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

Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30605, USA.
Experimental Cell Research (Impact Factor: 3.37). 10/2006; 312(15):2837-50. DOI: 10.1016/j.yexcr.2006.05.022
Source: PubMed

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.

0 Followers
 · 
340 Views
  • Source
    • "Pomgnt1. The enzyme O-mannose beta-1, 2-N-acetylgluco- saminyltransferase participates in O-mannosyl glycan synthesis and has been shown to function in diverse roles including reactive gliosis in the retina, neuroblastoma adhesion (Abbott et al. 2006), muscle-eye-brain disease (Manya et al. 2003, 2004) and the proliferation of myoblasts (Miyagoe-Suzuki "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Consortium for Functional Glycomics (CFG) was a large research initiative providing networking and resources for investigators studying the role of glycans and glycan-binding proteins in health and disease. Starting in 2001, six scientific cores were established to generate data, materials, and new technologies. By the end of funding in 2011, the Mouse Phenotype Core (MPC) submitted data to a website from the phenotype screen of thirty-six mutant mouse strains deficient in a gene for either a glycan-binding protein (GBP) or glycosyltransferase (GT). Each mutant strain was allotted three months for analysis and screened by standard phenotype assays used in the fields of immunology, histology, hematology, coagulation, serum chemistry, metabolism, and behavior. Twenty of the deficient mouse strains had been studied in other laboratories, and additional tests were performed on these strains to confirm previous observations and discover new data. The CFG constructed sixteen new homozygous mutant mouse strains and completed the initial phenotype screen of the majority of these new mutant strains. In total more than three hundred phenotype changes were observed, but considering the over one hundred assays performed on each strain, most of the phenotypes were unchanged. Phenotype differences include abnormal testis morphology in GlcNAcT9 and Siglec-H deficient mice, and lethality in Pomgnt1 deficient mice. The numerous altered phenotypes discovered, along with the consideration of the significant findings of normality, will provide a platform for future characterization to understand of the important role of glycans and GBPs in the mechanisms of health and disease.
    Glycobiology 10/2012; 23(3). DOI:10.1093/glycob/cws150 · 3.14 Impact Factor
  • Source
    • "For example , knockout of GnT-Va in mouse embryo fibroblasts caused the altered gene expression of other glycosyltransferases and membrane proteins such as β1 integrins (Guo et al. 2005) and galectins (Guo et al. 2008). Furthermore, the increased gene expression of integrins was also observed in SH-SY5Y cells after GnT-Vb knockdown by siRNA (Abbott et al. 2006). A study using GnT-Va null polyoma middle T-induced tumor cells (Lajoie et al. 2007) showed a reduction in the expression of caveolin-1, a coat protein of caveolae mediating endocytosis of some types of receptors. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Changes in the expression of N-glycan branching glycosyltransferases can alter cell surface receptor functions, involving their levels of cell surface retention, rates of internalization into the endosomal compartment, and subsequent intracellular signaling. To study in detail the regulation of signaling of the EGF receptor (EGFR) by GlcNAcbeta(1,6)Man branching, we utilized specific siRNA to selectively knockdown GnT-Va expression in the highly invasive human breast carcinoma line MDA-MB231, which resulted in the attenuation of its invasiveness-related phenotypes. Compared to control cells, ligand-induced downregulation of EGFR was significantly inhibited in GnT-Va-suppressed cells. This effect could be reversed by re-expression of GnT-Va, indicating that changes in ligand-induced receptor downregulation were dependent on GnT-Va activity. Knockdown of GnT-Va had no significant effect on c-Cbl mediated receptor ubiquitination and degradation, but did cause the inhibition of receptor internalization, showing that altered signaling and delayed ligand-induced downregulation of EGFR expression resulted from decreased EGFR endocytosis. Similar results were obtained with HT1080 fibrosarcoma cells treated with GnT-Va siRNA. Inhibited receptor internalization caused by the expression of GnT-Va siRNA appeared to be independent of galectin binding since decreased EGFR internalization in the knockdown cells was not affected by the treatment of the cells with lactose, a galectin inhibitor. Our results show that decreased GnT-Va activity due to siRNA expression in human carcinoma cells inhibits ligand-induced EGFR internalization, consequently resulting in delayed downstream signal transduction and inhibition of the EGF-induced, invasiveness-related phenotypes.
    Glycobiology 03/2009; 19(5):547-59. DOI:10.1093/glycob/cwp023 · 3.14 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Journal of Biological Chemistry 08/2007; 282(30):22150-62. DOI:10.1074/jbc.M611518200 · 4.57 Impact Factor
Show more

Preview

Download
1 Download
Available from