acetylglucosaminyltransferase III and the Bisected N-Glycans in GE11 Epithelial Cells INVOLVEMENT OF E-CADHERIN-MEDIATED CELL ADHESION

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 α1,6-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-cadherin-dependent 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.

Biochemical visualization of cell surface molecular clustering in living cells

Many plasma membrane-resident molecules cluster with other molecules to collaborate in a variety of biological events. We herein report a sensitive and simple method to identify components of cell surface molecular clusters in living cells. This method includes a recently established reaction, called the enzyme-mediated activation of radical source (EMARS), to label molecules within a limited distance (≈200–300 nm) from the probed molecule on which HRP is set. Because the size of this active area is close to that of the reported membrane clusters, it is suggested that the labeled molecules cluster with the probed molecule in the same membrane domain. A combination of the EMARS reaction and antibody array analysis demonstrated that many kinds of receptor tyrosine kinases (RTKs) formed clusters with β1 integrin in HeLa S3 cells. A similar antibody array analysis after the EMARS reaction with three HRP-labeled antibodies against growth factor receptors showed the patterns of biotinylated RTKs to be substantially different from each other. These results suggest that different types of cell surface molecular clusters can thus be distinguished using the EMARS reaction. Therefore, the present “biochemical visualization” method is expected to be a powerful tool to elucidate molecular clustering on the cell surface of living cells in various contexts.