遺傳疾病HPD／DRD : GTP 環形水解酵素突變株分析

Abstract

GTP cyclohydrolase (GCH) 基因突變會引發非典型苯酮尿症(phenylketonuria，PKU) 它以體隱性方式遺傳，及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 )以體顯性方式遺傳。GTP cyclohydrolase I 是催化tetrahydrobiopterin (BH4) 生合成的第一個步驟也是速率決定步驟。BH4是體內三種芳香性氨基酸單氧化酵素：笨丙氨酸氫氧酵素( phenylalanine hydroxylas)，酥氨酸氫氧酵索(tyrosine hydroxylase)，色氨酸氫氧酵素(tryptophan hydroxylase) 以及NO合成酵素 (Nitric oxide synthase) 的必要輔因數。 然而在HPD／DRD 病人體內 GCH 活性小於正當人的20％，至於HPD／DRD 病人體中 GCH 活性小於正常人的20％的機轉到目前為止仍不清楚。利用大腸桿菌及真核表現系統來研究存在HPD／DRD 病人體中突變基因GCHG201E的分子機轉。根據免疫共沈實驗結果，我們發現突變基因GCRG201E (在殘基201位置上的甘氨酸被麩氨酸取代) 其突變蛋白質GCRG201E可與正常蛋白質形成複合物。聯同轉染實驗與西方點潰法發現，此複合物不易偵測到。在pulse－chase實驗發現突變蛋白質 GCHG201E 的合成正常但其穩定性下降，顯示突變蛋白質 GCHG201E 具有 Dominant negative 作用，可能是因為造成正常蛋白質迅速分解，使之功能喪失。這或許可以解釋病人體中 GCH 活性小於正常人的20％。 除此之外，利用大腸桿菌表現突變蛋白質 GCHG201E，發現其無催化活性而且無法形成polymer，這或許可以解釋突變蛋白質 GCRG201E 活性的喪失。

Mutations in GTP cyclohydrolase(GCH) gene were found to cause both atypical phenylketonuria (PKU) with autosomal recessive trait and hereditary progressive dystonia(HPD)/DOPA responsive dystonia(DRD) with autosomal dominant trait. GCH is the initial an rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis. Three aromatic acid monooxygenases (phenylalanine hydroxylase, tyrosine hydroxylase，tryptophan hydroxylase) and NO synthase require BH4 as an essential cofactor. However, GTP cyclohydrolase activities in HPD/DRD patients were less than 20％ of those in normal individuals. The mechanism that the HPD/DRD patients have less than 20％ of the normal activity is not clear. The molecular mechanisms of mutant gene GCHG201E in HPD/DRD patients were studied using both E.coli and eukaryotic expression systems. In the co-immumoprecipitation experiment, we find mutant protein GCHG201E (Glycine residue is substituted with Glutamate at position 201) is able to interact with wild type protein. The amount of complex composed of wild type proteins and mutant proteins is fairly low as detected by cotransfection and western blot analysis. In pulse-chase experiment, the synthesis of mutant protein GCHG201E was normal, but the stability of the mutant protein GCHG201E dacreased, mutant protein GCHG201E exert its dominant negative effect on wild type protein possibly by rapid degradation. This may explain why the HPD/DRD patients have less than 20％ of the normal activity. Futhermore, mutant protein GCHG201E expressed in E.coli have no GTP cyclohydrolase activity and can not form polymer which may explain the activity loss of GCHG201E protein.