Opsin3糖基化位点的鉴定及糖基化修饰功能

Identification of the glycosylation sites of Opsin3 and its glycosylation modification function

摘要:

为鉴定Opsin3(OPN3)的糖基化位点，通过使用衣霉素(tunicamycin)以及N-糖酰胺酶F (PNGase F)处理发现OPN3存在糖基化修饰；通过预测以及蛋白质氨基酸位点点突变鉴定OPN3的糖基化位点；利用SNAP标签蛋白构建了SNAP-Opsin3(SNAP-OPN3)重组蛋白，使用SNAP-OPN3重组蛋白进行糖基化修饰对OPN3功能影响的研究。在表达SNAP-OPN3重组蛋白后，使用SNAP-tag的反应底物SNAP-Surface^® 549以及SNAP-Cell^® Oregon Green^®通过荧光共聚焦显微镜观察OPN3以及OPN3糖基化位点突变体是否能够成熟转运至细胞膜。结果表明，OPN3的N82位点为OPN3的糖基化位点；SNAP标签不影响OPN3功能的正常发挥；使用SNAP反应底物可以清楚地观察到糖基化位点突变的OPN3不能正常转运至细胞膜。本研究首次鉴定了OPN3的糖基化位点，并构建了SNAP-OPN3重组蛋白，发现SNAP标签并不影响OPN3的成熟转运，并利用SNAP底物验证了糖基化修饰影响OPN3蛋白的成熟转运。

Abstract:

Opsin3(OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain.In recent years,it has been reported that OPN3 is also highly expressed in adipose tissue,and the protein is associated with the production of skin melanin.We found that the N82 site is the glycosylation site of OPN3.SNAP-tag^TM has diverse functions and can be applied to a variety of different studies.By constructing a SNAP-tagged OPN3 recombinant protein,the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface^® 549 and SNAP-Cell^® OregonGreen^®,which provides a new method for studying the function of OPN3.It also shows that SNAP-tag does not affect the function of OPN3.Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.