A method for measurement of human asparagine synthetase (ASNS) activity and application to ASNS protein variants associated with ASNS deficiency.

IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Biology Methods and Protocols Pub Date : 2023-10-25 eCollection Date: 2023-01-01 DOI:10.1093/biomethods/bpad026
Mario C Chang, Stephen J Staklinski, Matthew E Merritt, Michael S Kilberg
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Abstract

Human asparagine synthetase (ASNS) catalyzes the conversion of aspartate to asparagine in an ATP-dependent reaction that utilizes glutamine as a nitrogen source while generating glutamate, AMP, and pyrophosphate as additional products. Asparagine Synthetase Deficiency (ASNSD) is an inborn error of metabolism in which children present with homozygous or compound heterozygous mutations in the ASNS gene. These mutations result in ASNS variant protein expression. It is believed that these variant ASNS proteins have reduced enzymatic activity or stability resulting in a lack of sufficient asparagine production for cell function. Reduced asparagine production by ASNS appears to severely hinder fetal brain development. Although a variety of approaches for assaying ASNS activity have been reported, we present here a straightforward method for the in vitro enzymatic analysis by detection of AMP production. Our method overcomes limitations in technical feasibility, signal detection, and reproducibility experienced by prior methods like high-performance liquid chromatography, ninhydrin staining, and radioactive tracing. After purification of FLAG-tagged R49Q, G289A, and T337I ASNS variants from stably expressing HEK 293T cells, this method revealed a reduction in activity of 90, 36, and 96%, respectively. Thus, ASNS protein expression and purification, followed by enzymatic activity analysis, has provided a relatively simple protocol to evaluate structure-function relationships for ASNS variants reported for ASNSD patients.

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测定人天冬酰胺合成酶(ASNS)活性的方法及其在与ASNS缺陷相关的ASNS蛋白变异中的应用
人天冬酰胺合成酶(ASNS)在atp依赖性反应中催化天冬氨酸转化为天冬酰胺,该反应利用谷氨酰胺作为氮源,同时产生谷氨酸、AMP和焦磷酸盐作为附加产物。天冬酰胺合成酶缺乏症(ASNSD)是一种先天性代谢错误,儿童在ASNS基因中存在纯合或复合杂合突变。这些突变导致ASNS变异蛋白的表达。据信,这些变异的ASNS蛋白降低了酶活性或稳定性,导致缺乏足够的天冬酰胺生产以维持细胞功能。ASNS减少天冬酰胺的产生似乎严重阻碍胎儿大脑发育。虽然已经报道了多种测定ASNS活性的方法,但我们在这里提出了一种直接的方法,通过检测AMP的产生进行体外酶分析。我们的方法克服了高效液相色谱、茚三酮染色和放射性示踪等先前方法在技术可行性、信号检测和重现性方面的局限性。从稳定表达HEK 293T细胞中纯化flag标记的R49Q、G289A和T337I ASNS变体后,该方法显示活性分别降低了90%、36%和96%。因此,ASNS蛋白的表达和纯化,以及随后的酶活性分析,为评估ASNSD患者ASNS变异的结构-功能关系提供了一个相对简单的方案。
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来源期刊
Biology Methods and Protocols
Biology Methods and Protocols Agricultural and Biological Sciences-Agricultural and Biological Sciences (all)
CiteScore
3.80
自引率
2.80%
发文量
28
审稿时长
19 weeks
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