Expression cloning and characterization of a cDNA encoding a novel membrane protein required for the formation of O-acetylated ganglioside: A putative acetyl-CoA transporter
Expression cloning and characterization of a cDNA encoding a novel membrane protein required for the formation of O-acetylated ganglioside: A putative acetyl-CoA transporter
复制标题
DOI：
10.1073/pnas.94.7.2897
复制
发表时间：
1997-04-01
影响因子：
11.1
通讯作者：
Hirabayashi, Y
中科院分区：
文献类型：
--
作者：
Kanamori, A;Nakayama, J;Hirabayashi, Y
通过使用稳定转染 GD3 合酶 (COS-1/CD3(+)) 的 COS-1 细胞作为受体细胞系进行表达克隆，我们分离了一个 cDNA，称为 AT-1，编码形成 O-乙酰化 (Ac) 神经节苷脂所需的新型蛋白质。该cDNA编码具有亮氨酸拉链基序的多跨膜结构域蛋白，由549个氨基酸组成，分子量为60.9 kDa，虽然O-Ac-GD3和O-Ac-GT3在受体细胞或仅用载体转染的细胞中几乎检测不到，但在含有AT-1的转染子中，当处理制备的半完整细胞时，它们的量显着增加 与链球菌溶血素 O 一起与 [Ac-C-14]-Ac-CoA 一起孵育，与模拟转染子相比，发现转染 AT-1 的细胞中放射性掺入增加，Northern 印迹分析显示在所有检查的组织中都有 3.3 和 4.3 kb 的两个主要转录本，使用 AT-1 蛋白特异性抗体进行的免疫组织化学研究表明，它最有可能在内质网中表达 基于这些结果，AT-1 编码的蛋白质被认为是参与 O-乙酰化过程的 Ac-CoA 转运蛋白。
By expression cloning using COS-1 cells stably transfected with GD3-synthase (COS-1/CD3(+)) as a recipient cell line, we have isolated a cDNA, termed AT-1, encoding a novel protein required for the formation of O-acetylated (Ac) gangliosides. The cDNA encodes a protein with multitransmembrane spanning domains with a leucine zipper motif, It consists of 549 amino acids and has a molecular mass of 60.9 kDa, Although both O-Ac-GD3 and O-Ac-GT3 were barely detectable in recipient cells or cells transfected with the vector alone, their amount increased significantly in transfectants containing AT-1, When semi-intact cells prepared by treatment with streptolysin O were incubated with [Ac-C-14]-Ac-CoA, increased incorporation of radioactivity was found in those cells transfected with AT-1 when compared with the mock transfectants, Northern blot analysis showed two major transcripts of 3.3 and 4.3 kb in all tissues examined, Immunohistochemical study with an antibody specific to the AT-1 protein suggested that it is most probably expressed in the endoplasmic reticulum membrane, Based on these results, the protein encoded by AT-1 is suggested to be an Ac-CoA transporter that is Involved in the process of O-acetylation.