Functional impact of galectins and aspects of N-glycosylation on epithelial-to-mesenchymal transition of retinal pigment epithelial cells and proliferative vitreoretinopathy formation

视网膜色素上皮细胞（RPE）的上皮向间质转化是增殖性玻璃体视网膜病变（PVR）发病的关键细胞事件，也是各种其他眼部病变的关键细胞事件。在早期PVR中，RPE细胞从高度分化转分化为肌成纤维细胞表型，该表型积极分裂和迁移，然后形成PVR中发现的牵引性纤维细胞膜。一些蛋白质，特别是那些位于质膜的蛋白质，如整合素或生长因子受体，被糖基化。这些聚糖又是碳水化合物结合蛋白（如凝集素）的配体。在EMT后，我们发现RPE细胞的多糖表达谱发生了深刻的变化，向高亲和力的聚集素糖配体的表达增强。此外，我们发现凝集素可以改变RPE细胞的粘附和迁移，这取决于RPE的糖基化、凝集素的表达水平和重组凝集素的存在。因此，重组半乳糖凝集素-1 （rGal-1）和rGal-3通过与EMT上调的糖型结合，可以高选择性地靶向肌成纤维细胞而非健康的RPE。在之前的研究中，我们发现了rpe特异性的rGal-1和rGal-3糖蛋白配体，包括PDGFRB、CD44和整合素，所有这些都被认为在EMT和PVR中发挥作用。这些糖蛋白与重组凝集素的相互作用诱导它们在细胞膜上聚集，这需要β1,6- n糖基化。初步数据表明，rGal-1和rGal-3也可以改变这些受体的内吞作用以及它们在细胞内的分布和运输。考虑到内吞作用是受体结合后的连续步骤，并且内吞作用可以导致受体失活也可以激活，我们假设依赖于糖蛋白受体的糖基化，半凝集素可能影响内吞作用，从而改变信号通路的活性，从而改变细胞功能。通过使用体外培养的人RPE细胞作为EMT的模型，目前的提议解决了PDGFRB和整合素-ß1内吞作用在RPE中对不同糖基化和内源性凝集素表达水平的潜在功能影响，因为它们发生在EMT中。本研究的最终目的是找出差异蛋白糖基化和凝集素结合对膜持久性、糖蛋白配体的内吞作用以及随后特定信号通路活性的功能影响。凝集素糖蛋白相互作用在内吞作用中的作用对于进一步了解EMT和PVR的形成具有重要意义，PVR是由转分化的RPE细胞驱动的。此外，更好地了解这一途径可能有助于开发利用单个糖蛋白的差异糖基化作为治疗靶点的新型治疗方法。

Epithelial-to-mesenchymal transition of retinal pigment epithelial cells (RPE) is a key cellular event in the onset of proliferative vitreoretinopathy (PVR), but also in various other ocular pathologies. In early PVR RPE cells transdifferentiate from a highly differentiated to a myofibroblastic phenotype, which is actively dividing and migratory and then gives rise to the formation of the tractive, fibrocellular membranes found in PVR.Several proteins especially those located at the plasma membrane, such as integrins or growth factor receptors, are glycosylated. These glycans are in turn ligands for carbohydrate-binding proteins such as galectins. Upon EMT we found profound changes in the glycan expression profile of RPE cells towards an enhanced expression of high affinity glycoligands for galectins. Furthermore, we showed that galectins can modify RPE cell adhesion and migration, which was dependent on RPE-glycosylation, galectin expression levels and the presence of recombinant galectins. Thus, via binding to glycoforms upregulated upon EMT, recombinant Galectin-1 (rGal-1) and rGal-3 may allow for targeting myofibroblastic but not healthy RPE with high selectivity.In previous investigations we identified RPE-specific glycoprotein ligands for rGal-1 and rGal-3, including PDGFRB, CD44 and integrins, all of which are attributed to play a role in EMT and PVR. Interaction of these glycoproteins with recombinant galectins induces their clustering at the cell membrane, which requires β1,6-N-glycosylation. Preliminary data indicate that rGal-1 and rGal-3 also modify endocytosis of these receptors as well as their intracellular distribution and trafficking. Considering that endocytosis is the consecutive step after receptor binding and that endocytosis can lead to receptor inactivation but also activation, we assume that depending on the glycosylation of the glycoprotein receptor galectins may influence endocytosis and thereby modify the activity of signaling pathways and hence cellular function. By using cultured human RPE cells as a model for EMT in vitro the current proposal addresses the potential functional implications of PDGFRB and integrin-ß1 endocytosis in the RPE with respect to differential glycosylation and endogenous galectin expression levels as they occur in EMT. The ultimate aim of this study is to single out the functional impact of differential protein glycosylation and galectin binding on membrane persistence as well as endocytosis of the glycoprotein ligands and subsequent activity of specific signaling pathways. The role of the galectin glycoprotein interaction in endocytosis is of major interest for a further understanding of EMT as well as PVR formation, which is driven by transdifferentiated RPE cells. Furthermore, a better understanding at this pathway could contribute to the development of novel therapeutic approaches exploiting differential glycosylation of individual glycoproteins as a therapeutic target.