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.