A common glycan alteration in transformed cells and human tumors is the highly elevated levels of N-linked ␤(1,6)glycans caused by increased transcription of Nacetylglucosaminyltransferase V (GnT-V). Here, we define the involvement of GnT-V in modulation of homotypic cell-cell adhesion in human fibrosarcoma HT1080 and mouse NIH3T3 cells. Increased GnT-V expression resulted in a significant decrease in the rates of calciumdependent cell-cell adhesion. Reduced cell-cell adhesion was blocked by function-blocking antibody against Ncadherin and abrogated by pre-treatment of cells with swainsonine, demonstrating the involvement of N-cadherin in the cell-cell adhesion and that changes in Nlinked ␤(1,6)glycan expression are responsible for the reduction in rates of adhesion, although this reduction could be mediated by the altered N-linked glycosylation of glycoproteins other than N-cadherin. Overexpression of GnT-V had no effect on the levels of cell surface expression of N-cadherin; however, it did cause a marked enhancement of both ␤(1,6) branching and poly-Nacetyllactosamine expression on N-cadherin. GnT-V overexpression resulted in decreased N-cadherin clustering on the cell surface induced by anti-N-cadherin antibody and affected the outside-in signal transduction pathway of ERK mediated by N-cadherin. Overexpression of GnT-V sensitized stimulation of tyrosine phosphorylation of catenins by growth factors and expression of v-src, which is consistent with its reduction of cell-cell adhesion. In vitro, GnT-V-overexpressing cells showed increased motility concomitant with increased phosphorylation of catenins. Moreover, GnT-V-deficient embryo fibroblasts from GnT-V homozygous null mice (GnT-V ؊/؊ ) express N-cadherin and showed significantly increased levels of N-cadherin-based cell-cell adhesion compared with those from GnT-V ؉/؊ mice. These levels of adhesion were inhibited significantly by transient expression of GnT-V, confirming the hypothesis that levels of GnT-V can regulate cadherin-associated homotypic cell-cell adhesion. Aberrant N-linked ␤(1,6) branching that occurs during oncogenesis can, therefore, lessen cell-cell adhesion, contributing to increased cellular motility and invasiveness.Changes in the expression of many cell surface adhesion receptors, including integrins, cadherins, CD44, and members of the immunoglobulin superfamily such as intercellular adhesion molecule, mediate cell-cell and cell-ECM 1 interactions that are clearly critical as cells undergo oncogenesis and show changes in motility and invasiveness (1-3). Recent studies demonstrate that aberrant glycosylation of several types of cell surface receptors results in dysfunctional intracellular signaling and altered cellular behavior. For example, mutations in an N-acetylglucosaminyltransferase, POMGnT-I, cause aberrant glycosylation of skeletal muscle dystrophin, resulting in dysfunctional neuromuscular junctions (4). Mutations in POMGnT-I have been shown to result in multiple phenotypes, some of which have been classified as "muscle-eye-b...

The effects of altering N-cadherin N-glycosylation on several cadherin-mediated cellular behaviors were investigated using small interfering RNA and site-directed mutagenesis. In HT1080 fibrosarcoma cells, small interfering RNA-directed knockdown of N-acetylglucosaminyltransferase V (GnT-V), a glycosyltransferase up-regulated by oncogene signaling, caused decreased expression of N-linked ␤(1,6)-branched glycans expressed on N-cadherin, resulting in enhanced N-cadherin-mediated cell-cell adhesion, but had no effect on N-cadherin expression on the cell surface. This effect on adhesion was accompanied by decreased cell migration and invasion, opposite of the effects observed when GnT-V was overexpressed in these cells (Guo, H. B., Lee, I., Kamar, M., and Pierce, M. (2003) J. Biol. Chem. 278, 52412-52424). A detailed study using site-directed mutagenesis demonstrated that three of the eight putative N-glycosylation sites in the N-cadherin sequence showed N-glycan expression. Moreover, all three of these sites, located in the extracellular domains EC2 and EC3, were shown by leucoagglutinating phytohemagglutinin binding to express at least some ␤(1,6)-branched glycans, products of GnT-V activity. Deletion of these sites had no effect on cadherin levels on the cell surface but led to increased stabilization of cell-cell contacts, cell-cell adhesion-mediated intracellular signaling, and reduced cell migration. We show for the first time that these deletions had little effect on formation of the N-cadherincatenin complex but instead resulted in increased N-cadherin cis-dimerization. Branched N-glycan expression at three sites in the EC2 and -3 domains regulates N-cadherin-mediated cell-cell contact formation, outside-in signaling, and cell migration and is probably a significant contributor to the increase in the migratory/invasive phenotype of cancer cells that results when GnT-V activity is up-regulated by oncogene signaling.Cadherins are single-pass transmembrane receptors that mediate calcium-dependent cell-cell adhesion at adherens junctions and play an essential role in regulating major cellular behaviors, including cell growth, motility, and differentiation (1, 2). Several cadherins, including E-cadherin and N-cadherin, have in common an extracellular domain with five segments of repeated sequences and regulate cell-cell adhesion in a homotypic manner through their association of amino-terminal extracellular domains, such as EC1 (3, 4). Calcium binding to the extracellular domain triggers a conformation that initiates the homotypic binding of cadherin between cells. The conserved cytoplasmic domain of cadherin interacts with various proteins, collectively termed catenins, that link cadherins to the actin-based cytoskeleton and promote strong cell-cell adhesion (5). Evidence indicates that the formation and tyrosine phosphorylation of the cadherin-catenin complex are critical for the maintenance of the stabilization of cell-cell adhesion (6, 7). It has been well documented that cadherins are implicated in the regul...

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 ␤(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.The expression of specific glycan structures on several cell surface adhesion molecules, growth factor receptors, and the GLUT2 transporter is associated with an alteration of their functions (1-5). For example, studies have shown that aberrant N-glycosylation on several cell surface receptors, including integrins and cadherins, is associated with changes in carcinoma progression and metastasis (6 -11). A glycan whose expression is often up-regulated during malignant transformation contains the ␤(1,6)-linked N-acetylglucosamine found on some N-glycans (12), synthesized by N-acetylglucosaminyltransferase V (GnT-V 2 or Mgat5, also called GnT-Va; EC 2.4.1.155), a key enzyme in the processing of multiantennary N-glycans during glycoprotein biosynthesis whose expression is regulated by oncogene expression (13)(14)(15)(16)(17)(18). Studies have demonstrated the association o...

SummaryWe present direct evidence that the N-glycosylation state of neural cadherin impacts the intrinsic kinetics of cadherin-mediated intercellular binding. Micropipette manipulation measurements quantified the effect of N-glycosylation mutations on intercellular binding dynamics. The wild-type protein exhibits a two-stage binding process in which a fast, initial binding step is followed by a short lag and second, slower transition to the final binding stage. Mutations that ablate N-glycosylation at three sites on the extracellular domains 2 and 3 of neural cadherin alter this kinetic fingerprint. Glycosylation does not affect the affinities between the adhesive Nterminal domains, but instead modulates additional cadherin interactions, which govern the dynamics of intercellular binding. These results, together with previous findings that these hypo-glycosylation mutations increase the prevalence of cis dimers on cell membranes, suggest a binding mechanism in which initial adhesion is followed by additional cadherin interactions, which enhance binding but are modulated by N-glycosylation. Given that oncogene expression drives specific changes in N-glycosylation, these results provide insight into possible mechanisms altering cadherin function during tumor progression.

The expression of an enzyme, GnT-V, that catalyzes a specific posttranslational modification of a family of glycoproteins, namely a branched N-glycan, is transcriptionally up-regulated during breast carcinoma oncogenesis. To determine the molecular basis of how early events in breast carcinoma formation are regulated by GnT-V, we studied both the early stages of mammary tumor formation by using 3D cell culture and a her-2 transgenic mouse mammary tumor model. Overexpression of GnT-V in MCF-10A mammary epithelial cells in 3D culture disrupted acinar morphogenesis with impaired hollow lumen formation, an early characteristic of mammary neoplastic transformation. The disrupted acinar morphogenesis of mammary tumor cells in 3D culture caused by her-2 expression was reversed in tumors that lacked GnT-V expression. Moreover, her-2-induced mammary tumor onset was significantly delayed in the GnT-V null tumors, evidence that the lack of the posttranslational modification catalyzed by GnT-V attenuated tumor formation. Inhibited activation of both PKB and ERK signaling pathways was observed in GnT-V null tumor cells. The proportion of tumorinitiating cells (TICs) in the mammary tumors from GnT-V null mice was significantly reduced compared with controls, and GnT-V null TICs displayed a reduced ability to form secondary tumors in NOD/ SCID mice. These results demonstrate that GnT-V expression and its branched glycan products effectively modulate her-2-mediated signaling pathways that, in turn, regulate the relative proportion of tumor initiating cells and the latency of her-2-driven tumor onset. T he amplification and overexpression of her-2/erbB2, a member of the epidermal growth factor (EGF) receptor family, play a pivotal role in the development of several different types of cancers, including breast carcinoma (1, 2). Oncogenesis observed in mouse mammary epithelia induced by her-2 expression shares similarities with that of human breast carcinoma (3). Her-2 signaling is activated through its interactions with other EGF family receptors after they bind ligands, including epidermal growth factor (EGF) and neuregulin (NRG) (4). Ligand-induced phosphorylation of this family of receptors recruits various docking proteins and signaling molecules that convey proliferative and survival signals via MAPK and PI3K/PKB pathways (5). In a reconstituted basement membrane culture system (3D culture), activation or overexpression of the her-2 receptor in a nontransformed mammary epithelial cell line (MCF-10A) elicits a multiacinar phenotype characterized by excessive cell proliferation and filling of the acinar luminal space that results from inhibited apoptosis and altered apicobasal polarization (6, 7). These in vitro alterations caused by her-2 overexpression are linked to the phenotypes observed for human breast ductal carcinoma in situ (DCIS) with erbB2 amplification (8). Recent studies have shown that her-2 regulates the mammary epithelial stem/ progenitor cell population that drives tumorigenesis and progression (9, 10), and th...

Background: GnT-V levels are implicated in regulating cancer stem cells and tumor development. Results: GnT-V levels via altered Wnt signaling regulate the compartment of colon cancer stem cells and tumor formation. Conclusion: GnT-V levels modulate Wnt signaling that regulate colon adenoma progression. Significance: A specific post-translational modification regulates Wnt signaling and colon cancer progression.

An N-linked glycan often increased during oncogenic transformation contains ␤(1,6)-linked GlcNAc, synthesized by the N-acetylglucosaminyltransferase V (GnT-V). The progression of polyoma middle T-antigen oncoprotein-induced mammary carcinomas in GnT-V null mice was significantly retarded compared with that observed in wild-type mice. The matrix adhesion of mouse embryonic fibroblasts (MEF) from GnT-V null and wildtype mice was investigated to understand the mechanism by which deletion of GnT-V could retard tumor progression. GnT-V null MEF displayed enhanced adhesion to and spreading on fibronectin-coated plates with concomitant inhibition of cell migration. GnT-V null MEF also showed increased focal adhesion kinase tyrosine phosphorylation, consistent with decreased cell motility on fibronectin-coated plates. Expression of GnT-V cDNA in the null MEF reversed these abnormal characteristics, indicating the direct involvement of N-glycosylation events in these phenotypic changes. The ␣5␤1 fibronectin receptors exhibited increased clustering on the null MEF cell surfaces, consistent with previous studies that observed less integrin clustering in cells overexpressing GnT-V. Most surprisingly, GnT-V null MEF displayed increased expression levels of both ␣5 and ␤1 subunits in lysates and on the cell surface. Increased ␣5␤1 expression in the null MEF was because of increased ␣5␤1 transcript levels that declined after re-expression of GnT-V cDNA, confirming that increased ␣5␤1 expression in null MEF was because of changes in GnT-V expression. The increased null MEF transcripts were shown to be caused at least in part by increased integrin promoter activity. Moreover, increased ␣5␤1 integrin transcripts in GnT-V null MEF were not due to a differential response to fibronectin; rather, they appeared to be mediated by activation of a protein kinase C signaling pathway. These results demonstrate that deletion of MEF GnT-V resulted in enhanced integrin clustering and activation of ␣5␤1 transcription by protein kinase C signaling, which in turn up-regulated levels of cell surface ␣5␤1 fibronectin receptors that resulted in increased matrix adhesion and inhibition of migration.Tumor progression is associated with changes in glycan structures on the cell surface. A common glycan up-regulated during malignant transformation is ␤(1,6)-GlcNAc on N-glycans 1 (1), synthesized by N-acetylglucosaminyltransferase V (GnT-V or Mgat5, EC 2.4.1.155), a key enzyme in the processing of multiantennary N-glycans during glycoprotein biosynthesis (2, 3). The increase in GnT-V activity and its cell surface products during oncogenesis results from increased transcription driven by activation of the Ras-Ets and protein kinase B signaling pathways (4 -6). Studies have demonstrated the association of increased GnT-V activity and its glycan products with enhanced cell invasiveness and in some cases metastatic potential (7-9). Consequently, mice that lack GnT-V expression because of a targeted deletion (GnT-V or Mgat5(Ϫ/Ϫ)) have been used to study the e...

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi alpha-mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc beta1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [3H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc beta1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin alpha5beta1 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated alpha5, but not beta1, and antisense GnT-V decreased both alpha5 and beta1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.