The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site—WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM? Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with α2–6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily α-fucosylated complex type chains with α2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells. Published in 2004.     

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.     

An N-Glycosylation Site on the��-Propeller Domain of the Integrin ��5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification by��1,4-N-Acetylglucosaminyltransferase III

Recently we reported that N-glycans on the β-propeller domain of the integrin α5 subunit (S-3,4,5) are essential for α5β1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type α5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the α5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type α5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the α5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin α5β1 is well known as a fibronectin (FN)³ receptor. The interaction between integrin α5 and FN is essential for cell migration, cell survival, and development (5–8). In addition, integrins are N-glycan carrier proteins. For example, α5β1 integrin contains 14 and 12 putative N-glycosylation sites on the α5 and β1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin α5β1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature α5β1 integrin appeared on the cell surface, and FN-dependent adhesion was greatly reduced (9). Treatment of purified integrin α5β1 with N-glycosidase F, which cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine N-glycan residues of N-linked glycoproteins, prevented the inherent association between subunits and blocked α5β1 binding to FN (10).A growing body of evidence indicates that the presence of the appropriate oligosaccharide can modulate integrin activation. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of GlcNAc to mannose that is β1,4-linked to an underlying N-acetylglucosamine, producing what is known as a “bisecting” GlcNAc linkage as shown in Fig. 1B. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways and contributes to inhibition of metastasis. The introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional processing and elongation of N-glycans. These reactions, which are catalyzed in vitro by other glycosyltransferases, such as N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the formation of β1,6 GlcNAc branching structures (Fig. 1B) and plays important roles in tumor metastasis, do not proceed because the enzymes cannot utilize the bisected N-glycans as a substrate. Introduction of the bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in decreased in ligand binding and down-regulation of cell adhesion and migration (11–13). Contrary to the functions of GnT-III, overexpression of GnT-V promoted integrin α5β1-mediated cell migration on FN (14). These observations clearly demonstrate that the alteration of N-glycan structure affected the biological functions of integrin α5β1. Similarly characterization of the carbohydrate moieties in integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that expression of β1,6 GlcNAc branched structures was higher in metastatic cells compared with non-metastatic cells, confirming the notion that the β1,6 GlcNAc branched structure confers invasive and metastatic properties to cancer cells. In fact, Partridge et al. (15) reported that GnT-V-modified N-glycans containing poly-N-acetyllactosamine, the preferred ligand for galectin-3, on surface receptors oppose their constitutive endocytosis, promoting intracellular signaling and consequently cell migration and tumor metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its modification by GnT-III and GnT-V. A, schematic diagram of potential N-glycosylation sites on the α5 subunit. Putative N-glycosylation sites are indicated by triangles, and point mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q, N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q). B, illustration of the reaction catalyzed by GnT-III and GnT-V. Square, GlcNAc; circle, mannose. TM, transmembrane domain.In addition, sialylation on the non-reducing terminus of N-glycans of α5β1 integrin plays an important role in cell adhesion. Colon adenocarcinomas express elevated levels of α2,6 sialylation and increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively correlated with metastasis and poor survival. Therefore, ST6GalI-mediated hypersialylation likely plays a role in colorectal tumor invasion (16, 17). In fact, oncogenic ras up-regulated ST6GalI and, in turn, increased sialylation of β1 integrin adhesion receptors in colon epithelial cells (18). However, this is not always the case. The expression of hyposialylated integrin α5β1 was induced by phorbol esterstimulated differentiation in myeloid cells in which the expression of the ST6GalI was down-regulated by the treatment, increasing FN binding (19). A similar phenomenon was also observed in hematopoietic or other epithelial cells. In these cells, the increased sialylation of the β1 integrin subunit was correlated with reduced adhesiveness and metastatic potential (20–22). In contrast, the enzymatic removal of α2,8-linked oligosialic acids from the α5 integrin subunit inhibited cell adhesion to FN (23). Collectively these findings suggest that the interaction of integrin α5β1 with FN is dependent on its N-glycosylation and the processing status of N-glycans.Because integrin α5β1 contains multipotential N-glycosylation sites, it is important to determine the sites that are crucial for its biological function and regulation. Recently we found that N-glycans on the β-propeller domain (sites 3, 4, and 5) of the integrin α5 subunit are essential for α5β1 heterodimerization, cell surface expression, and biological function (24). In this study, to further investigate the underlying molecular mechanism of GnT-III-regulated biological functions, we characterized the N-glycans on the α5 subunit in detail using genetic and biochemical approaches and found that site-4 is a key site that can be specifically modified by GnT-III.     

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.     

N-glycan of ErbB family plays a crucial role in dimer formation and tumor promotion

More and more evidence indicates that N-glycan regulates signal transduction by modulating receptor functions. Previous studies suggested that glycosylation of EGFR is involved in dimerization and endocytosis. We further determined the role of N-glycosylation of ErbB family. A series of human ErbB3 mutants that lack each of the 10 N-glycosylation sites were prepared and transfected to Flp-In-CHO cells for stable expression. A crosslinking study showed that Asn 418 to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin, and the heterodimer formation with ErbB2 was also increased. The N418Q mutant of ErbB3 co-expressed with ErbB2 promoted downstream signaling, anchorage-independent cell growth and the tumor growth in athymic mice. These findings suggest that the specific N-glycan in domain III of ErbB family plays an essential role in regulating receptor dimerization and transforming activity. We assume that the N-glycans affect the conformation of ErbB family, which is crucial for their activity. Together with findings from other laboratories, it is suggested that N-glycosylation controls ErbB signaling by various mechanisms.     

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.     

N-Glycosylation of the I-like Domain of ��1 Integrin Is Essential for ��1 Integrin Expression and Biological Function: IDENTIFICATION OF THE MINIMAL N-GLYCOSYLATION REQUIREMENT FOR ��5��1*

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.Integrin is a heterodimeric glycoprotein that consists of both an α and a β subunit (1). The interaction between integrin and the extracellular matrix is essential to both physiologic and pathologic events, such as cell migration, development, cell viability, immune homeostasis, and tumorigenesis (2, 3). Among the integrin superfamily, β1 integrin can combine with 12 distinct α subunits (α1–11, αv) to form heterodimers, thereby acquiring a wide variety of ligand specificity (1, 4). Integrins are thought to be regulated by inside-out signaling mechanisms that provoke conformational changes, which modulate the affinity of integrin for the ligand (5). However, an increasing body of evidence suggests that cell-surface carbohydrates mediate a variety of interactions between integrin and its extracellular environment, thereby affecting integrin activity and possibly tumor metastasis as well (6–8).Guo et al. (9) reported that an increase in β1–6-GlcNAc sugar chains on the integrin β1 subunit stimulated cell migration. In addition, elevated sialylation of the β1 subunit, because of Ras-induced STGal-I transferase activity, also induced cell migration (10, 11). Conversely, cell migration and spreading were reduced by the addition of a bisecting GlcNAc, which is a product of N-acetylglucosaminyltransferase III (GnT-III),² to the α5β1 and α3β1 integrins (12, 13). Alterations of N-glycans on integrins might also regulate their cis interactions with membrane-associated proteins, including the epidermal growth factor receptor, the galectin family, and the tetraspanin family of proteins (14–19).In addition to the positive and negative regulatory effects of N-glycan, several research groups have reported that N-glycans must be present on integrin α5β1 for the αβ heterodimer formation and proper integrin-matrix interactions. Consistent with this hypothesis, in the presence of the glycosylation inhibitor, tunicamycin, normal integrin-substrate binding and transport to the cell surface are inhibited (20). Moreover, treatment of purified integrin with N-glycosidase F blocked both the inherent association of the subunits and the interaction between integrin and fibronectin (FN) (21). These results suggest that N-glycosylation is essential to the functional expression of α5β1. However, because integrin α5β1 contains 26 potential N-linked glycosylation sites, 14 in the α subunit and 12 in the β subunit, identification of the sites that are essential to its biological functions is key to understanding the molecular mechanisms by which N-glycans alter integrin function. Recently, our group determined that N-glycosylation of the β-propeller domain on the α5 subunit is essential to both heterodimerization and biological functions of the subunit. Furthermore, we determined that sites 3–5 are the most important sites for α5 subunit-mediated cell spreading and migration on FN (22). The purpose of this study was to clarify the roles of N-glycosylation of the β1 subunit. Therefore, we performed combined substitutions in the putative N-glycosylation sites by replacement of asparagine residues with glutamine residues. We subsequently introduced these mutated genes into β1-deficient epithelial cells (GE11). The results of these mutation experiments revealed that the N-glycosylation sites on the I-like domain of the β1 subunit, sites number 4–6 (S4-6), are essential to both heterodimer formation and biological functions, such as cell spreading.     

Development and application of mass spectrometric methods for the analysis of progranulin N-glycosylation

PGRN is a modular protein with 7 1/2 repeats of the granulin domain separated by short spacer sequences. Elevated expression of PGRN is associated with cancer growth, while mutations of PGRN cause frontotemporal lobar degeneration (FTLD), an early onset form of dementia. PGRN is a glycoprotein, containing five N-glycosylation consensus sequons, three of which fall within granulin domains. A method tailored to enable detailed analysis of the PGRN oligosaccharides and glycopeptides has been developed. The approach involves in-gel deglycosylation using peptide-N-glycosidase F (PNGase F) followed by permethylation of the released oligosaccharides. Permethylation was applied for rapid sample clean-up and to improve sensitivity of MS detection and mass spectrometric fragmentation. Reversed-phase monolithic LC–ESI–MS/MS was used for analysis of permethylated oligosaccharides, enabling structural characterization of released N-linked glycans in one chromatographic run. In-gel tryptic digestion was further applied to the gel pieces containing deglycosylated protein, for N-glycosylation site determination. In addition, glycopeptides were produced using in-solution pronase digestion to identify species of N-glycan attached at particular sites. The method developed was applied to progranulin (PGRN) to characterize the structures of the released glycans and to identify the sites of glycosylation. Glycosylation of four out of five potential PGRN N-glycosylation consensus sites was demonstrated (the final one remains undetermined), with one of the four observed to be partially occupied. Two of the observed glycosylation sites occur within granulin domains, which may have important implications for understanding the structural basis of PGRN action.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site--WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily alpha-fucosylated complex type chains with alpha2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells.     

N-cadherin as a key regulator of collective cell migration in a 3D environment

Cell migration is a critical step of normal developmental processes and disease progression. Often, migrating cells interact and maintain contact with neighboring cells. However, the precise roles of cell-cell adhesion in cell migration have thus far been poorly defined. Often in aggressive cancers, N-cadherin is prominently upregulated, yet, these highly motile cells have limited cell-cell adhesion when plated on a stiff 2D substrate. But, the same cells in a 3D matrix migrate as a multicellular cluster. This new observation suggests that N-cadherin-mediated cell-cell adhesion supports cell interactions between migrating cells in a more physiologically relevant 3D matrix, but not on a 2D substrate. While N-cadherin is an integral part of neural synapses, the ectopic expression of N-cadherin in transformed epithelial cells plays an equally important part in initiating pro-migratory signaling, and providing strong yet flexible cell cohesion essential for persistent cell migration in a 3D matrix. The 3D cell migration analysis for studying cell-to-cell interactions exposes the roles of N-cadherin in multicellular migration, and reveals novel insights into cell migration-dependent normal and pathological processes.     

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.     

N-cadherin as a key regulator of collective cell migration in a 3D environment

Cell migration is a critical step of normal developmental processes and disease progression. Often, migrating cells interact and maintain contact with neighboring cells. However, the precise roles of cell-cell adhesion in cell migration have thus far been poorly defined. Often in aggressive cancers, N-cadherin is prominently upregulated, yet, these highly motile cells have limited cell-cell adhesion when plated on a stiff 2D substrate. But, the same cells in a 3D matrix migrate as a multicellular cluster. This new observation suggests that N-cadherin-mediated cell-cell adhesion supports cell interactions between migrating cells in a more physiologically relevant 3D matrix, but not on a 2D substrate. While N-cadherin is an integral part of neural synapses, the ectopic expression of N-cadherin in transformed epithelial cells plays an equally important part in initiating pro-migratory signaling, and providing strong yet flexible cell cohesion essential for persistent cell migration in a 3D matrix. The 3D cell migration analysis for studying cell-to-cell interactions exposes the roles of N-cadherin in multicellular migration, and reveals novel insights into cell migration-dependent normal and pathological processes.     

Role of E-cadherin N-glycosylation profile in a mammary tumor model

Modifications in cell surface glycosylation affecting cell adhesion are common characteristics of transformed cells. This study characterizes the N-glycosylation profile of E-cadherin in models of canine mammary gland adenoma and carcinoma evaluating the importance of these glycosylation modifications in the malignant phenotype.Our results show that the pattern of E-cadherin N-glycosylation in mammary carcinoma is characterized by highly branched N-glycans, increase in sialylation and an expression of few high mannose structures. Detailed mass spectrometry analysis demonstrated a new N-glycosylation site containing a potential complex type N-glycan in E-cadherin from a mammary carcinoma cell line.Our study demonstrates the importance of E-cadherin N-glycans in the process of tumor development and in the transformation to the malignant phenotype.     

Characterizing the Link between Glycosylation State and Enzymatic Activity of the Endo-β1,4-glucanase KORRIGAN1 from Arabidopsis thaliana

Defects in N-glycosylation and N-glycan processing frequently cause alterations in plant cell wall architecture, including changes in the structure of cellulose, which is the most abundant plant polysaccharide. KORRIGAN1 (KOR1) is a glycoprotein enzyme with an essential function during cellulose biosynthesis in Arabidopsis thaliana. KOR1 is a membrane-anchored endo-β1,4-glucanase and contains eight potential N-glycosylation sites in its extracellular domain. Here, we expressed A. thaliana KOR1 as a soluble, enzymatically active protein in insect cells and analyzed its N-glycosylation state. Structural analysis revealed that all eight potential N-glycosylation sites are utilized. Individual elimination of evolutionarily conserved N-glycosylation sites did not abolish proper KOR1 folding, but mutations of Asn-216, Asn-324, Asn-345, and Asn-567 resulted in considerably lower enzymatic activity. In contrast, production of wild-type KOR1 in the presence of the class I α-mannosidase inhibitor kifunensine, which abolished the conversion of KOR1 N-glycans into complex structures, did not affect the activity of the enzyme. To address N-glycosylation site occupancy and N-glycan composition of KOR1 under more natural conditions, we expressed a chimeric KOR1-Fc-GFP fusion protein in leaves of Nicotiana benthamiana. Although Asn-108 and Asn-133 carried oligomannosidic N-linked oligosaccharides, the six other glycosylation sites were modified with complex N-glycans. Interestingly, the partially functional KOR1 G429R mutant encoded by the A. thaliana rsw2-1 allele displayed only oligomannosidic structures when expressed in N. benthamiana, indicating its retention in the endoplasmic reticulum. In summary, our data indicate that utilization of several N-glycosylation sites is important for KOR1 activity, whereas the structure of the attached N-glycans is not critical.     

N-Glycosylation and N-Glycan Moieties of CTB Expressed in Rice Seeds

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.     

Galectin-3 Protein Regulates Mobility of N-cadherin and GM1 Ganglioside at Cell-Cell Junctions of Mammary Carcinoma Cells

Galectin-3 binding to cell surface glycoproteins, including branched N-glycans generated by N-acetylglucosaminyltransferase V (Mgat5) activity, forms a multivalent, heterogeneous, and dynamic lattice. This lattice has been shown to regulate integrin and receptor tyrosine kinase signaling promoting tumor cell migration. N-cadherin is a homotypic cell-cell adhesion receptor commonly overexpressed in tumor cells that contributes to cell motility. Here we show that galectin-3 and N-cadherin interact and colocalize with the lipid raft marker GM1 ganglioside in cell-cell junctions of mammary epithelial cancer cells. Disruption of the lattice by deletion of Mgat5, siRNA depletion of galectin-3, or competitive inhibition with lactose stabilizes cell-cell junctions. It also reduces, in a p120-catenin-dependent manner, the dynamic pool of junctional N-cadherin. Proteomic analysis of detergent-resistant membranes (DRMs) revealed that the galectin lattice opposes entry of many proteins into DRM rafts. N-cadherin and catenins are present in DRMs; however, their DRM distribution is not significantly affected by lattice disruption. Galectin lattice integrity increases the mobile fraction of the raft marker, GM1 ganglioside binding cholera toxin B subunit Ctb, at cell-cell contacts in a p120-catenin-independent manner, but does not affect the mobility of either Ctb-labeled GM1 or GFP-coupled N-cadherin in nonjunctional regions. Our results suggest that the galectin lattice independently enhances lateral molecular diffusion by direct interaction with specific glycoconjugates within the adherens junction. By promoting exchange between raft and non-raft microdomains as well as molecular dynamics within junction-specific raft microdomains, the lattice may enhance turnover of N-cadherin and other glycoconjugates that determine junctional stability and rates of cell migration.     

Developmental Expression of the Neuron-specific N-Acetylglucosaminyltransferase Vb (GnT-Vb/IX) and Identification of Its in Vivo Glycan Products in Comparison with Those of Its Paralog, GnT-V

The severe phenotypic effects of altered glycosylation in the congenital muscular dystrophies, including Walker-Warburg syndrome, muscle-eye-brain disease, Fukuyama congenital muscular dystrophy, and congenital muscular dystrophy 1D, are caused by mutations resulting in altered glycans linked to proteins through O-linked mannose. A glycosyltransferase that branches O-Man, N-acetylglucosaminyltransferase Vb (GnT-Vb), is highly expressed in neural tissues. To understand the expression and function of GnT-Vb, we studied its expression during neuromorphogenesis and generated GnT-Vb null mice. A paralog of GnT-Vb, N-acetylglucosaminyltransferase (GnT-V), is expressed in many tissues and brain, synthesizing N-linked, β1,6-branched glycans, but its ability to synthesize O-mannosyl-branched glycans is unknown; conversely, although GnT-Vb can synthesize N-linked glycans in vitro, its contribution to their synthesis in vivo is unknown. Our results showed that deleting both GnT-V and GnT-Vb results in the total loss of both N-linked and O-Man-linked β1,6-branched glycans. GnT-V null brains lacked N-linked, β1,6-glycans but had normal levels of O-Man β1,6-branched structures, showing that GnT-Vb could not compensate for the loss of GnT-V. By contrast, GnT-Vb null brains contained normal levels of N-linked β1,6-glycans but low levels of some O-Man β1,6-branched glycans. Therefore, GnT-V could partially compensate for GnT-Vb activity in vivo. We found no apparent change in α-dystroglycan binding of glycan-specific antibody IIH6C4 or binding to laminin in GnT-Vb null mice. These results demonstrate that GnT-V is involved in synthesizing branched O-mannosyl glycans in brain, but the function of these branched O-mannosyl structures is unresolved using mice that lack these glycosyltransferases.