title: Epithelial-to-Mesenchymal Transition of RPE Cells In Vitro Confers Increased β1,6-N-Glycosylation and Increased Susceptibility to Galectin-3 Binding.
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.
authors.affiliation: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Claudia S
authors.initials: CS
authors.lastname: Priglinger
authors.affiliation: Research Unit Protein Science, Helmholtz Zentrum Munich, German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Jara
authors.initials: J
authors.lastname: Obermann
authors.affiliation: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Christoph M
authors.initials: CM
authors.lastname: Szober
authors.affiliation: Research Unit Protein Science, Helmholtz Zentrum Munich, German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Merl-Pham
authors.affiliation: Research Unit Protein Science, Helmholtz Zentrum Munich, German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Uli
authors.initials: U
authors.lastname: Ohmayer
authors.affiliation: Research Unit Protein Science, Helmholtz Zentrum Munich, German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Behler
authors.affiliation: Research Unit Protein Science, Helmholtz Zentrum Munich, German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Gruhn
authors.affiliation: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Kreutzer
authors.affiliation: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Wertheimer
authors.affiliation: Protein Expression and Purification Facility, Institute of Structural Biology, Helmholtz Zentrum Munich, German Research Center for Environmental Health (GmbH), Neuherberg, Germany.
authors.forename: Arie
authors.initials: A
authors.lastname: Geerlof
authors.affiliation: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Siegfried G
authors.initials: SG
authors.lastname: Priglinger
authors.affiliation: Research Unit Protein Science, Helmholtz Zentrum Munich, German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Stefanie M
authors.initials: SM
authors.lastname: Hauck
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D058750:Epithelial-Mesenchymal Transition; D037502:Galectin 3 / Q000378:metabolism*; D054794:Glycomics; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008875:Middle Aged; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D013552:Swine; D018630:Vitreoretinopathy, Proliferative / Q000378:metabolism; D055815:Young Adult
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D037502:Galectin 3; D011134:Polysaccharides; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C016467:Thomsen-Friedenreich antigen; C000601122:Mgat5 protein, human; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase