高度表現於GTP環形水解酵素non-DN細胞中之新奇基因的選殖與表現

Abstract

GTP cyclohydrolase I(GCH）的突變會導致遺傳性漸進式肌緊張不足（Hereditary progressive dystonia, HPD)/對多巴反應肌緊張不足（Dopa-responsive dystonia, DRD）。GCH是體內重要輔因數BH4生合成的第一個步驟，也是速率決定步驟酵素。在病人體內,GCH的酵素活性約只有正常人的2-20%，在細胞模式的研究中，發現突變的蛋白質具有dominant negative作用，而影響正常蛋白質的作用。 R13是在non-dominant negative細胞中表現較多的基因，經由北方墨點分析確定後，經基因庫做序列比對，發現它位於人類第20對染色體而此基因由於使用不同的?動子和alternative splicing，推測三種不同mRNA及蛋白質的產生，依蛋白質的長度將之命名為，R13-A (559aa)，R13-B (374aa）及R13-C (197aa)，此篇論文即是對這三種可能的蛋白質做分析探討。 在基因選殖的過程中，我們發現R13-C這種蛋白質並不存在即所預期的stop codon並不存在，而R13-B可能如生物資訊預期的有轉譯後的修飾，R13-A經由細胞螢光染色法，發現它不存在於細胞核中，而詳細的存在位置需要進一步的探討。

Hereditary progressive dystonia (HPD)/DOPA responsive dystonia (DRD) is caused by mutation of the GTP cyclohydrolase I (GCH) gene which is responsible for the first and rate limiting step of tetrahydrobioterin (BH4) synthesis. BH4 plays an important and essential role in aromatic amino acid monooxygeneases and nitric oxide synthase. HPD/DRD patients showed very low (about 2-20% of normal values) GCH activities compared with normal individuals. Dominant negative mechanism hypothesis for HPD/DRD has been proposed and was examined in the cell model. To understand the molecular regulation different between DN and non-DN cells and to look more details about dominant negative mechanism, we performed PCR-selected cDNA subtraction method to selectively amplify differentially expressed genes. More than thirty clones differentially expressed clones were found in DN and non-DN cells. Clone R13, expressed mainly in non-DN cells as confirmed by northern blot analysis, was a novel gene with unknown function. Deduced amino acids encoded by R13 gene were obtained from the GenBank database. There were three forms of deduced R13 proteins resulted from alternative splicing and different promoter usage. The three proteins were designated as R13-A, R13-B, and R13-C. R13-A may localize in the cytosol or membrane bound using immunocytochemistry. R13-B may have post-translational processing, the predicted signal peptide might be cleaved. The stop codon resided in R13-C was not detected. The function of R13 on the GCH protein was discussed.