Integrin-dependent neuroblastoma cell adhesion and migration on laminin is regulated by expression levels of two enzymes in the O-mannosyl-linked glycosylation pathway, PomGnT1 and GnT-Vb

doi:10.1016/j.yexcr.2006.05.022

Experimental Cell Research

Volume 312, Issue 15, 10 September 2006, Pages 2837-2850

https://doi.org/10.1016/j.yexcr.2006.05.022 Get rights and content

Abstract

O-mannosyl-linked glycans constitute a third of all brain O-linked glycoproteins, and yet very little is understood about their functions. Several congenital muscular dystrophies with central nervous system defects are caused by genetic disruptions in glycosyltransferases responsible for the synthesis of O-mannosyl glycans. The glycosyltransferase GnT-Vb, also known as GnT-IX, is expressed abundantly in the brain and testis and is proposed to be the enzyme that branches O-mannosyl-linked glycans. In this study, we show in a human neuronal model that GnT-Vb expression enhances neurite outgrowth on laminin. GnT-Vb has been shown to perform both N-linked and O-mannosyl-linked glycosylation. To determine if the effect on neurite outgrowth was due to N-linked or O-mannosyl-linked glycosylation by GnT-Vb we suppressed the expression of glycosyltransferases important for the elongation of both N-linked and O-mannosyl-linked glycans using RNA interference. Our results suggest that GnT-Vb and PomGnT1, enzymes involved in the O-mannosyl glycosylation pathway, play an active role in modulating integrin and laminin-dependent adhesion and migration of human neuronal cells.

Introduction

The genomic analysis of various neuronal disorders over the past few years has shed light on the critical functions of protein glycosylation during the development of the nervous system. Human congenital disorders such as Walker Warburg syndrome (WWS), muscle–eye–brain disease (MEB), and Fukuyama congenital muscular dystrophy (FCMD) are caused by inactivation of glycosyltransferases that function in the O-mannosyl glycan processing pathway [1], [2], [3], [4]. These disorders are characterized by muscular dystrophy with neuronal abnormalities. Particularly interesting are the defects in neuronal migration and nerve–muscle adhesion noted in muscle–eye–brain disease that are caused by inactivating mutations in the peptide-O-mannose β-1,2-acetylglucosminyltransferase known as PomGnT1 [3], the glycosyltransferase that transfers N-acetylglucosamine (GlcNAc) in beta(1,2) linkage onto Ser/Thr-O-mannosyl residues, allowing extension of O-mannosyl glycans. O-linked mannose structures comprise approximately 25% of the total O-linked glycans in brain [5]. These structures have been characterized from rabbit and sheep brain and partial structural information has been obtained for the J1 glycoprotein from the mouse nervous system [6], [7], [8], as well as alpha-dystroglycan in smooth muscle [9].

The glycosyltransferase termed GnT-Vb, also known as GnT-IX, is a recently discovered enzyme that is selectively expressed in neuronal cells and participates in the synthesis of O-mannosyl glycans [10], [11], [12], [13]. This enzyme catalyzes the transfer of GlcNAc in β(1,6) linkage to the peptide-O-linked-Man, but only if the β1,2-linked GlcNAc formed by PomGnT1 is present (Fig. 2A). Studies in vitro reveal that GnT-Vb prefers O-mannosyl acceptors, but can also transfer to the Man (β1,2)GlcNAc found in non-galactosylated N-linked glycans during biosynthesis [10]. However, its homolog, GnT-Va, is only capable of transferring GlcNAc in β(1,6) linkage to N-linked glycans. GnT-Vb activity is expected to be required for the expression of the majority of both O-linked polylactosamine and HNK-glycans in brain [10], both of which function in neuronal cell adhesion. Since GnT-Vb shows selective expression during neuromorphogenesis in the mouse (Matthews, unpublished data), it may function in neuronal cell adhesion and migration.

Neuroblastoma cells of the N-type (neuronal), such as SH-SY5Y, are typically loosely adherent to substrates, express neuronal cell surface markers, can be induced to differentiate, and are inherently tumorigenic in nude mice [14], [15], [16], [17]. In the present study, SH-SY5Y cells were used to study the potential role of GnT-Vb glycosylation in the regulation of cell adhesion, differentiation, and migration. We find that increased GnT-Vb expression leads to enhanced neurite outgrowth in SH-SY5Y cells plated on laminin. RNA interference studies show that reducing the expression levels of two glycosyltransferases acting in the O-mannosyl glycan pathway, GnT-Vb and POMGnT-I, using small interfering RNAs [18], leads to increased adhesion to laminin, as well as reduced migration, and impaired neurite outgrowth on this extracellular matrix glycoprotein. The phenotype observed in GnT-Vb suppressed cells could be rescued by expression of a form of GnT-Vb not targeted for RNA interference. We also show that suppression of GnT-Vb expression leads to significant changes in β1 integrin transcript and protein levels. Overall, these data indicate that GnT-Vb and PomGnT1 expression levels play an active role in modulating integrin and laminin-dependent adhesion and migration of human neuronal cells.

Section snippets

Antibodies and chemicals

ERK1/2 monoclonal antibody and all HRP-labeled anti-mouse and anti-rabbit IgG were obtained from Santa Cruz Biotechnology. Polyclonal antibody against β1 integrin was obtained from Chemicon. The HA epitope antibody was obtained from Roche. Laminin from human placenta was obtained from Sigma. All trans retinoic acid was obtained from Sigma. NHS-LC–biotin was a product of Pierce, and streptavidin–HRP was obtained from Vector Laboratory.

Plasmids

Targets for RNA interference were selected using the

GnT-Vb expression levels regulate neurite outgrowth of SH-SY5Y neuroblastoma cells

The SH-SY5Y cell line has been extensively used to study neuronal differentiation [20], [21], [22]. Notably, SH-SY5Y cells plated on laminin can be induced to differentiate and extend neurites by adding micromolar amounts of all trans retinoic acid (ATRA). To test if GnT-Vb-mediated glycosylation functions in human neuroblastoma cell differentiation in vitro, GnT-Vb was cloned into the expression vector pCDNA 3.1-HA and constitutively expressed in SH-SY5Y cells. There were no noticeable

Discussion

In this study, we demonstrate that the expression levels of GnT-Vb regulate neurite outgrowth, cell migration, and cell adhesion in the neuronal model SH-SY5Y neuroblastoma cells. The novel findings in this study include: (1) GnT-Vb expression increases retinoic acid-induced neurite outgrowth on laminin, while targeted RNA interference of GnT-Vb using 2 different targeting sequences reduces neurite outgrowth; (2) suppression of both GnT-Vb and PomGnT1, but not GnT-Va increases β1

Acknowledgments

We thank Dr. Steven K. Akiyama of NIEHS, National Institutes of Health, Research Triangle Park, NC, for the kind gift of function-blocking antibody against β1 integrin (mAb13); Dr. Huabei Guo for the technical help with the cell adhesion and migration assays; and Drs. Michael Tiemeyer and Rick Matthews for the excellent advice and editorial review.

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Branched O-mannosyl glycans in the corpus callosum in the brain are involved in remyelination inhibition in the demyelination model, suggesting that the modulation of O-mannosyl glycan production is a potential candidate for therapeutic strategies against demyelinating diseases. It has been reported that the expression levels of GnT-IX modulate the integrin-dependent cell adhesion of neuroblastoma cells, and migration on laminin and integrin (Abbott, Troupe, Lee, & Pierce, 2006). The knockdown of GnT-IX in the study was likely related to O-mannosyl glycans because similar effects were also observed for the knockdown for POMGnT1.
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The timing and localization of GnT-Vb raise the possibility that it could also contribute to these disorders. Consistent with this hypothesis, we demonstrated previously that overexpression of GnT-Vb in a neuroblastoma cell line decreased adhesion to and increased motility on laminin (51). However, this study found no obvious changes in the migration or positioning of neurons in either GnT-Vb null or GnT-V/GnT-Vb double null brains.
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Research ArticleIntegrin-dependent neuroblastoma cell adhesion and migration on laminin is regulated by expression levels of two enzymes in the O-mannosyl-linked glycosylation pathway, PomGnT1 and GnT-Vb

Abstract

Introduction

Section snippets

Antibodies and chemicals

Plasmids

GnT-Vb expression levels regulate neurite outgrowth of SH-SY5Y neuroblastoma cells

Discussion

Acknowledgments

Dev. Cell

Am. J. Hum. Genet.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Carbohydr. Res.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Mech Dev.

Int. J. Biochem. Cell Biol.

Exp. Neurol.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Exp. Cell Res.

Cell

Mol. Cell. Neurosci.

Research Article
Integrin-dependent neuroblastoma cell adhesion and migration on laminin is regulated by expression levels of two enzymes in the O-mannosyl-linked glycosylation pathway, PomGnT1 and GnT-Vb