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Molecular Characterization of Carbamoyl-Phosphate Synthetase (CPS1) Deficiency Using Human Recombinant CPS1 as a Key Tool

Authors

  • Carmen Diez-Fernandez,

    1. Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain
    2. Centro de Investigación Príncipe Felipe, Valencia, Spain
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    • These authors contributed equally to this work.

  • Ana I. Martínez,

    1. Centro de Investigación Príncipe Felipe, Valencia, Spain
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    • These authors contributed equally to this work.

  • Satu Pekkala,

    1. Centro de Investigación Príncipe Felipe, Valencia, Spain
    Current affiliation:
    1. Department of Health Sciences, University of Jyväskylä, Finland
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  • Belén Barcelona,

    1. Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain
    2. Centro de Investigación Príncipe Felipe, Valencia, Spain
    3. Group 739, CIBERER, ISCIII, Spain
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  • Isabel Pérez-Arellano,

    1. Centro de Investigación Príncipe Felipe, Valencia, Spain
    2. Group 739, CIBERER, ISCIII, Spain
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  • Ana María Guadalajara,

    1. Centro de Investigación Príncipe Felipe, Valencia, Spain
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  • Marshall Summar,

    1. Childrens National Medical Center, Washington, District of Columbia
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  • Javier Cervera,

    Corresponding author
    1. Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain
    2. Centro de Investigación Príncipe Felipe, Valencia, Spain
    3. Group 739, CIBERER, ISCIII, Spain
    • Correspondence to: Vicente Rubio, Instituto de Biomedicina de Valencia, C/ Jaime Roig 11, Valencia-46010, Spain. E-mail: rubio@ibv.csic.es; Javier Cervera, Instituto de Biomedicina de Valencia, C/Jaime Roig 11, Valencia-46010, Spain. E-mail: cervera@ibv.csic.es

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    • These authors contributed equally to this work.

  • Vicente Rubio

    Corresponding author
    1. Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain
    2. Group 739, CIBERER, ISCIII, Spain
    • Correspondence to: Vicente Rubio, Instituto de Biomedicina de Valencia, C/ Jaime Roig 11, Valencia-46010, Spain. E-mail: rubio@ibv.csic.es; Javier Cervera, Instituto de Biomedicina de Valencia, C/Jaime Roig 11, Valencia-46010, Spain. E-mail: cervera@ibv.csic.es

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    • These authors contributed equally to this work.


  • 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

ABSTRACT

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.

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