STRUCTURE AND FUNCTION IN RHODOPSIN - THE ROLE OF ASPARAGINE-LINKED GLYCOSYLATION
STRUCTURE AND FUNCTION IN RHODOPSIN - THE ROLE OF ASPARAGINE-LINKED GLYCOSYLATION
复制标题
DOI：
10.1073/pnas.91.9.4024
复制
发表时间：
1994-04-26
影响因子：
11.1
通讯作者：
KHORANA, HG
中科院分区：
文献类型：
--
作者：
KAUSHAL, S;RIDGE, KD;KHORANA, HG
视紫红质是视杆细胞的暗光感受器，是一种完整的膜蛋白，在ASN-2和ASN-15处糖基化。在这里，我们报告了糖基化在视紫红质折叠和功能中的作用的实验。非糖化视蛋白是在天冬酰胺连接的糖基化抑制剂衣霉素存在下，通过在COS-1细胞中表达野生型牛视蛋白基因而制备的。非糖化视蛋白折叠正确，棕榈酰化正常，转运至细胞表面，并形成具有11顺式视网膜特征的视紫红质发色团(最大波长500 nm)。然而，在与糖基化视紫红质相当的浓度水平下，非糖基化视紫红质对G(T)的光依赖激活作用显著降低。第2和15位氨基酸替换和同源三肽共有序列[Asn-2--&Gt；Gln，Gly-3-&Gt；Cys(Pro)，Thr-4-&Gt；Lys，Asn-15-&Gt；Ala(Cys，Glu，Lys，Gin，Arg)，Lys-16-&Gt；Cys(Arg)，Thr-17-&Gt；Met(Val)]表明，Asn-2、Gly-3和Thr-4处的替换对视紫红质的折叠、细胞转运和/或功能没有显著影响，而Asn-15和Lys-16处的替换导致折叠不良并不能转运到细胞表面。此外，带有ASN-15和Thr-17氨基酸替换的突变色素对G(T)的激活作用很差。我们得出结论：ASN-19糖基化在信号转导中起重要作用。
Rhodopsin, the dim light photoreceptor of the rod cell, is an integral membrane protein that is glycosylated at Asn-2 and Asn-15. Here we report experiments on the role of the glycosylation in rhodopsin folding and function. Nonglycosylated opsin was prepared by expression of a wild-type bovine opsin gene in COS-1 cells in the presence of tunicamycin, an inhibitor of asparagine-linked glycosylation. The nonglycosylated opsin folded correctly as shown by its normal palmitoylation, transport to the cell surface, and the formation of the characteristic rhodopsin chromophore (lambda(max) 500 nm) with 11-cis-retinal. However, the nonglycosylated rhodopsin showed strikingly low light-dependent activation of G(T) at concentration levels comparable with those of glycosylated rhodopsin. Amino acid replacements at positions 2 and 15 and the cognate tripeptide consensus sequence [Asn-2 --> Gln, Gly-3 --> Cys (Pro), Thr-4 --> Lys, Asn-15 --> Ala (Cys, Glu, Lys, Gin, Arg), Lys-16 --> Cys (Arg), Thr-17 --> Met (Val)] showed that the substitutions at Asn-2, Gly-3, and Thr-4 had no significant effect on the folding, cellular transport, and/or function of rhodopsin, whereas those at Asn-15 and Lys-16 caused poor folding and were defective in transport to the cell surface. Further, mutant pigments with amino acid replacements at Asn-15 and Thr-17 activated G(T) very poorly. We conclude that Asn-19 glycosylation is important in signal transduction.