Use of pure recombinant human enzymes to assess the disease-causing potential of missense mutations in urea cycle disorders, applied to N-acetylglutamate synthase deficiency

IF 4.2 2区 医学 Q1 ENDOCRINOLOGY & METABOLISM Journal of Inherited Metabolic Disease Pub Date : 2024-05-13 DOI:10.1002/jimd.12747
Nadine Gougeard, Enea Sancho-Vaello, M. Leonor Fernández-Murga, Borja Martínez-Sinisterra, Badr Loukili-Hassani, Johannes Häberle, Clara Marco-Marín, Vicente Rubio
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Abstract

N-acetylglutamate synthase (NAGS) makes acetylglutamate, the essential activator of the first, regulatory enzyme of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1). NAGS deficiency (NAGSD) and CPS1 deficiency (CPS1D) present identical phenotypes. However, they must be distinguished, because NAGSD is cured by substitutive therapy with the N-acetyl-L-glutamate analogue N-carbamyl-L-glutamate, while curative therapy of CPS1D requires liver transplantation. Since their differentiation is done genetically, it is important to ascertain the disease-causing potential of CPS1 and NAGS genetic variants. With this goal, we previously carried out site-directed mutagenesis studies with pure recombinant human CPS1. We could not do the same with human NAGS (HuNAGS) because of enzyme instability, leading to our prior utilization of a bacterial NAGS as an imperfect surrogate of HuNAGS. We now use genuine HuNAGS, stabilized as a chimera of its conserved domain (cHuNAGS) with the maltose binding protein (MBP), and produced in Escherichia coli. MBP-cHuNAGS linker cleavage allowed assessment of the enzymatic properties and thermal stability of cHuNAGS, either wild-type or hosting each one of 23 nonsynonymous single-base changes found in NAGSD patients. For all but one change, disease causation was accounted by the enzymatic alterations identified, including, depending on the variant, loss of arginine activation, increased KmGlutamate, active site inactivation, decreased thermal stability, and protein misfolding. Our present approach outperforms experimental in vitro use of bacterial NAGS or in silico utilization of prediction servers (including AlphaMissense), illustrating with HuNAGS the value for UCDs of using recombinant enzymes for assessing disease-causation and molecular pathogenesis, and for therapeutic guidance.

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利用纯重组人酶评估尿素循环障碍中错义突变的致病潜力,并将其应用于 N-乙酰谷氨酸合成酶缺乏症。
N-乙酰谷氨酸合成酶(NAGS)制造乙酰谷氨酸,而乙酰谷氨酸是尿素循环的第一个调节酶--氨基甲酰基磷酸合成酶 1(CPS1)的重要激活剂。NAGS 缺乏症(NAGSD)和 CPS1 缺乏症(CPS1D)表现出相同的表型。不过,它们必须加以区分,因为 NAGSD 可通过 N-乙酰-L-谷氨酸类似物 N-氨基甲酰-L-谷氨酸的替代治疗治愈,而 CPS1D 的治愈治疗则需要肝移植。由于它们的分化是通过基因完成的,因此确定 CPS1 和 NAGS 基因变异的致病潜力非常重要。为此,我们先前对纯重组人 CPS1 进行了定点诱变研究。由于酶的不稳定性,我们无法对人类 NAGS(HuNAGS)进行同样的研究,这导致我们之前使用细菌 NAGS 作为 HuNAGS 的不完全替代物。现在,我们使用真正的 HuNAGS,将其保守结构域(cHuNAGS)与麦芽糖结合蛋白(MBP)嵌合稳定,并在大肠杆菌中生产。通过 MBP-cHuNAGS 连接器裂解,可以评估野生型 cHuNAGS 的酶特性和热稳定性,或评估 NAGSD 患者中发现的 23 种非同义单碱基变化中每一种变化的宿主 cHuNAGS 的酶特性和热稳定性。除一种变异外,其他所有变异的致病原因都是由所发现的酶学改变引起的,包括精氨酸活化丧失、谷氨酸 Km 值升高、活性位点失活、热稳定性降低和蛋白质错误折叠。我们目前的方法优于体外使用细菌 NAGS 的实验方法或利用预测服务器(包括 AlphaMissense)进行的硅学研究,通过 HuNAGS 说明了使用重组酶评估致病原因和分子发病机制以及提供治疗指导对 UCDs 的价值。
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来源期刊
Journal of Inherited Metabolic Disease
Journal of Inherited Metabolic Disease 医学-内分泌学与代谢
CiteScore
9.50
自引率
7.10%
发文量
117
审稿时长
4-8 weeks
期刊介绍: The Journal of Inherited Metabolic Disease (JIMD) is the official journal of the Society for the Study of Inborn Errors of Metabolism (SSIEM). By enhancing communication between workers in the field throughout the world, the JIMD aims to improve the management and understanding of inherited metabolic disorders. It publishes results of original research and new or important observations pertaining to any aspect of inherited metabolic disease in humans and higher animals. This includes clinical (medical, dental and veterinary), biochemical, genetic (including cytogenetic, molecular and population genetic), experimental (including cell biological), methodological, theoretical, epidemiological, ethical and counselling aspects. The JIMD also reviews important new developments or controversial issues relating to metabolic disorders and publishes reviews and short reports arising from the Society''s annual symposia. A distinction is made between peer-reviewed scientific material that is selected because of its significance for other professionals in the field and non-peer- reviewed material that aims to be important, controversial, interesting or entertaining (“Extras”).
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