Dandoy-Dron Françoise, Griffond Bernadette, Mishal Zohar, Tovey Michael G, Dron Michel
Laboratoire d'Oncologie Virale, CNRS UPR 9045, 7 rue G. Môquet BP-8, 94801 Villejuif Cedex, France.
Eur J Neurosci. 2003 Nov;18(9):2449-59. doi: 10.1046/j.1460-9568.2003.03009.x.
Scrapie responsive gene one (Scrg1) is a novel transcript discovered through identification of the genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies. Scrg1 mRNA is distributed principally in the central nervous system and the cDNA sequence predicts a small cysteine-rich protein 98 amino acids in length, with a N-terminal signal peptide. In this study, we have generated antibodies against the predicted protein and revealed expression of a predominant immunoreactive protein of 10 kDa in mouse brain by Western blot analysis. We have established CAD neuronal cell lines stably expressing Scrg1 to determine its subcellular localization. Several lines of evidence show that the protein is targeted to dense-core vesicles in these cells. (i) Scrg1 is detected by immunocytochemistry as very punctate signals especially in the Golgi apparatus and tips of neurites, suggesting a vesicular localization for the protein. Moreover, Scrg1 exhibits a high degree of colocalization with secretogranin II, a dense-core vesicle marker and a very limited colocalization with markers for small synaptic vesicles. (ii) Scrg1 immunoreactivity is associated with large secretory granules/dense-core vesicles, as indicated by immuno-electron microscopy. (iii) Scrg1 is enriched in fractions of sucrose density gradient where synaptotagmin V, a dense-core vesicle-associated protein, is also enriched. The characteristic punctate immunostaining of Scrg1 is observed in N2A cells transfected with Scrg1 and for the endogenous protein in cultured primary neurons, attesting to the generality of the observations. Our findings strongly suggest that Scrg1 is associated with the secretory pathway of neuronal cells.
瘙痒病反应基因1（Scrg1）是通过鉴定与可传播性海绵状脑病中观察到的神经退行性变化相关或导致这些变化的基因而发现的一种新转录本。Scrg1 mRNA主要分布于中枢神经系统，其cDNA序列预测编码一个长度为98个氨基酸的富含半胱氨酸的小蛋白，带有一个N端信号肽。在本研究中，我们制备了针对该预测蛋白的抗体，并通过蛋白质印迹分析揭示了小鼠脑中一种主要的10 kDa免疫反应性蛋白的表达。我们建立了稳定表达Scrg1的CAD神经元细胞系以确定其亚细胞定位。多条证据表明该蛋白定位于这些细胞中的致密核心囊泡。（i）通过免疫细胞化学检测到Scrg1为非常点状的信号，尤其是在高尔基体和神经突末端，提示该蛋白定位于囊泡。此外，Scrg1与致密核心囊泡标记物分泌粒蛋白II高度共定位，与小突触囊泡标记物的共定位非常有限。（ii）免疫电子显微镜显示Scrg1免疫反应性与大分泌颗粒/致密核心囊泡相关。（iii）Scrg1在蔗糖密度梯度分馏物中富集，突触结合蛋白V（一种与致密核心囊泡相关的蛋白）也在该分馏物中富集。在用Scrg1转染的N2A细胞以及培养的原代神经元中的内源性蛋白中均观察到Scrg1特征性的点状免疫染色，证明了这些观察结果的普遍性。我们的发现强烈提示Scrg1与神经元细胞的分泌途径相关。
