Effect of Cysteamine on Mutant ASL Proteins with Cysteine for Arginine Substitutions
- 159 Downloads
Cysteamine is used to treat cystinosis via the modification of cysteine residues substituting arginine in mutant proteins.
We investigated the effect of cysteamine on mutant argininosuccinate lyase (ASL), the second most common defect in the urea cycle.
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.
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.
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.
Keywords293T Cell Cysteamine Cystinosis Urea Cycle Disorder Transfected 293T Cell
Compliance with Ethical Standards
Conflict of interest
The authors CI, VR, AVP, LH, HB, JMN, and JH declare that they have no conflicts of interest.
This work was supported by the Swiss National Science Foundation (Grants 310030_153196/1 to JH and 310031_134926 to AVP) and a grant from Schweizerische Mobiliar Genossenschaft Jubiläumsstiftung to AVP.
- 2.Häberle J, Shahbeck N, Ibrahim K, Schmitt B, Scheer I, O’Gorman R, et al. Glutamine supplementation in a child with inherited GS deficiency improves the clinical status and partially corrects the peripheral and central amino acid imbalance. Orphanet J Rare Dis. 2012;7:48. doi: 10.1186/1750-1172-7-48.CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Aly AM, Arai M, Hoyer LW. Cysteamine enhances the procoagulant activity of Factor VIII-East Hartford, a dysfunctional protein due to a light chain thrombin cleavage site mutation (arginine-1689 to cysteine). J Clin Invest. 1992;89(5):1375–81. doi: 10.1172/JCI115725.CrossRefPubMedPubMedCentralGoogle Scholar
- 15.Hu L, Pandey AV, Eggimann S, Rüfenacht V, Moslinger D, Nuoffer JM, et al. Understanding the role of argininosuccinate lyase transcript variants in the clinical and biochemical variability of the urea cycle disorder argininosuccinic aciduria. J Biol Chem. 2013;288(48):34599–611. doi: 10.1074/jbc.M113.503128.CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Engel K, Vuissoz JM, Eggimann S, Groux M, Berning C, Hu L, et al. Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in-vitro enzyme activity with clinical phenotype in argininosuccinic aciduria. J Inherit Metab Dis. 2012;35(1):133–40. doi: 10.1007/s10545-011-9357-x.CrossRefPubMedGoogle Scholar
- 20.Vriend G. WHAT IF: a molecular modeling and drug design program. J Mol Graph. 1990;8(1):52–6, 29.Google Scholar
- 24.Kleijer WJ, Garritsen VH, Linnebank M, Mooyer P, Huijmans JG, Mustonen A, et al. Clinical, enzymatic, and molecular genetic characterization of a biochemical variant type of argininosuccinic aciduria: prenatal and postnatal diagnosis in five unrelated families. J Inherit Metab Dis. 2002;25(5):399–410.CrossRefPubMedGoogle Scholar
- 28.Jezegou A, Llinares E, Anne C, Kieffer-Jaquinod S, O’Regan S, Aupetit J, et al. Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy. Proc Natl Acad Sci USA. 2012;109(50):E3434–43. doi: 10.1073/pnas.1211198109.CrossRefPubMedPubMedCentralGoogle Scholar
- 30.Smolin LA, Clark KF, Thoene JG, Gahl WA, Schneider JA. A comparison of the effectiveness of cysteamine and phosphocysteamine in elevating plasma cysteamine concentration and decreasing leukocyte free cystine in nephropathic cystinosis. Pediatr Res. 1988;23(6):616–20. doi: 10.1203/00006450-198806000-00018.CrossRefPubMedGoogle Scholar
- 31.Hua Long L, Halliwell B. Oxidation and generation of hydrogen peroxide by thiol compounds in commonly used cell culture media. Biochem Biophys Res Commun. 2001;286(5):991–4. doi: 10.1006/bbrc.2001.5514.