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B, WGA immunoprecipitation reactions from GnT-Vb-expressing cell lysates were treated with the indicated enzymes before being analyzed by Western blot using the Cat antibody. The fact that GnT-Vb and PomGnT1 suppression resulted in a similar phenotype suggested that O -mannosyl glycans played a role in adhesion and migration of these cells on laminin.

In this study, we aimed to define potential acceptor substrates for GnT-Vb that could be regulating cell adhesion and migration. The neuroblastoma cell line SH-SY5Y has a neural crest lineage; the HNK-1 epitope is highly expressed in neural crest cells 37 — 39 and plays a role in modulating adhesion and migration to laminin 16 , Our results showed that the Cat antibody exhibited a very low but detectable level of reactivity in mock-transfected cells, and a large increase in binding in the GnT-Vb-expressing cells Fig.

The largest increase for both Cat and CD57 antibody binding occurred in a diffuse band migrating above kDa Fig. To determine whether the Cat reactivity of this large glycoprotein was due primarily to N -linked or O -linked glycans, we performed serial digestions using sialidase and O -glycanase, followed by N -glycanase digestion. Although a size shift of the band was apparent after each digestion, demonstrating that some glycans were removed by the enzymatic treatments, the reactivity with Cat remained. The marked resistance of the epitope to enzymatic deglycosylation using these enzymes suggests that this band represents a glycoprotein s that expresses the O -mannosyl-linked HNK-1 epitope.

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GnT-Vb expression increases cell migration and reduces calcium-dependent cell-cell adhesion. Experiment shown is representative of three separate experiments. The HNK-1 epitope has been reported to play a role in cell migration during the development of the nervous system.

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Therefore, we examined the effect of GnT-Vb expression on cell migration induced in response to a scratch wound. Because suppression of GnT-Vb expression inhibited migration most strongly on laminin 35 , we examined the effect of increased GnT-Vb expression on laminin-coated chamber slides.

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Confluent cells plated on laminin in serum-free medium were scraped with a yellow pipette tip, and cells were allowed to migrate for 4 and 6 h. Inspection of the cells at the 4-h time point revealed a greater number of GnT-Vb-expressing cells had migrated into the scratch wound compared with mock cells Fig. At the 6-h time point the mock cells have begun to migrate and display a ruffled edge.

However, at 6 h the GnT-Vb-expressing cells have spanned the wound, demonstrating a significant effect of GnT-Vb expression on the rate of migration Fig. Next, we explored the possibility that GnT-Vb expression may influence cell-cell adhesion, considering that several proteins reported to express the HNK-1 epitope are cell adhesion molecules. Single-cell suspensions of mock and GnT-Vb cells were made, and aggregation assays were performed as described After 30 min, phase contrast microscopy revealed a significant reduction of cell aggregation in the GnT-Vb-expressing cells compared with mock cells Fig.

These results suggest that GnT-Vb glycosylation regulates homotypic cell-cell adhesion. The large molecular weight of the most intensely staining band Fig. No significant level of secreted Catreactive phosphacan could be detected from either cell type data not shown.

The results shown are representative of three separate experiments. C, densitometry analysis of dimerization experiments. D, galectin-1 cell-surface half-life was measured as described for A , except that anti-galectin-1 antibody was used following streptavidin magnetic bead precipitation. The rationale for these experiments is based on previous studies for receptor protein-tyrosine phosphatases showing that clustering of the extracellular domains and subsequent dimerization can inhibit phosphatase activity 42 , Galectin-1 binding was measured by flow cytometry of intact cells using streptavidin phycoerythrin detection.

Exogenous galectin-1 binding was elevated in GnT-Vb-expressing cells compared with mock cells after incubation in sucrose Fig. This experiment demonstrates that GnT-Vb glycosylation increases galectin-1 cell surface ligands and that 10 m m LacNAc is sufficient to completely inhibit galectin-1 binding. These results agree with those of the cell-surface half-life experiments described above. Bound biotinylated galectin-1 was detected using streptavidin-phycoerythrin. Data presented have been subtracted for secondary-only fluorescence and are representative of two separate experiments.

We have reported that expression levels of N -acetylglucosaminyltransferase Vb modulate integrin-dependent neuroblastoma cell-matrix adhesion and migration on laminin, using siRNA specific for this enzyme These effects were shown to be due to changes in O -mannosyl glycan expression, because the effects were also observed when expression of POMGnT-1 was knocked down using siRNA.

The HNK-1 epitope is a terminal sulfoglucuronyl carbohydrate structure that plays important roles in neural cell adhesion and migration 3 and has been shown to be expressed on O -mannosyl-linked glycans In this study, increased GnT-Vb expression led to increased levels of a major Cat antibody-reactive glycoprotein with a concomitant decrease in cell-cell adhesion and increase in laminin-dependent migration. The Cat antibody binds to a predominant glycoprotein that most likely expresses O -mannosyl-linked glycans because of the resistance of the Cat epitope to N -glycanase and several other glycosidases.

The possibility that the Cat epitope may also react with the HNK-1 epitope expressed on other glycans resistant to enzymatic de-glycosylation cannot be excluded, however. Our results show an increase in the number of cell surface ligands for galectin-1 binding after GnT-Vb expression; galectin-1 is the predominant lectin expressed in SH-SY5Y cells. We do not know of a study that has measured the affinity of galectin-1 for a glycan containing LacNAc or poly-LacNAc that terminates in the HNK-1 epitope, although it is clear that galectin-1 has affinity for some sulfated glycans Several studies using T-cells have shown that galectin-1 binding to, and clustering of, CD45 results in decreased phosphatase activity 50 — Interestingly, immune cells and neural cells are both cell types that communicate via synapse formation.

Both galectin-1 and GnT-Vb are expressed within the adult mouse subventricular zone during neuromorphogenesis where neurons are actively dividing and migrating Our model links these proteins in a mechanism capable of regulating key signaling events that reduce cell-cell adhesion and promote cell migration during brain development. Changes in glycan expression and galectin binding regulate specific glycoprotein cell-surface retention and affect function 42 , 44 , 45 , These effects are suggestive of the clustering reported for galectins in vitro 59 , In one case, the Glut-2 transporter on mouse pancreatic islet cells displayed significantly reduced cell-surface retention, and subsequent poor uptake of glucose in animals that lack a specific glycosyltransferase, GnT-Iva, that is active in N -linked biosynthesis In this case Glut-2 surface transporter retention on the cell surface is down-regulated by elimination or reduction of GnT-IVa expression, which in turns causes reduced binding by galectin-9, the galectin expressed at highest levels in this cell type.

Moreover, in the case of the Glut-2 receptor, increased glycosylation by GnT-IVa results in increased galectin-9 binding, increased cell-surface retention, and high levels of glucose transport. Increased GnT-Va glycosylation of some growth factor receptors has been reported to result in higher levels of galectin-3 binding which, in turn, causes increased cell-surface retention, increased tyrosine phosphorylation, and increased receptor signaling via these phosphorylated residues By contrast, however, inhibiting GnT-Va activity with siRNA expression in a human breast carcinoma cell line stimulated by epidermal growth factor resulted in increased cell-surface retention, lowered ERK phosphorylation, and increased SHP-2 phosphatase activity Increased GnT-Va in all cases, however, does appear to result in decreased cadherin-mediated cell-cell and integrin-mediated cell-matrix adhesion, promoting increased cell migration and an invasive phenotype.

Intriguingly, specific acceptor glycosylation by both paralogs, GnT-Va and GnT-Vb, appear to regulate cell-cell interactions, although by distinct mechanisms.

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We thank Mabel Pang and Linda Baum for providing recombinant galectin We acknowledge Julie Nelson for help with the flow cytometry, Amanda Anderson for technical assistance, and Huabei Guo for help with cell-cell adhesion assays. We also thank Drs. Ron Schnaar and Linda Baum for helpful discussions and advice during the preparation of this manuscript. Matthews, unpublished results. The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this fact. Advertisement Hide.

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