Yelin R, Steiner-Mordoch S, Aroeti B, Schuldiner S
Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel.
J Neurochem. 1998 Dec;71(6):2518-27. doi: 10.1046/j.1471-4159.1998.71062518.x.
The role of N-glycosylation in the expression, ligand recognition, activity, and intracellular localization of a rat vesicular monoamine transporter (rVMAT1) was investigated. The glycosylation inhibitor tunicamycin induced a dose-dependent decrease in the rVMAT1-mediated uptake of [3H]serotonin. Part of this effect was due to a general toxic effect of the drug. Therefore, to assess the contribution of each of the glycosylation sites to the transporter activity, the three putative N-glycosylation sites were mutated individually, in combination, and in toto ("triple" mutant). Mutation of each glycosylation site caused a minor and additive decrease in activity, up to the triple mutant, which retained at least 50% of the wild-type activity. No significant differences were found either in the time dependence of uptake or the apparent affinity for ligands of the triple mutant compared with the wild-type protein. It is interesting that in contrast to plasma-membrane neurotransmitter transporters, the unglycosylated form of rVMAT1 distributed in the cell as the wild-type protein. Pro43 is a highly conserved residue located at the beginning of the large loop in which all the potential glycosylation sites are found. A Pro43Leu mutant transporter was inactive. It is remarkable that despite the presence of glycosylation sites, the mutant transporter was not glycosylated. Moreover, the distribution pattern of the Pro43Leu mutant clearly differed from that of the wild type. In contrast, a Pro43Gly mutant displayed an activity practically identical to the wild-type protein. As this replacement generated a protein with wild-type characteristics, we suggest that the conformation conferred by the amino acid at this position is essential for activity.
研究了N-糖基化在大鼠囊泡单胺转运体（rVMAT1）的表达、配体识别、活性及细胞内定位中的作用。糖基化抑制剂衣霉素可导致rVMAT1介导的[3H]5-羟色胺摄取呈剂量依赖性降低。该效应部分归因于药物的一般毒性作用。因此，为评估每个糖基化位点对转运体活性的贡献，分别、联合及全部（“三重”突变体）突变了三个假定的N-糖基化位点。每个糖基化位点的突变均导致活性轻微且呈累加性降低，直至三重突变体，其仍保留至少50%的野生型活性。与野生型蛋白相比，三重突变体在摄取的时间依赖性或对配体的表观亲和力方面均未发现显著差异。有趣的是，与质膜神经递质转运体不同，未糖基化形式的rVMAT1在细胞内的分布与野生型蛋白相同。Pro43是位于大环起始处的一个高度保守残基，所有潜在的糖基化位点均位于该大环中。Pro43Leu突变体转运体无活性。值得注意的是，尽管存在糖基化位点，但该突变体转运体并未发生糖基化。此外，Pro43Leu突变体的分布模式与野生型明显不同。相反地，Pro43Gly突变体表现出与野生型蛋白几乎相同的活性。由于这种替换产生了具有野生型特征的蛋白，我们认为该位置氨基酸赋予的构象对活性至关重要。
