Galectin Binding to Mgat5-Modified N-Glycans Regulates Fibronectin Matrix Remodeling in Tumor Cells

Abstract

Oncogenic signaling stimulates the dynamic remodeling of actin microfilaments and substrate adhesions, essential for cell spreading and motility. Transformation is associated with increased expression of β1,6GlcNAc-branched N-glycans, products of Golgi β1,6-acetylglucosaminyltransferase V (Mgat5) and the favored ligand for galectins. Herein we report that fibronectin fibrillogenesis and fibronectin-dependent cell spreading are deficient in Mgat5^−/− mammary epithelial tumor cells and inhibited in Mgat5^+/+ cells by blocking Golgi N-glycan processing with swainsonine or by competitive inhibition of galectin binding. At an optimum dosage, exogenous galectin-3 added to Mgat5^+/+ cells activates focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), recruits conformationally active α5β1-integrin to fibrillar adhesions, and increases F-actin turnover. RGD peptide inhibits PI3K-dependent fibronectin matrix remodeling and fibronectin-dependent cell motility, while galectin-3 stimulates and overrides the inhibitory effects of RGD. Antibodies to the galectin-3 N-terminal oligomerization domain stimulate α5β1 activation and recruitment to fibrillar adhesions in Mgat5^+/+ cells, an effect that is blocked by disrupting galectin-glycan binding. Our results demonstrate that fibronectin polymerization and tumor cell motility are regulated by galectin-3 binding to branched N-glycan ligands that stimulate focal adhesion remodeling, FAK and PI3K activation, local F-actin instability, and α5β1 translocation to fibrillar adhesions.

We are particularly grateful to Martin Humphries for his kind gift of the SNAKA51 monoclonal antibody.

This study was supported by a grant from the Canadian Institutes of Health Research (CIHR) to I.R.N. and J.W.D. and NIH grant CA-46120 to A.R. I.R.N. is an Investigator of the CIHR, and J.W.D. holds a Canada Research Chair. A.L. is the recipient of an FCAR en santé studentship, and J.G.G. holds a doctoral fellowship from the Ministère de la Recherche et des Technologies for his doctoral studies to be submitted jointly to the Université de Montréal and the Université Louis Pasteur de Strasbourg (UMR CNRS 7034).