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

半乳糖凝集素和 N-糖基化方面对视网膜色素上皮细胞上皮间质转化和增殖性玻璃体视网膜病变形成的功能影响

• 批准号: 181475910
• 负责人: Privatdozent Dr. Andreas Ohlmann
• 金额: --
• 依托单位: Augenklinik (Innenstadt)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/181475910?language=en
• 关键词: Functional; impact; galectins; aspects; glycosylation

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.

视网膜色素上皮细胞（RPE）的上皮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理学的关键细胞事件。在早期PVR中，RPE细胞从高度分化的表型转分化为成肌纤维细胞表型，成肌纤维细胞表型活跃地分裂和迁移，然后引起PVR中发现的牵引性纤维细胞膜的形成。这些聚糖又是碳水化合物结合蛋白如半乳糖凝集素的配体。在EMT后，我们发现RPE细胞的聚糖表达谱发生了深刻的变化，朝向半乳糖凝集素的高亲和力糖配体的表达增强。此外，我们发现半乳糖凝集素可以改变RPE细胞的粘附和迁移，这取决于RPE糖基化，半乳糖凝集素的表达水平和重组半乳糖凝集素的存在。因此，通过结合糖型上调EMT，重组半乳糖凝集素-1（rGal-1）和rGal-3可以允许靶向肌纤维母细胞，但不是健康的RPE与高选择性在以前的调查中，我们确定了RPE特异性糖蛋白配体的rGal-1和rGal-3，包括PDGFRB，CD 44和整合素，所有这些都归因于发挥作用的EMT和PVR。这些糖蛋白与重组半乳糖凝集素的相互作用诱导它们在细胞膜上聚集，这需要β 1，6-N-糖基化。初步数据表明，rGal-1和rGal-3也修改这些受体的内吞作用以及它们的细胞内分布和运输。考虑到内吞作用是受体结合后的连续步骤，并且内吞作用可导致受体失活，但也可导致活化，我们假设取决于糖蛋白受体半乳糖凝集素的糖基化，半乳糖凝集素可影响内吞作用，从而改变信号传导途径的活性，从而改变细胞功能。通过使用培养的人RPE细胞作为体外EMT的模型，本发明提出了RPE中PDGFRB和整联蛋白-β 1内吞作用关于差异糖基化和内源性半乳糖凝集素表达水平的潜在功能意义，因为它们发生在EMT中。本研究的最终目的是挑出差异蛋白糖基化和半乳糖凝集素结合对膜持久性以及糖蛋白配体的内吞作用和随后的特定信号传导途径的活性的功能影响。内吞作用中半乳糖凝集素糖蛋白相互作用的作用是进一步了解EMT以及PVR形成的主要兴趣，这是由转分化的RPE细胞驱动的。此外，更好地了解这一途径可能有助于开发新的治疗方法，利用个别糖蛋白的差异糖基化作为治疗靶点。