Syntaxin-3 regulates Tight Junction Assembly in Human Retinal Pigment Epithelium

Abstract The retinal pigment epithelium (RPE) consists of polarized epithelial cells, serving as a support system for photoreceptor maintenance, where the polarity is contributed by the distribution of syntaxins (STX) within the cell. STX3, a known regulator of apical trafficking in epithelial cells, was previously understood to be absent in human RPE cells, with its functions thought to be compensated by STX1A. However, our results on SNARE mRNA expression profile in RPE detected the presence of 2 splice variants of STX3. Further investigation in donor retina, primary hRPE, and ARPE-19 cells revealed detectable levels of STX3 mRNA and protein. STX3 knockdown in ARPE19 resulted in a significant reduction of tight junction (TJ) proteins, compromising TJ assembly, highlighting the critical role of STX3 in maintaining RPE integrity. In addition, immunoprecipitation followed by LC-MS/MS analysis revealed that STX3 and STX1A have a distinct novel protein interactome in RPE. This study identified unique and shared interactants for STX3 and STX1A, suggesting a broader role for RPE beyond its traditional photoreceptor support function. This further emphasises the biological significance of STX1A and STX3 in maintaining retinal homeostasis, which could facilitate the development of novel therapeutic strategies for retinal disorders. Significance This study identified the presence of STX3 in the human RPE cells, which was previously reported to be absent. Further, we demonstrated that STX3 knockdown in ARPE19 cells disrupted TJ assembly, highlighting its potential role in preserving RPE cell polarity and structural integrity, challenging the notion that STX3 functions were thought to be compensated by STX1A. Moreover, immunoprecipitation followed by LC-MS/MS analysis in RPE identified the protein interaction networks of both STX1A and STX3. Interestingly, unique and shared interactants, including proteins associated with neuronal plasticity, indicated unidentified functions of STX3 and STX1A in RPE. This suggests that they might perform both overlapping and distinct functions for maintaining RPE cell integrity and thus retinal homeostasis. Overall, our preliminary findings challenge the established view that STX3 is non-existent in RPE cells and initiate new directions for exploring the multifaceted and potentially non-redundant functions of STX3 in RPE.