Misawa H, Matsuura J, Oda Y, Takahashi R, Deguchi T
Department of Neurology, Tokyo Metropolitan Institute for Neuroscience, Fuchu City, Japan.
Brain Res Mol Brain Res. 1997 Mar;44(2):323-33. doi: 10.1016/s0169-328x(96)00231-8.
Choline acetyltransferase (ChAT, EC 2.3.1.6) is the biosynthetic enzyme for acetylcholine. We have previously shown that multiple ChAT mRNA species with different 5'-noncoding regions are expressed in the rat and mouse. However, the diversity of ChAT mRNA species in human has not completely been elucidated. In this work N1- and N2-type ChAT cDNAs were cloned from a human brain cDNA library and the N-exon located in the human ChAT gene. Polymerase chain reaction analysis indicates that four species of ChAT mRNAs (R-, N1-, N2- and M-types) are produced in human brain and spinal cord. In all human transcripts, the ATG initiation codon in the rat, mouse and pig was replaced by ACG, which does not serve as an initiation codon for translation. In vitro translation and mammalian expression analyses revealed that N1-, N2- and R-type mRNAs give rise to a single 69 kDa enzyme, while M-type mRNA produces both 82 and 69 kDa enzymes. The translation efficiency of M-type mRNA was lower than that of the other mRNA species. Moreover, the translation efficiency of human ChAT mRNAs was considerably lower than that of rat ChAT mRNA, suggesting that the ATG codons for human ChAT are unfavorable for translation initiation compared with the initiation codon for rat ChAT. These results provide rational explanations for the previous reports that human ChAT protein purified from the brain and placenta had 66-70 kDa molecular mass, and that ChAT activity in a single motor neuron of human was far lower than that of other vertebrates. Sequencing of monkey ChAT gene showed that the initiation ATG in rodent ChAT was also replaced by ACA in the monkey.
胆碱乙酰转移酶（ChAT，EC 2.3.1.6）是合成乙酰胆碱的酶。我们之前已经表明，具有不同5'-非编码区的多种ChAT mRNA在大鼠和小鼠中表达。然而，人类ChAT mRNA种类的多样性尚未完全阐明。在这项工作中，从人脑cDNA文库和人ChAT基因中的N外显子克隆了N1型和N2型ChAT cDNA。聚合酶链反应分析表明，人脑和脊髓中产生四种ChAT mRNA（R型、N1型、N2型和M型）。在所有人类转录本中，大鼠、小鼠和猪中的ATG起始密码子被ACG取代，而ACG不作为翻译的起始密码子。体外翻译和哺乳动物表达分析表明，N1型、N2型和R型mRNA产生单一的69 kDa酶，而M型mRNA产生82 kDa和69 kDa两种酶。M型mRNA的翻译效率低于其他mRNA种类。此外，人类ChAT mRNA的翻译效率远低于大鼠ChAT mRNA，这表明与大鼠ChAT的起始密码子相比，人类ChAT的ATG密码子不利于翻译起始。这些结果为先前的报道提供了合理的解释，即从人脑和胎盘中纯化的人类ChAT蛋白具有66 - 70 kDa的分子量，并且人类单个运动神经元中的ChAT活性远低于其他脊椎动物。猴ChAT基因测序表明，啮齿动物ChAT中的起始ATG在猴中也被ACA取代。
