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.

Autor: Gougeard N; Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain.; Group 739, Centro de Investigación Biomédica en Red de Enfermedades Raras, (CIBERER-ISCIII) at the IBV-CSIC, Valencia, Spain., Sancho-Vaello E; Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain., Fernández-Murga ML; Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain., Martínez-Sinisterra B; Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain., Loukili-Hassani B; Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain., Häberle J; University Children's Hospital Zurich and Children's Research Centre, Zurich, Switzerland., Marco-Marín C; Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain.; Group 739, Centro de Investigación Biomédica en Red de Enfermedades Raras, (CIBERER-ISCIII) at the IBV-CSIC, Valencia, Spain., Rubio V; Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain.; Group 739, Centro de Investigación Biomédica en Red de Enfermedades Raras, (CIBERER-ISCIII) at the IBV-CSIC, Valencia, Spain.
Jazyk: angličtina
Zdroj: Journal of inherited metabolic disease [J Inherit Metab Dis] 2024 Nov; Vol. 47 (6), pp. 1194-1212. Date of Electronic Publication: 2024 May 13.
DOI: 10.1002/jimd.12747
Abstrakt: 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 K m Glutamate , 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.
(© 2024 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)
Databáze: MEDLINE
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