Molecular cloning of a cDNA for a putative choline co-transporter from Limulus CNS.
Molecular cloning of a cDNA for a putative choline co-transporter from Limulus CNS.
复制标题
鲎 CNS 推定胆碱协同转运蛋白 cDNA 的分子克隆。
DOI：
10.1016/s0378-1119(01)00421-8
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发表时间：
2001
期刊：
影响因子：
3.5
通讯作者：
Townsel,JG
中科院分区：
文献类型：
--
作者：
Wang,Y;Cao,Z;Newkirk,RF;Ivy,MT;Townsel,JG
据充分记载，钠依赖性、半胆碱-3敏感性、高亲和力胆碱共转运蛋白在乙酰胆碱的生物合成中是限速的，并且对于胆碱能传递是必需的。直到最近，这种传送器还无法克隆。Okuda等人（2000.自然神经科学。3，120 - 125）最近报道了在秀丽隐杆线虫（CHO-1）和大鼠（CHT 1）中成功克隆胆碱共转运蛋白。我们在此报告的克隆胆碱转运蛋白的马蹄蟹，美洲鲎。通过使用一系列简并引物，从CHO-1和CHT 1的共有序列中选择，我们产生了两个探针，用于搜索从中枢神经系统（CNS）组织产生的鲎cDNA文库。鲎同源物的全长核苷酸序列由3368 bp组成，其中包括一个开放阅读框（ORF）和两个非翻译区（NTR），该开放阅读框预测了一个579个氨基酸的蛋白质，一个在3 '端，另一个在5'端。其氨基酸序列与大鼠CHT 1的同源性为46%，与C. elegans和最近克隆的人共转运蛋白（hCHT; Apparsundaram等，2000.生物化学、生物物理通信资源276，862 - 867; Okuda和Haga，2000. FEBS Lett. 484，92 - 97）。亲水性图分析预测鲎胆碱共转运蛋白（LChCoT）具有13个跨膜结构域（TMD），N-末端定向于细胞外，C-末端定向于细胞内。北方印迹分析使用的cDNA探针设计的LChCoT的cDNA序列显示其分布特异性的中枢神经系统结构。另一方面，在非神经组织中未发现。LChCoT是钠依赖性葡萄糖转运蛋白家族（SLGT）的成员，其克隆的成功将有助于研究其生理调控，包括胞内转运。
It is well documented that the sodium dependent, hemicholinium-3 sensitive, high affinity choline co-transporter is rate limiting in the biosynthesis of acetylcholine and is essential to cholinergic transmission. Until recently this transporter had eluded cloning. Okuda et al. (2000. Nature Neurosci. 3, 120–125) recently reported the successful cloning of the choline co-transporter in Caenorhabditis elegans (CHO-1) and rat (CHT1). We report herein the cloning of the choline co-transporter in the horseshoe crab, Limulus polyphemus. Through the use of a series of degenerate primers selected from consensus sequences of CHO-1 and CHT1, we generated two probes that were used to search a Limulus cDNA library produced from central nervous system (CNS) tissue. The full length nucleotide sequence of the Limulus homolog consists of 3368 bp which includes an open reading frame (ORF) that predicts a protein of 579 amino acids and two non-translation regions (NTR), one at the 3′ end and the other at the 5′ end. The amino acid sequence has 46% identity with rat CHT1 and 50% identity with both CHO-1 in C. elegans and the recently cloned human co-transporter (hCHT; Apparsundaram et al., 2000. Biochem. Biophys. Res. Commun. 276, 862–867; Okuda and Haga, 2000. FEBS Lett. 484, 92–97). Hydropathy plot analysis predicts the Limulus choline co-transporter (LChCoT) to have thirteen transmembrane domains (TMD), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analyses using cDNA probes designed from LChCoT cDNA sequences revealed its distribution specifically in central nervous system structures. On the other hand it was not found in non-nervous tissues. The successful cloning of LChCoT, which was shown to be a member of the sodium-dependent glucose transporter family (SLGT), should prove useful in the determination of its physiological regulation, including its intracellular trafficking.