Corinne Inauen1, Véronique Rüfenacht1, Amit V Pandey2, Liyan Hu1,3, Henk Blom4, Jean-Marc Nuoffer5,6, Johannes Häberle7,8,9. 1. Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zürich, Steinwiesstr. 75, 8032, Zurich, Switzerland. 2. Pediatric Endocrinology, University Children's Hospital and Department of Clinical Research, University of Bern, Bern, Switzerland. 3. Affiliated with the Neuroscience Center Zürich (ZNZ), Zurich, Switzerland. 4. Department of General Pediatrics, Adolescent Medicine and Neonatology, University Medical Centre Freiburg, Freiburg, Germany. 5. University Institute of Clinical Chemistry, University of Bern, Bern, Switzerland. 6. University Children's Hospital, University of Bern, Bern, Switzerland. 7. Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zürich, Steinwiesstr. 75, 8032, Zurich, Switzerland. johannes.haeberle@kispi.uzh.ch. 8. Affiliated with the Neuroscience Center Zürich (ZNZ), Zurich, Switzerland. johannes.haeberle@kispi.uzh.ch. 9. Affiliated with the Zürich Center for Integrative Human Physiology (ZIHP), Zurich, Switzerland. johannes.haeberle@kispi.uzh.ch.
Abstract
INTRODUCTION: Cysteamine is used to treat cystinosis via the modification of cysteine residues substituting arginine in mutant proteins. OBJECTIVES: We investigated the effect of cysteamine on mutant argininosuccinate lyase (ASL), the second most common defect in the urea cycle. METHODS: In an established mammalian expression system, 293T cell lysates were produced after transfection with all known cysteine for arginine mutations in the ASL gene (p.Arg94Cys, p.Arg95Cys, p.Arg168Cys, p.Arg379Cys, and p.Arg385Cys), allowing testing of the effect of cysteamine over 48 h in the culture medium as well as for 1 h immediately prior to the enzyme assay. RESULTS: Cysteamine at low concentrations showed no effect on 293T cell viability, ASL protein expression, or ASL activity when applied during cell culture. However, incubation of transfected cells with 0.05 mM cysteamine immediately before the enzyme assay resulted in increased ASL activity of p.Arg94Cys, p.Arg379Cys, and p.Arg385Cys by 64, 20, and 197 %, respectively, and this result was significant (p < 0.01). Cell lysates carrying p.Arg385Cys and treated with cysteamine recover enzyme activity that is similar to the untreated designed mutation p.Arg385Lys, providing circumstantial evidence for the assumed cysteamine-induced change of a cysteine to a lysine analogue. CONCLUSION: Since 12 % of all known genotypes in ASL deficiency are affected by a cysteine for arginine mutation, we conclude that the potential of cysteamine or of related substances as remedy for this disease should be investigated further.
INTRODUCTION:Cysteamine is used to treat cystinosis via the modification of cysteine residues substituting arginine in mutant proteins. OBJECTIVES: We investigated the effect of cysteamine on mutant argininosuccinate lyase (ASL), the second most common defect in the urea cycle. METHODS: In an established mammalian expression system, 293T cell lysates were produced after transfection with all known cysteine for arginine mutations in the ASL gene (p.Arg94Cys, p.Arg95Cys, p.Arg168Cys, p.Arg379Cys, and p.Arg385Cys), allowing testing of the effect of cysteamine over 48 h in the culture medium as well as for 1 h immediately prior to the enzyme assay. RESULTS:Cysteamine at low concentrations showed no effect on 293T cell viability, ASL protein expression, or ASL activity when applied during cell culture. However, incubation of transfected cells with 0.05 mM cysteamine immediately before the enzyme assay resulted in increased ASL activity of p.Arg94Cys, p.Arg379Cys, and p.Arg385Cys by 64, 20, and 197 %, respectively, and this result was significant (p < 0.01). Cell lysates carrying p.Arg385Cys and treated with cysteamine recover enzyme activity that is similar to the untreated designed mutation p.Arg385Lys, providing circumstantial evidence for the assumed cysteamine-induced change of a cysteine to a lysine analogue. CONCLUSION: Since 12 % of all known genotypes in ASL deficiency are affected by a cysteine for arginine mutation, we conclude that the potential of cysteamine or of related substances as remedy for this disease should be investigated further.
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