These authors contributed equally to this work.
Molecular Characterization of Carbamoyl-Phosphate Synthetase (CPS1) Deficiency Using Human Recombinant CPS1 as a Key Tool
Article first published online: 28 MAY 2013
© 2013 WILEY PERIODICALS, INC.
Volume 34, Issue 8, pages 1149–1159, August 2013
How to Cite
Diez-Fernandez, C., Martínez, A. I., Pekkala, S., Barcelona, B., Pérez-Arellano, I., Guadalajara, A. M., Summar, M., Cervera, J. and Rubio, V. (2013), Molecular Characterization of Carbamoyl-Phosphate Synthetase (CPS1) Deficiency Using Human Recombinant CPS1 as a Key Tool. Hum. Mutat., 34: 1149–1159. doi: 10.1002/humu.22349
Contract grant sponsors: Fundación Alicia Koplowitz 2011; Valencian Government (Prometeo 2009/051); Science Department of the Spanish Government (BFU2011-30407 and SAF2010-17933); FPU (Spanish Government) Fellowship; CIPF-Bancaixa Fellowship.
Communicated by David S. Rosenblatt
- Issue published online: 15 JUL 2013
- Article first published online: 28 MAY 2013
- Accepted manuscript online: 3 MAY 2013 02:21PM EST
- Manuscript Accepted: 18 APR 2013
- Manuscript Received: 8 NOV 2012
- Fundación Alicia Koplowitz 2011
- Valencian Government (Prometeo 2009/051)
- Science Department of the Spanish Government. Grant Numbers: BFU2011-30407, SAF2010-17933
- FPU (Spanish Government) Fellowship
- CIPF-Bancaixa Fellowship
- urea cycle;
- CPS1 deficiency;
The urea cycle disease carbamoyl-phosphate synthetase deficiency (CPS1D) has been associated with many mutations in the CPS1 gene [Häberle et al., 2011. Hum Mutat 32:579–589]. The disease-causing potential of most of these mutations is unclear. To test the mutations effects, we have developed a system for recombinant expression, mutagenesis, and purification of human carbamoyl-phosphate synthetase 1 (CPS1), a very large, complex, and fastidious enzyme. The kinetic and molecular properties of recombinant CPS1 are essentially the same as for natural human CPS1. Glycerol partially replaces the essential activator N-acetyl-l-glutamate (NAG), opening possibilities for treating CPS1D due to NAG site defects. The value of our expression system for elucidating the effects of mutations is demonstrated with eight clinical CPS1 mutations. Five of these mutations decreased enzyme stability, two mutations drastically hampered catalysis, and one vastly impaired NAG activation. In contrast, the polymorphisms p.Thr344Ala and p.Gly1376Ser had no detectable effects. Site-limited proteolysis proved the correctness of the working model for the human CPS1 domain architecture generally used for rationalizing the mutations effects. NAG and its analogue and orphan drug N-carbamoyl-l-glutamate, protected human CPS1 against proteolytic and thermal inactivation in the presence of MgATP, raising hopes of treating CPS1D by chemical chaperoning with N-carbamoyl-l-glutamate.