Epithelial-to-Mesenchymal Transition of RPE Cells In Vitro Confers Increased β1,6-N-Glycosylation and Increased Susceptibility to Galectin-3 Binding.

Claudia S Priglinger,Christoph M Szober,Uli Ohmayer,Thomas C Kreutzer,Arie Geerlof,Fabian Gruhn,Jara Obermann,Juliane Merl-Pham,Christian Wertheimer,Stefanie M Hauck,Jennifer Behler,Siegfried G Priglinger

doi:10.1371/journal.pone.0146887

Claudia S Priglinger, Christoph M Szober + Show 10 more

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https://doi.org/10.1371/journal.pone.0146887

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Abstract

Epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells is a crucial event in the onset of proliferative vitreoretinopathy (PVR), the most common reason for treatment failure in retinal detachment surgery. We studied alterations in the cell surface glycan expression profile upon EMT of RPE cells and focused on its relevance for the interaction with galectin-3 (Gal-3), a carbohydrate binding protein, which can inhibit attachment and spreading of human RPE cells in a dose- and carbohydrate-dependent manner, and thus bares the potential to counteract PVR-associated cellular events. Lectin blot analysis revealed that EMT of RPE cells in vitro confers a glycomic shift towards an abundance of Thomsen-Friedenreich antigen, poly-N-acetyllactosamine chains, and complex-type branched N-glycans. Using inhibitors of glycosylation we found that both, binding of Gal-3 to the RPE cell surface and Gal-3-mediated inhibition of RPE attachment and spreading, strongly depend on the interaction of Gal-3 with tri- or tetra-antennary complex type N-glycans and sialylation of glycans but not on complex-type O-glycans. Importantly, we found that β1,6 N-acetylglucosaminyltransferase V (Mgat5), the key enzyme catalyzing the synthesis of tetra- or tri-antennary complex type N-glycans, is increased upon EMT of RPE cells. Silencing of Mgat5 by siRNA and CRISPR-Cas9 genome editing resulted in reduced Gal-3 binding. We conclude from these data that binding of recombinant Gal-3 to the RPE cell surface and inhibitory effects on RPE attachment and spreading largely dependent on interaction with Mgat5 modified N-glycans, which are more abundant on dedifferentiated than on the healthy, native RPE cells. Based on these findings we hypothesize that EMT of RPE cells in vitro confers glycomic changes, which account for high affinity binding of recombinant Gal-3, particularly to the cell surface of myofibroblastic RPE. From a future perspective recombinant Gal-3 may disclose a therapeutic option allowing for selectively targeting RPE cells with pathogenic relevance for development of PVR.

Highlights

Proliferative vitreoretinopathy (PVR) is the major cause of treatment failure in retinal detachment surgery even after primarily successful re-attachment of the retina
Phaseolus vulgaris leukoagglutinin (PHA-L), which has a high specificity for complex-type β1,6-branched tri- and tetrantennary N-glycans only weakly bound to extracts form native retinal pigment epithelial (RPE) cells, whereas strong signals were visible in the cell lysates derived from myofibroblastic RPE cells, consistent with an increase in β1,6-N-glycosylation in myofibroblastic, cultured RPE (Fig 1C)
We demonstrate here for the first time that human RPE cells upon epithelial-to-mesenchymal transition (EMT) acquire a unique glycan expression profile that is distinct from healthy, native RPE cells and provide evidence that myofibroblastic RPE cells express an abundance of non-sialylated Thomsen Friedenreich (TF) antigen as well as complex-type β1,6-branched triand tetraantennary N-glycans (β1,6(GlcNAc)-branched complex-type N-glycans), glycan chain extension with poly-N-acetyllactosamine, and terminal N-acetyllactosamines when compared to native RPE cells

Summary

Introduction

Proliferative vitreoretinopathy (PVR) is the major cause of treatment failure in retinal detachment surgery even after primarily successful re-attachment of the retina. Pharmacologic prevention of PVR membrane formation has primarily been based on antiproliferative strategies or the inhibition of growth factors and inflammation [1,2,3,4]. Studies targeting growth factors or inflammatory cytokines showed that PVR is a primarily cell driven disease that cannot be controlled by the inhibition of single growth factors alone. A need remains for nontoxic agents that will block cellular activities such as RPE attachment or spreading in PVR and, even more importantly, selectively target the cells of relevance for the pathogenesis of the disease. In the present study we focus on such a concept

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