Understanding carbamoyl phosphate synthetase (CPS1) deficiency by using the recombinantly purified human enzyme: Effects of CPS1 mutations that concentrate in a central domain of unknown function

Abstract

Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is an inborn error of the urea cycle that is due to mutations in the CPS1 gene. In the first large repertory of mutations found in CPS1D, a small CPS1 domain of unknown function (called the UFSD) was found to host missense changes with high frequency, despite the fact that this domain does not host substrate-binding or catalytic machinery. We investigate here by in vitro expression studies using baculovirus/insect cells the reasons for the prominence of the UFSD in CPS1D, as well as the disease-causing roles and pathogenic mechanisms of the mutations affecting this domain. All but three of the 18 missense changes found thus far mapping in this domain in CPS1D patients drastically decreased the yield of pure CPS1, mainly because of decreased enzyme solubility, strongly suggesting misfolding as a major determinant of the mutations negative effects. In addition, the majority of the mutations also decreased from modestly to very drastically the specific activity of the fraction of the enzyme that remained soluble and that could be purified, apparently because they decreased V max . Substantial although not dramatic increases in K m values for the substrates or for N-acetyl-L-glutamate were observed for only five mutations.Similarly, important thermal stability decreases were observed for three mutations. The results indicate a disease-causing role for all the mutations, due in most cases to the combined effects of the low enzyme level and the decreased activity. Our data strongly support the value of the present expression system for ascertaining the disease-causing potential of CPS1 mutations, provided that the CPS1 yield is monitored. The observed effects of the mutations have been rationalized on the basis of an existing structural model of CPS1. This model shows that the UFSD, which is in the middle of the 1462-residue multidomain CPS1 protein, plays a key integrating role for creating the CPS1 multidomain architecture leading us to propose here a denomination of "Integrating 3 Domain" for this CPS1 region. The majority of these 18 mutations distort the interaction of this domain with other CPS1 domains, in many cases by causing improper folding of structural elements of the Integrating Domain that play key roles in these interactions.

The Study of Carbamoyl Phosphate Synthetase 1 Deficiency Sheds Light on the Mechanism for Switching On/Off the Urea Cycle

“…Unlike the case for clinical mutations mapping in the integrating domain (Díez-Fernández et al, 2014), which drastically reduced CPS1 production in the present expression system, none of the five ASD clinical mutations tested here, (R1371L, T1391M, L1398V, P1439L and P1462R; in bold type in Table 1) decreased importantly CPS1 production or purification ( Fig. 2A).…”

“…Furthermore, NCG stabilizes CPS1 (Díez-Fernández et al, 2013), opening the way to testing whether NCG might be beneficial for patients with mutations causing CPS1 destabilization. These mutations can also be identified with the present system (Díez-Fernández et al, 2014), although in the case of the ASD only few clinical mutations cause substantial destabilization. Nevertheless, these mutations reveal that the ASD contributes to CPS1 stability.…”

Cellular Serpents and Dreaming Spires: New Frontiers in Arginine and Pyrimidine Biology

“…Carmen Díez Fernandez identified a domain of unknown function (UFSD) in CPS1 which contains many potentially pathological missense mutations. Finally, she concluded by suggesting that the UFSD may be involved in the cross talk between N-acetyl glutamate (NAG) and ATP binding sites (Diez-Fernandez et al, 2013;Diez-Fernandez et al, 2014).…”

Novel Pathogenic Variant (c.580C>T) in the CPS1 Gene in a Newborn With Carbamoyl Phosphate Synthetase 1 Deficiency Identified by Whole Exome Sequencing

“…Urea cycle disorders (UCDs) result from an inherited deficiency caused by pathogenic variations in one or more of the genes encoding the necessary enzymes [2]. With the aid of the allosteric activator N-acetylglutamate, synthesized by NAGS, CPS1 catalyzes the first step in the urea cycle, converting ammonia to carbamoyl phosphate [3]. Although UCD diagnosis is primarily based on the biochemical measurement of intermediary metabolites, such measurements cannot be used to distinguish between NAGS deficiency and CPS1D; genetic pathogenic variant testing is needed to distinguish these conditions [4].…”

“…CPS1 is a large gene located on 2q35, spanning >120 kb, encompassing 4,500 coding nucleotides over 38 coding exons [13]. More than 240 CPS1 pathogenic variations have been reported to be widely distributed among the coding exons in CPS1 pathogenic variants as described in the Leiden Open Variation Database (LOVD, http://www.LOVD.nl/CPS1) and the Human Gene Mutation Database (HGMD, http://www.hgmd.org/).…”