AN ARGININE TO GLUTAMINE MUTATION IN RESIDUE 109 OF HUMAN ORNITHINE TRANSCARBAMYLASE COMPLETELY ABOLISHES ENZYMATIC-ACTIVITY IN COS1 CELLS
AN ARGININE TO GLUTAMINE MUTATION IN RESIDUE 109 OF HUMAN ORNITHINE TRANSCARBAMYLASE COMPLETELY ABOLISHES ENZYMATIC-ACTIVITY IN COS1 CELLS
复制标题
DOI：
10.1172/jci114360
复制
发表时间：
1989-12-01
影响因子：
15.9
通讯作者：
NUSSBAUM, RL
中科院分区：
文献类型：
--
作者：
LEE, JT;NUSSBAUM, RL
鸟氨酸转氨甲酰酶（OTC）是氨解毒为尿素的重要酶，其缺乏是人类尿素生成最常见的先天性错误。在之前检查的24个OTC缺陷病例中，三个无关个体均表现出OTC基因中对应密码子109的Taq I位点缺失，表明该Taq I位点可能容易发生突变。其中两名患者表现出相同的 C .fwdarw。 T 转换（反义链中）将 Arg109 转化为 Gln。尽管这些研究表明错义突变与 OTC 缺陷表型之间存在很强的关联，但无法牢固地建立因果关系。我们通过在体外重建突变来研究这种关系。全长人类 OTC cDNA 被克隆到基于 SV40 的表达载体中，并在 Cos1 细胞系中高水平重复表达。通过对该野生型序列进行定点诱变，我们构建了包含C.fwdarw的错义突变。 T 过渡。 Cos1 的电穿孔和瞬时测定表明，突变型 OTC 的比活性比野生型低 100 倍。该结果证实，导致 Gln 错义的 Taq I 改变是影响上述患者的 OTC 缺乏的原因。
Ornithine transcarbamylase (OTC) is an important enzyme in the detoxification of ammonia to urea, and its deficiency is the most common inborn error of ureagenesis in humans. Among 24 cases of OTC deficiency previously examined, three unrelated individuals all showed loss of a Taq I site in the OTC gene correponding to codon 109, suggesting that this Taq I site may be prone to mutation. Two of these patients demonstrated the same C .fwdarw. T transition (in antisense strand) converting Arg109 to Gln. Although these studies implied a strong association between the missense mutation and OTC-deficient phenotype, a causal relationship could not be firmly established. We have investigated this relationship by reconstructing the mutation in vitro. A full-length human OTC cDNA was cloned into an SV40-based expression vector and has been reproducibly expressed at high levels in the cell line Cos1. By site-directed mutagenesis of this wild type sequence, we constructed a missense mutation which contains the C .fwdarw. T transition. Electroporation and transient assay in Cos1 indicated that the specific activity of mutant OTC was 100-fold lower than that of wild type. This result confirms that the Taq I alteration leading to the Gln missense is responsible for the OTC deficiency affecting the above patients.