Title: Proteome-wide Identification of Glycosylation-dependent Interactors of Galectin-1 and Galectin-3 on Mesenchymal Retinal Pigment Epithelial (RPE) Cells
Abstract: Identification of interactors is a major goal in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here, we aim to identify novel interacting binding partners for the β-galactoside-binding proteins galectin-1 (Gal-1) and galectin-3 (Gal-3) relevant in the context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion, and epithelial-to-mesenchymal transition of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes. Gal-1 and Gal-3 bind in a dose- and carbohydrate-dependent manner to mesenchymal RPE cells and inhibit cellular processes like attachment and spreading. Yet knowledge about glycan-dependent interactors of Gal-1 and Gal-3 on RPE cells is very limited, although this is a prerequisite for unraveling the influence of galectins on distinct cellular processes in RPE cells. We identify here 131 Gal-3 and 15 Gal-1 interactors by galectin pulldown experiments combined with quantitative proteomics. They mainly play a role in multiple binding processes and are mostly membrane proteins. We focused on two novel identified interactors of Gal-1 and Gal-3 in the context of PVR: the low-density lipoprotein receptor LRP1 and the platelet-derived growth factor receptor β PDGFRB. Addition of exogenous Gal-1 and Gal-3 induced cross-linking with LRP1/PDGFRB and integrin-β1 (ITGB1) on the cell surface of human RPE cells and induced ERK/MAPK and Akt signaling. Treatment with kifunensine, an inhibitor of complex-type N-glycosylation, weakened the binding of Gal-1 and Gal-3 to these interactors and prevented lattice formation. In conclusion, the identified specific glycoprotein ligands shed light into the highly specific binding of galectins to dedifferentiated RPE cells and the resulting prevention of PVR-associated cellular events. Identification of interactors is a major goal in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here, we aim to identify novel interacting binding partners for the β-galactoside-binding proteins galectin-1 (Gal-1) and galectin-3 (Gal-3) relevant in the context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion, and epithelial-to-mesenchymal transition of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes. Gal-1 and Gal-3 bind in a dose- and carbohydrate-dependent manner to mesenchymal RPE cells and inhibit cellular processes like attachment and spreading. Yet knowledge about glycan-dependent interactors of Gal-1 and Gal-3 on RPE cells is very limited, although this is a prerequisite for unraveling the influence of galectins on distinct cellular processes in RPE cells. We identify here 131 Gal-3 and 15 Gal-1 interactors by galectin pulldown experiments combined with quantitative proteomics. They mainly play a role in multiple binding processes and are mostly membrane proteins. We focused on two novel identified interactors of Gal-1 and Gal-3 in the context of PVR: the low-density lipoprotein receptor LRP1 and the platelet-derived growth factor receptor β PDGFRB. Addition of exogenous Gal-1 and Gal-3 induced cross-linking with LRP1/PDGFRB and integrin-β1 (ITGB1) on the cell surface of human RPE cells and induced ERK/MAPK and Akt signaling. Treatment with kifunensine, an inhibitor of complex-type N-glycosylation, weakened the binding of Gal-1 and Gal-3 to these interactors and prevented lattice formation. In conclusion, the identified specific glycoprotein ligands shed light into the highly specific binding of galectins to dedifferentiated RPE cells and the resulting prevention of PVR-associated cellular events. Galectins are widely expressed across different species and organs and share homology in the amino acid sequence of their carbohydrate recognition domain (CRD)1 (1.Barondes S.H. Castronovo V. Cooper D.N. Cummings R.D. Drickamer K. Feizi T. Gitt M.A. Hirabayashi J. Hughes C. Kasai K. galectins: a family of animal β-galactoside-binding lectins.Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1011) Google Scholar, 2.Cooper D.N. Barondes S.H. God must love galectins; he made so many of them.Glycobiology. 1999; 9: 979-984Crossref PubMed Scopus (273) Google Scholar, 3.Hirabayashi J. Kasai K. 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Galectin-3 (Gal-3) is the only known chimera-type galectin of the human lectin family (1.Barondes S.H. Castronovo V. Cooper D.N. Cummings R.D. Drickamer K. Feizi T. Gitt M.A. Hirabayashi J. Hughes C. Kasai K. galectins: a family of animal β-galactoside-binding lectins.Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1011) Google Scholar, 9.Gabius H.J. Animal lectins.Eur. J. Biochem. 1997; 243: 543-576Crossref PubMed Google Scholar, 10.Leffler H. Carlsson S. Hedlund M. Qian Y. Poirier F. Introduction to galectins.Glycoconj. J. 2002; 19: 433-440Crossref PubMed Scopus (472) Google Scholar). It consists of a C-terminal domain to bind specific carbohydrate branches and an N-terminal domain, which enables Gal-3 to multimerize (1.Barondes S.H. Castronovo V. Cooper D.N. Cummings R.D. Drickamer K. Feizi T. Gitt M.A. Hirabayashi J. Hughes C. 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Platelet-derived growth factor receptor kinase inhibitor AG1295 and inhibition of experimental proliferative vitreoretinopathy.Jpn. J. Ophthalmol. 2003; 47: 158-165Crossref PubMed Scopus (0) Google Scholar). However, PVR is a multifactorial, mostly cell-driven process, which requires a multimodal concept (24.Pastor J.C. de la Rúa E.R. Martín F. Proliferative vitreoretinopathy: risk factors and pathobiology.Prog. Retin. Eye Res. 2002; 21: 127-144Crossref PubMed Scopus (272) Google Scholar). Identification of a pharmacological agent that is able to govern several cellular processes simultaneously is necessary to treat PVR. Therefore, Gal-1 and Gal-3 bear a high potential to counteract PVR-associated cellular events. However, although the cell-surface proteins on RPE cells targeted by specific galectins are largely unknown, this in-depth knowledge is a prerequisite to unravel the possible influence of galectins on the signal transduction mechanisms associated with PVR processes. In many other cell types, several interactors for Gal-1 or Gal-3 have been identified; these include, among others, lysosomal membrane-associated glycoproteins (LAMPs)-1 and -2, neural cell adhesion molecule, cell adhesion molecule L1, CD43, CD45, CD71, mucin-1, and receptors for distinct growth factors like the epidermal growth factor (EGF), transforming growth factor β (TGF-β), or vascular endothelial growth factor (VEGF) (5.Römer C.E. Elling L. Galectins: Structures, Binding Properties and Function in Cell Adhesion.INTECH Open Access Publisher. 2011; : 1-28Google Scholar, 40.Stillman B.N. Hsu D.K. Pang M. Brewer C.F. Johnson P. Liu F.-T. Baum L.G. Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death.J. Immunol. 2006; 176: 778-789Crossref PubMed Google Scholar, 41.Yu L.-G. Andrews N. Zhao Q. McKean D. Williams J.F. Connor L.J. Gerasimenko O.V. Helens J. Hirabayashi J. Kasai K. Rhodes J.M. 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The purpose of this study was to identify novel and specific glycoprotein ligands for Gal-1 and Gal-3 on the surface of mesenchymal RPE cells by an affinity capture quantitative LC-MS/MS-based approach. From the 131 and 15 specific interactors identified for Gal-3 and Gal-1, respectively, we focused on two novel interactors for functional validation of the PVR-relevant cellular behavior: the low density lipoprotein receptor-related protein (LRP1) and the platelet-derived growth factor receptor β (PDGFRB). Addition of Gal-1 and Gal-3 induced clustering with the identified glycoprotein receptors LRP1 and PDGFRB together with ITGB1 on RPE cell surfaces, validating their potential to influence cellular effects. Relevance of glycosylation of these interactors for the functional galectin binding and the cross-linking activity was also analyzed. Human donor cadaver eyes were received by the Eye Bank of the Department of Ophthalmology at the Linz General Hospital (Linz, Austria) or at the Ludwig-Maximilians-University (LMU) (Munich, Germany) and were processed within 24 h after death as described in Priglinger et al. (42.Priglinger C.S. Szober C.M. Priglinger S.G. Merl J. Euler K.N. Kernt M. Gondi G. Behler J. Geerlof A. Kampik A. Ueffing M. Hauck S.M. Galectin-3 induces clustering of CD147 and integrin-β1 transmembrane glycoprotein receptors on the RPE cell surface.PLoS One. 2013; 8: e70011Crossref PubMed Scopus (30) Google Scholar) and Priglinger et al. (31.Priglinger C.S. Obermann J. Szober C.M. Merl-Pham J. Ohmayer U. Behler J. Gruhn F. Kreutzer T.C. Wertheimer C. Geerlof A. Priglinger S.G. Hauck S.M. Epithelial-to-mesenchymal transition of RPE cells in vitro confers increased β-1,6-N-glycosylation and increased susceptibility to galectin-3 binding.PLoS One. 2016; 11: e0146887Crossref PubMed Scopus (14) Google Scholar). The securing processes of the human tissue were humane, complied with the Declaration of Helsinki, and were approved by the relatives. The ethics committees of the hospital of the LMU, Munich, and of the Land Oberoesterreich authorized the procedure of isolation of RPE cells from human cadaver eyes, which were enucleated by an ophthalmologist in accordance with the standard operating procedures of the institution. After removal of the cornea for cornea transplantation, the front segment of the eye and the vitreous body were removed. The inner part of the rest of the eye was filled with phosphate-buffered saline (PBS, Gibco), and the retina was aspirated. To get rid of the remaining retina and photoreceptors, the eye was refilled with pre-warmed 1 mm EDTA in PBS (37 °C), pH 7.4, and incubated for 15–20 min at room temperature. PBS, 1 mm EDTA was aspirated, and the eyecup was filled with dissociation buffer (3 mm l-cysteine, 1 μg/μl BSA in PBS, 1 mm EDTA) containing 45 μg of papain (Worthington) per 1 ml of dissociation buffer. After incubation for 23 min at 37 °C, the solution within the eye was gently agitated with a pipette to dispense as many RPE cells as possible. The loosened RPE cells were transferred in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal calf serum (FCS; Gibco) and centrifuged for 5 min at 930 rpm at room temperature. The resuspended RPE cells were cultivated in DMEM, 10% FCS at 37 °C and 5% CO2 with or without 10 μm kifunensine for up to 4 weeks (Sigma-Aldrich). Primary human RPE cells of passage 4–7 were used for experiments. For some experiments, the human ARPE-19 cell line (ATCC® CRL-2302TM) was used (55.Kuznetsova A.V. Kurinov A.M. Aleksandrova M.A. Cell models to study regulation of cell transformation in pathologies of retinal pigment epithelium.J. Ophthalmol. 2014; 2014: 801787Crossref PubMed Scopus (45) Google Scholar). ARPE-19 cells were cultivated under the same cell culture conditions like the primary RPE cells. For preparation of protein lysates, RPE cells were washed with ice-cold PBS, collected, and lysed in RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1%(w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, Complete 1×). Human Gal-1 and Gal-3 were cloned in the bacterial pETM-11 expression vector as described previously (31.Priglinger C.S. Obermann J. Szober C.M. Merl-Pham J. Ohmayer U. Behler J. Gruhn F. Kreutzer T.C. Wertheimer C. Geerlof A. Priglinger S.G. Hauck S.M. Epithelial-to-mesenchymal transition of RPE cells in vitro confers increased β-1,6-N-glycosylation and increased susceptibility to galectin-3 binding.PLoS One. 2016; 11: e0146887Crossref PubMed Scopus (14) Google Scholar, 42.Priglinger C.S. Szober C.M. Priglinger S.G. Merl J. Euler K.N. Kernt M. Gondi G. Behler J. Geerlof A. Kampik A. Ueffing M. Hauck S.M. Galectin-3 induces clustering of CD147 and integrin-β1 transmembrane glycoprotein receptors on the RPE cell surface.PLoS One. 2013; 8: e70011Crossref PubMed Scopus (30) Google Scholar). The single modification was that His6-tagged human galectin-1 was dialyzed against PBS containing 5 mm β-mercaptoethanol at the end of the purification process. For biotinylation, 150 mm β-lactose was added to 1 mg of purified Gal-1 and Gal-3, and the proteins were dialyzed for 2 h at 4 °C against 0.1 m sodium hydrogen carbonate with 50 mm β-lactose, pH 9.2, followed by a 1-h biotinylation at room temperature with 100 μg of biotinamidohexanoic acid N-hydroxysuccinimide ester according to the manufacturer's instructions (Sigma-Aldrich). The biotinylated galectins were dialyzed overnight at 4 °C against PBS. Activity of biotinylated galectins was determined semi-quantitatively by hemagglutination assays, adapted from Nowak et al. (56.Nowak T.P. Haywood P.L. Barondes S.H. Developmentally regulated