Potential of N-glycan in cell adhesion and migration as either a positive or negative regulator

Glycosylation is one of the most abundant posttranslational modification reactions, and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharide structure (glycan) are associated with many physiological and pathological events, including cell adhesion, migration, cell growth, cell differentiation and tumor invasion. Glycosylation reactions are catalyzed by the action of glycosyltransferases, which add sugar chains to various complex carbohydrates such as glycoproteins, glycolipids and proteoglycans. Functional glycomics, which uses sugar remodeling by glycosyltransferases, is a promising tool for the characterization of glycan functions. Here, we will focus on the positive and negative regulation of biological functions of integrins by the remodeling of N-glycans with N-acetylglucosaminyltransferase III (GnT-III) and N-acetylglucosaminyltransferase V (GnT-V), which catalyze branched N-glycan formations, bisecting GlcNAc and β1,6 GlcNAc, respectively. Typically, integrins are modified by GnT-III, which inhibits cell migration and cancer metastasis. In contrast, integrins modified by GnT-V promote cell migration and cancer invasion.     

N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on alpha3beta1 integrin-mediated cell migration

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta1,6-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 alpha3beta1 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 beta1,6-GlcNAc-branched N-glycans on integrin alpha3 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 beta1,6-GlcNAc-branched N-glycans on the alpha3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the alpha3 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.     

N-Acetylglucosaminyltransferase V regulates TGF-β response in hepatic stellate cells and the progression of steatohepatitis

N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in tumor metastasis and carcinogenesis. Although the expression of GnT-V is induced in chronic liver diseases, the biological meaning of GnT-V in the diseases remains unknown. The aim of this study was to investigate the effects of GnT-V on the progression of chronic hepatitis, using GnT-V transgenic (Tg) mice fed a high fat and high cholesterol (HFHC) diet, an experimental model of murine steatohepatitis. Although enhanced hepatic lymphocytes infiltration and fibrosis were observed in wild-type (WT) mice fed the HFHC diet, they were dramatically prevented in Tg mice. In addition, the gene expression of inflammatory Th1 cytokines in the liver was significantly decreased in Tg mice than WT mice. Inhibition of liver fibrosis was due to the dysfunction of hepatic stellate cells (HSCs), which play pivotal roles in liver fibrosis through the production of transforming growth factor (TGF)-β1. Although TGF-β1 signaling was enhanced in Tg mouse-derived HSCs (Tg-HSCs) compared with WT mouse-derived HSCs (WT-HSCs), collagen expression was significantly reduced in Tg-HSCs. As a result from DNA microarray, cyclooxygenase-2 (COX2) expression, known as a negative feedback signal for TGF-β1, was significantly elevated in Tg-HSCs compared with WT-HSCs. Prostaglandin E2 (PGE2), the product of COX2, production was also significantly elevated in Tg-HSCs. COX2 inhibition by celecoxib decreased PGE2 and increased collagen expression in Tg-HSCs. In conclusion, GnT-V prevented steatohepatitis progression through modulating lymphocyte and HSC functions.     

Silencing of Discoidin Domain Receptor-1 (DDR1) Concurrently Inhibits Multiple Steps of Metastasis Cascade in Gastric Cancer

Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by interacting with the surrounding collagen matrix. In this study, we investigated the expression and role of DDR1 in human gastric cancer metastasis. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1-expressing MKN74 gastric cancer cells and corresponding DDR1-silenced cells. The effects of DDR1 on tumor growth and metastasis were examined in orthotopically implanted and experimental liver metastasis models in nude mice. The expression of DDR1 in surgical specimens was analyzed by immunohistochemistry. DDR1 was expressed in human gastric cancer cell lines, and its expression in human gastric tumors was associated with poor prognosis. Among seven gastric cancer cell lines, MKN74 expressed the highest levels of DDR1. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumors significantly reduced angiogenesis and lymphangiogenesis, thereby leading to reductions in lymph node metastasis and liver metastasis. In a model of experimental liver metastasis, DDR1-silenced cells almost completely inhibited liver colonization and metastasis. DDR1 deficiency led to reduced expression of the genes encoding vascular endothelial growth factor (VEGF)-A, VEGF-C, and platelet-derived growth factor-B. These results suggest that DDR1 is involved in gastric cancer tumor progression and that silencing of DDR1 inhibits multiple steps of the gastric cancer metastasis process.     

Caveolin-1 regulates the functional localization of N-acetylglucosaminyltransferase III within the golgi apparatus

In an investigation of the mechanism underlying the functional sublocalization of glycosyltransferases within the Golgi apparatus, caveolin-1 was identified as a possible cellular factor. Caveolin-1 appears to regulate the localization of N-acetylglucosaminyltransferase III (GnT-III) in the intra-Golgi subcompartment. Structural analyses of total cellular N-glycans indicated that the overexpression of GnT-III in human hepatoma cells, in which caveolin-1 is not expressed, failed to reduce branch formation, whereas expression of caveolin-1 led to a dramatic decrease in the extent of branching with no enhancement in GnT-III activity. Because the addition of a bisecting GlcNAc by GnT-III to the core beta-Man in N-glycans prevents the action of GnT-IV and GnT-V, both of which are involved in branch formation, this result suggests that caveolin-1 facilitates the prior action of GnT-III, relative to the other GnTs, on the nascent sugar chains in the Golgi apparatus and that GnT-III is redistributed in the earlier Golgi subcompartment by caveolin-1. Indeed, when caveolin-1 was expressed in human hepatoma cells, it was found to be co-localized with GnT-III, as evidenced by the fractionation of Triton X-100-insoluble cellular membranes by density gradient ultracentrifugation. Caveolin-1 may modify the biosynthetic pathway of sugar chains via the regulation of the intra-Golgi subcompartment localization of this key glycosyltransferase.     

Post-translational Glycoprotein Modifications Regulate Colon Cancer Stem Cells and Colon Adenoma Progression in Apcmin/+ Mice through Altered Wnt Receptor Signaling

Deletion of GnT-V (MGAT5), which synthesizes N-glycans with β(1,6)-branched glycans, reduced the compartment of cancer stem cells (CSC) in the her-2 mouse model of breast cancer, leading to delay of tumor onset. Because GnT-V levels are also commonly up-regulated in colon cancer, we investigated their regulation of colon CSC and adenoma development. Anchorage-independent cell growth and tumor formation induced by injection of colon tumor cells into NOD/SCID mice were positively associated with GnT-V levels, indicating regulation of proliferation and tumorigenicity. Using Apc^min/+ mice with different GnT-V backgrounds, knock-out of GnT-V had no significant effect on the number of adenoma/mouse, but adenoma size was significantly reduced and accompanied increased survival of Apc^min/+ mice with GnT-V deletion (p < 0.01), suggesting an inhibition in the progression of colon adenoma caused by deletion of GnT-V. Decreased expression levels of GnT-V down-regulated the population of colon (intestine) CSC, affecting their ability for self-renewal and tumorigenicity in NOD/SCID mice. Furthermore, altered nuclear translocation of β-catenin and expression of Wnt target genes were positively associated with expression levels of GnT-V, indicating the regulation of canonical Wnt/β-catenin signaling. By overexpressing the Wnt receptor, FZD-7, in colon cancer cells, we found that FZD-7 receptors expressed N-linked β(1,6) branching, indicating that FZD-7 can be modified by GnT-V. The aberrant Wnt signaling observed after modulating GnT-V levels is likely to result from altered N-linked β(1,6) branching on FZD-7, thereby affecting Wnt signaling, the compartment of CSC, and tumor progression.     

Roles of N-acetylglucosaminyltransferase III in epithelial-to-mesenchymal transition induced by transforming growth factor β1 (TGF-β1) in epithelial cell lines

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.     

E-cadherin and adherens-junctions stability in gastric carcinoma: Functional implications of glycosyltransferases involving N-glycan branching biosynthesis,N-acetylglucosaminyltransferases III and V

Background

E-cadherin is a cell–cell adhesion molecule and the dysfunction of which is a common feature of more than 70% of all invasive carcinomas, including gastric cancer. Mechanisms behind the loss of E-cadherin function in gastric carcinomas include mutations and silencing at either the DNA or RNA level. Nevertheless, in a high percentage of gastric carcinoma cases displaying E-cadherin dysfunction, the mechanism responsible for E-cadherin dysregulation is unknown. We have previously demonstrated the existence of a bi-directional cross-talk between E-cadherin and two major N-glycan processing enzymes, N-acetylglucosaminyltransferase-III or -V (GnT-III or GnT-V).

Methods

In the present study, we have characterized the functional implications of the N-glycans catalyzed by GnT-III and GnT-V on the regulation of E-cadherin biological functions and in the molecular assembly and stability of adherens-junctions in a gastric cancer model. The results were validated in human gastric carcinoma samples.

Results

We demonstrated that GnT-III induced a stabilizing effect on E-cadherin at the cell membrane by inducing a delay in the turnover rate of the protein, contributing for the formation of stable and functional adherens-junctions, and further preventing clathrin-dependent E-cadherin endocytosis. Conversely, GnT-V promotes the destabilization of E-cadherin, leading to its mislocalization and unstable adherens-junctions with impairment of cell–cell adhesion.

Conclusions

This supports the role of GnT-III on E-cadherin-mediated tumor suppression, and GnT-V on E-cadherin-mediated tumor invasion.

General significance

These results contribute to fill the gap of knowledge of those human carcinoma cases harboring E-cadherin dysfunction, opening new insights into the molecular mechanisms underlying E-cadherin regulation in gastric cancer with potential translational clinical applications.     

Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation,and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels

N-Acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of the bisecting GlcNAc branch on N-glycans, is usually described as a metastasis suppressor. Overexpression of GnT-III inhibited migration in multiple types of tumor cells. However, these results seem controversial to the clinical observations for the increased expression of GnT-III in human hepatomas, glioma, and ovarian cancers. Here, we present evidence that these inconsistencies are mainly attributed to the different expression pattern of cell sialylation. In detail, we show that overexpression of GnT-III significantly inhibits α2,3-sialylation but not α2,6-sialylation. The migratory ability of cells without or with a low level of α2,6-sialylation is consistently suppressed after GnT-III overexpression. In contrast, the effects of GnT-III overexpression are variable in tumor cells that are highly α2,6-sialylated. Overexpression of GnT-III promotes the cell migration in glioma cells U-251 and hepatoma cells HepG2, although it has little influence in human breast cancer cell MDA-MB-231 and gastric cancer cell MKN-45. Interestingly, up-regulation of α2,6-sialylation by overexpressing β-galactoside α2,6-sialyltranferase 1 in the α2,6-hyposialylated HeLa-S3 cells abolishes the anti-migratory effects of GnT-III. Conversely, depletion of α2,6-sialylation by knock-out of β-galactoside α2,6-sialyltranferase 1 in α2,6-hypersialylated HepG2 cells endows GnT-III with the anti-migratory ability. Taken together, our data clearly demonstrate that high expression of α2,6-sialylation on the cell surface could affect the anti-migratory role of GnT-III, which provides an insight into the mechanistic roles of GnT-III in tumor metastasis.     

All-Trans-Retinoic Acid Modulates ICAM-1 N-Glycan Composition by Influencing GnT-III Levels and Inhibits Cell Adhesion and Trans-Endothelial Migration

Changes in the expression of glycosyltransferases directly influence the oligosaccharide structures and conformations of cell surface glycoproteins and consequently cellular phenotype transitions and biological behaviors. In the present study, we show that all-trans-retinoic acid (ATRA) modulates the N-glycan composition of intercellular adhesion molecule-1 (ICAM-1) by manipulating the expression of two N-acetylglucosaminyltransferases, GnT-III and GnT-V, via the ERK signaling pathway. Exposure of various cells to ATRA caused a remarkable gel mobility down-shift of ICAM-1. Treatment with PNGase F confirmed that the reduction of the ICAM-1 molecular mass is attributed to the decreased complexity of N-glycans. We noticed that the expression of the mRNA encoding GnT-III, which stops branching, was significantly enhanced following ATRA exposure. In contrast, the level of the mRNA encoding GnT-V, which promotes branching, was reduced following ATRA exposure. Silencing of GnT-III prevented the molecular mass shift of ICAM-1. Moreover, ATRA induction greatly inhibited the adhesion of SW480 and U937 cells to the HUVEC monolayer, whereas knock-down of GnT-III expression effectively restored cell adhesion function. Treatment with ATRA also dramatically reduced the trans-endothelial migration of U937 cells. These data indicate that the alteration of ICAM-1 N-glycan composition by ATRA-induced GnT-III activities hindered cell adhesion and cell migration functions simultaneously, pinpointing a unique regulatory role of specific glycosyltransferases in the biological behaviors of tumor cells and a novel function of ATRA in the modulation of ICAM-1 N-glycan composition.     

Alterations in glycosyltransferases during myeloid and monocytoid differentiation of HL-60 cells.

HL-60, a human promyelocytic leukemia cell line, can be differentiated to myeloid lineage by all- trans retinoic acid (ATRA), dimethylsulfoxide (DMSO) and n -butyric acid (n -BA), or to monocytoid(monocytic/macrophagic) lineage by phorbol-12-myristate-13-acetate (PMA) and ganglioside GM(3). The activity alterations of N -acetylglucosaminyltransferase III and V (GnT-III, GnT-V) as well as alpha-1,6-fucosyl-tranferase (alpha1,6 Fuc T) were studied during the differentiation of HL-60 cells by the above-mentioned five inducers using the fluorescence (PA)-labeled glycan-HPLC method for GnT assays and biotin-labeled glycan-LCA affinity chromatography combined with the HRP-avidin colorimetric method for alpha1,6 Fuc T assay. It was observed that after 3 days, all three enzymes decreased in HL-60 cells induced by 1 micromol/l ATRA and 0.6 mmol/l n-BA, while GnT-III and alpha1,6 Fuc T increased, but GnT-V still decreased after induction by 1% DMSO. GnT-V and alpha1,6 Fuc T declined, while GnT-III was elevated after induction by 0.1 micromol/l PMA for 3 days. In contrast, GnT-III increased after the treatment with 50 micromol/l GM(3)for 3 or 6 days, but GnT-V was not appreciably changed and alpha1,6 FucT was elevated after 6 days of GM(3)treatment. It may be concluded that the decrease of GnT-V is the common change in myeloid differentiation and the increase of GnT-III is the general alteration in monocytoid differentiation. The changes in the activities of glycosyltransferases were consistent with the structural changes in surface N -glycans previously found in our laboratory, i.e. that the antennary number of N -glycans decreased during myeloid differentiation by ATRA, and the amount of bisecting GlcNAc in N -glycans increased during monocytoid differentiation by PMA.     

Prometastatic effect of N-acetylglucosaminyltransferase V is due to modification and stabilization of active matriptase by adding beta 1-6 GlcNAc branching

Oligosaccharide moieties of glycoproteins are structurally altered during development, carcinogenesis, and malignant transformations. It is well known that beta1-6 GlcNAc branching, a product of UDP-GlcNAc alpha-mannoside beta1-6-N-acetylglucosaminyltransferase (GnT-V), is associated with malignant transformation as the results of such alterations. However, the mechanism by which beta1-6 GlcNAc branching is linked to metastasis remains unclear, because the identification of specific glycoprotein(s) that are glycosylated by GnT-V and its biological function have not been examined. We herein report that matriptase, which activates both urokinase-type plasminogen activator and hepatocyte growth factor, is a target protein for GnT-V. The overexpression of GnT-V in gastric cancer cells leads to severe peritoneal dissemination in athymic mice, which can be attributed to the increased expression of matriptase. This increase was due to the acquired resistance of matriptase to degradation, since it is glycosylated by GnT-V and a corresponding increase in the active form. These results indicate that this process is a key element in malignant transformation, as the direct result of oligosaccharide modification.     

Transforming growth factor-β signaling: Tumorigenesis and targeting for cancer therapy

Transforming growth factor (TGF)-β is a multitasking cytokine such that its aberrant expression is related to cancer progression and metastasis. TGF-β is produced by a variety of cells within the tumor microenvironment (TME), and it is responsible for regulation of the activity of cells within this milieu. TGF-β is a main inducer of epithelial–mesenchymal transition (EMT), immune evasion, and metastasis during cancer progression. TGF-β exerts most of its functions by acting on TβRI and TβRII receptors in canonical (Smad-dependent) or noncanonical (Smad-independent) pathways. Members of mitogen-activated protein kinase, phosphatidylinositol 3-kinase/protein kinase B, and nuclear factor κβ are involved in the non-Smad TGF-β pathway. TGF-β acts by complex signaling, and deletion in one of the effectors in this pathway may influence the outcome in a diverse way by taking even an antitumor role. The stage and the type of tumor (contextual cues from cancer cells and/or the TME) and the concentration of TGF-β are other important factors determining the fate of cancer (progression or repression). There are a number of ways for targeting TGF-β signaling in cancer, among them the special focus is on TβRII suppression.     

肿瘤浸润转移分子机制的研究进展

肿瘤浸润转移是多因素参与、多步骤完成的生物化学变化过程。人们已经逐渐认识到浸润转移不仅与肿瘤细胞有关,更是肿瘤细胞和肿瘤组织微环境复杂的相互作用的结果,其过程涉及多个分子作用机制和信号转导途径,包括细胞和细胞的黏附分子、细胞外基质降解、生长因子、趋化因子和淋巴血管生成因子等。本文综述了肿瘤浸润转移的分子机制。     

Hypothyroidism Enhances Tumor Invasiveness and Metastasis Development

Background

Whereas there is increasing evidence that loss of expression and/or function of the thyroid hormone receptors (TRs) could result in a selective advantage for tumor development, the relationship between thyroid hormone levels and human cancer is a controversial issue. It has been reported that hypothyroidism might be a possible risk factor for liver and breast cancer in humans, but a lower incidence of breast carcinoma has been also reported in hypothyroid patients

Methodology/Principal Findings

In this work we have analyzed the influence of hypothyroidism on tumor progression and metastasis development using xenografts of parental and TRβ1–expressing human hepatocarcinoma (SK-hep1) and breast cancer cells (MDA-MB-468). In agreement with our previous observations tumor invasiveness and metastasis formation was strongly repressed when TRβ–expressing cells were injected into euthyroid nude mice. Whereas tumor growth was retarded when cells were inoculated into hypothyroid hosts, tumors had a more mesenchymal phenotype, were more invasive and metastatic growth was enhanced. Increased aggressiveness and tumor growth retardation was also observed with parental cells that do not express TRs.

Conclusions/Significance

These results show that changes in the stromal cells secondary to host hypothyroidism can modulate tumor progression and metastatic growth independently of the presence of TRs on the tumor cells. On the other hand, the finding that hypothyroidism can affect differentially tumor growth and invasiveness can contribute to the explanation of the confounding reports on the influence of thyroidal status in human cancer.     

Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes: inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase

Changes in the expression of glycosyltransferases that branch N-linked glycans can alter the function of several types of cell surface receptors and a glucose transporter. To study in detail the mechanisms by which aberrant N-glycosylation caused by altered N-acetylglucosaminyltransferase V(GnT-V, GnT-Va, and Mgat5a) expression can regulate the invasiveness-related phenotypes found in some carcinomas, we utilized specific small interfering RNA (siRNA) to selectively knock down GnT-V expression in the highly metastatic and invasive human breast carcinoma cell line, MDA-MB231. Knockdown of GnT-V by siRNA expression had no effect on epidermal growth factor receptor expression levels but lowered expression of N-linked beta(1,6)-branching on epidermal growth factor receptor, as expected. Compared with control cells, knockdown of GnT-V caused significant inhibition of the morphological changes and cell detachment from matrix that is normally seen after stimulation with epidermal growth factor (EGF). Decreased expression of GnT-V caused a marked inhibition of EGF-induced dephosphorylation of focal adhesion kinase (FAK), consistent with the lack of cell morphology changes in the cells expressing GnT-V siRNA. The attenuation of EGF-mediated phosphorylation and activation of the tyrosine phosphatase SHP-2 was dramatically observed in GnT-V knockdown cells, and these effects could be rescued by reintroduction of GnT-V into these cells, indicating that reduced EGF-mediated activation of SHP-2 was GnT-V related. Concomitantly, knockdown of GnT-V caused reduced EGF-mediated ERK signaling and tumor cell invasiveness-related phenotypes, including effects on actin rearrangement and cell motility. No changes in EGF binding were observed, however, after knockdown of GnT-V. Our results demonstrate that decreased GnT-V activity due to siRNA expression in human breast carcinoma cells resulted in an inhibition of EGF-stimulated SHP-2 activation and, consequently, caused attenuation of the dephosphorylation of FAK induced by EGF. These effects suppressed EGF-mediated downstream signaling and invasiveness-related phenotypes and suggest GnT-V as a potential therapeutic target.     

Potential of N-glycan in cell adhesion and migration as either a positive or negative regulator

Glycosylation is one of the most abundant posttranslational modification reactions, and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharide structure (glycan) are associated with many physiological and pathological events, including cell adhesion, migration, cell growth, cell differentiation and tumor invasion. Glycosylation reactions are catalyzed by the action of glycosyltransferases, which add sugar chains to various complex carbohydrates such as glycoproteins, glycolipids and proteoglycans. Functional glycomics, which uses sugar remodeling by glycosyltransferases, is a promising tool for the characterization of glycan functions. Here, we will focus on the positive and negative regulation of biological functions of integrins by the remodeling of N-glycans with N-acetylglucosaminyltransferase III (GnT-III) and N-acetylglucosaminyltransferase V (GnT-V), which catalyze branched N-glycan formations, bisecting GlcNAc and β1,6 GlcNAc, respectively. Typically, integrins are modified by GnT-III, which inhibits cell migration and cancer metastasis. In contrast, integrins modified by GnT-V promote cell migration and cancer invasion.Key words: integrin, E-cadherin, GnT-III, GnT-V, N-glycosylation, glycosyltransferaseProtein glycosylation encompasses N-glycans, O-glycans and Glycosaminoglycans. N-glycans are linked to asparagine residues of proteins, which is a specific subset residing in the Asn-X-Ser/Thr motif, whereas O-glycans are attached to a subset of serines and threonines (Fig. 1).¹ An increasing body of evidence indicates that glycans in glycoproteins are involved in the regulation of cellular functions including cell-cell communication and signal transduction.^2,3 In fact, most receptors on the cell surface are N-glycosylated—integrins and epithelial growth factor receptors; and transforming growth factor β receptors. Here, we focus mainly on the modification of N-glycans of integrin α3β1 and α5β1 to address the important roles of N-glycans in cell adhesion and migration.Open in a separate windowFigure 1Two major types of protein glycosylation. N-glycans are covalently linked to asparagine (Asn) residue of proteins, specifically the Asn-X-Ser/Thr motif. In contrast, O-glycans are attached to a subset of glycosidically linked hydroxyl groups of the amino acids serine (Ser) and threonine (Thr).Previous studies indicate that the presence of the appropriate oligosaccharide can modulate integrin activation. When human fibroblasts were cultured in the presence of l-deoxymannojirimycin, an inhibitor of α-mannosidase II, which prevents N-linked oligosaccharide processing, immature α5β1 integrin appeared at the cell surface, and fibronectin (FN)-dependent adhesion was greatly reduced.⁴ In addition, the treatment of purified integrin α5β1 with N-glycosidase F, which cleaves between the innermost GlcNAc and asparagine residues of N-glycans from N-linked glycoproteins, resulted in the blockage of α5β1 binding to FN and the inherent association of both subunits,⁵ suggesting that N-glycosylation is essential for functional integrin α5β1. The production of glycoprotein glycans is catalyzed by various glycosyltransferases. N-Acetylglucosaminyltransferase III (GnT-III) transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to a β1, 4 mannose in N-glycans to form a “bisecting” GlcNAc linkage, as shown in Figure 2. Bisecting GlcNAc linkage is found in various hybrid and complex N-glycans. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways. Introduction of a bisecting GlcNAc suppresses further processing and elongation of N-glycans catalyzed by N-acetylglucosaminyltransferase V (GnT-V), which is strongly associated with cancer metastasis, since GnT-V cannot utilize the bisected oligosaccharide as a substrate.^6–8 It has also been reported that GnT-V activity and β1, 6 branched N-glycan levels are increased in highly metastatic tumor cell lines.^9,10 When NIH3T3 cells were transformed with the oncogenic Ras gene, cell spreading on FN was greatly enhanced due to an increase in β1, 6 GlcNAc branched tri- and tetra-antennary oligosaccharides in α5β1 integrins.⁹ Similarly, the characterization of N-glycans of integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that β1, 6 GlcNAc branched structures were expressed at high levels in metastatic cells compared with non-metastatic cells.¹⁰ Cancer metastasis was consistently, and significantly, suppressed in GnT-V knockout mice.¹¹Open in a separate windowFigure 2Glycosylation reactions catalyzed by the action of glycosyltransferase GnT-III and GnT-V. The remodeled N-glycans regulate cell adhesion and migration. Enhanced expression of GnT-V in epithelial cells results in a loss of cell-cell adhesion, increasing integrin-mediated cell migration. In contrast, overexpression of GnT-III strengthens cell-cell interaction and downregulates integrin-mediated cell migration, which may contribute to the suppression of cancer metastasis. The β1,6GlcNAc branching is preferentially modified by polylactosamine and other sugar motifs such as sialyl Lewis X, which also contribute to promotion of cancer metastasis. It is worth mentioning that GnT-III could be proposed as an antagonistic of GnT-V, since GnT-V cannot utilize the bisected oligosaccharide as a substrate.To explore the possible mechanisms involved in increased β1, six branched N-glycans on cancer cells, Guo et al. found that cell migration toward FN and invasion through the matrigel were both substantially stimulated in cells in which the expression of GnT-V was induced.¹² Increased branched sugar chains inhibited the clustering of integrin α5β1 and the organization of F-actin into extended microfilaments in cells plated on FN-coated plates, which supports the hypothesis that the degree of adhesion of cells to their extracellular matrix (ECM) substrate is a critical factor in regulating the rate of cell migration, i.e., migration is maximal under conditions of intermediate levels of cell adhesion.¹³ Conversely, GnT-V null mouse embryonic fibroblasts (MEF) displayed enhanced cell adhesion to, and spreading on, FN-coated plates with the concomitant inhibition of cell migration. The restoration of GnT-V cDNA in the null MEF reversed these abnormal characteristics, indicating the direct involvement of N-glycosylation events in these phenotypic changes.In contrast to GnT-V, the overexpression of GnT-III resulted in an inhibition of α5β1 integrin-mediatedcell spreading and migration, and the phosphorylation of the focal adhesion kinase.¹⁴ The affinity of the binding of integrin α5β1 to FN was significantly reduced as a result of the introduction of a bisecting GlcNAc to the α5 subunit. In addition, overexpression of GnT-III in highly metastatic melanoma cells reduced β1, six branching in cell-surface N-glycans and increased bisected N-glycans.¹⁵ Therefore, GnT-III has been proposed as an antagonistic of GnT-V, thereby contributing to the suppression of cancer metastasis. In fact, the opposing effects of GnT-III and GnT-V have been observed for the same target protein, integrin α3β1.¹⁶ GnT-V stimulates α3β1 integrin-mediated cell migration, while overexpression of GnT-III inhibits GnT-V-induced cell migration. The modification of the α3 subunit by GnT-III supersedes modification by GnT-V. As a result, GnT-III inhibits GnT-V-induced cell migration. These results strongly suggest that remodeling of glycosyltransferase-modified N-glycan structures either positively or negatively modulates cell adhesion and migration.In addition, sialylation on the non-reducing terminus of N-glycans of α5β1 integrin plays an important role in cell adhesion. The increased sialylation of the β1 integrin subunit was correlated with a decreased adhesiveness and metastatic potential.^17–19 On the other hand, the enzymatic removal of α2, eight-linked oligosialic acids from the α5 integrin subunit inhibited cell adhesion to FN,²⁰ supporting the observation that the N-glycans of α and β integrin subunits play distinct roles in cell-ECM interactions.²¹ Collectively, these findings suggest that the interaction of integrin α5β1 with FN is dependent on N-glycosylation and the processing status of N-glycans.Although alteration of the oligosaccharide portion on integrin α5β1 could affect cis- and trans-interactions caused by GnT-III, ST6GalI and GnT-V, as described above, the molecular mechanism remains unclear. Considering integrin α5β1 contains 26 potential N-linked glycosylation sites (14 in the α subunit and 12 in the β subunit), the determination of those crucial N-glycosylation sites for its biological function is, therefore, quite important for an understanding of the underlying mechanism. We sequentially mutated either one or a combination of asparagine residues in the putative N-glycosylation sites of glutamine residues, and found that N-glycosylation on the β-propeller domain of the α5 subunit (in particular sites number 3–5) is essential for its hetero-dimer formation and its biological functions such as cell spreading and cell migration, as well as for the proper folding of the α5 subunit.²² On the other hand, N-glycans on β1 integrin also play important roles in the regulation of its biological functions^23,24 (and our unpublished data). Very recently, we also found that GnT-III specifically modifies one of the important glycosylation sites, which results in functional regulation (unpublished data). We postulate that these important sites may participate in supramolecular complex formation on the cell surface, which controls intracellular signal transduction.It also is worth noting that N-glycans regulate cell-ECM association as well as cell-cell adhesion. Overexpression of GnT-III slowed E-cadherin turnover, resulting in increased E-cadherin expression on the surface of B16 melanoma cells.²⁵ E-cadherin engagement at cell-cell contacts is known to suppress cell migration, and that effect has been best described in the context of tumorigenesis.²⁶ Conversely, the disruption of E-cadherin-mediated cell adhesion appears to be a central event in the transition from non-invasive to invasive carcinomas. Interestingly, we recently found that E-cadherin-mediated cell-cell interaction upregulated GnT-III expression,^27,28 suggesting that regulation of GnT-III and E-cadherin expression may exist as a positive feedback loop. Taken together, the overexpression of GnT-III inhibits cell migration by at least two mechanisms: an enhancement in cell-cell adhesion and a downregulation of cell-ECM adhesion (Fig. 2).Indeed, glycosylation defects in humans and their links to disease have shown that the mammalian glycome contains a significant amount of biological information.²⁹ The mammalian glycome repertoire is estimated to be between hundreds and thousands of glycan structures and could be larger than its proteome counterpart. Nevertheless, characterization of the biological functions of each glycan could one day make a significant contribution to the diagnosis and treatment of disease.