Abstract
Classical homocystinuria is caused by mutations in the cystathionine β-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B6. Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.
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Acknowledgments
The authors would like to thank Alena Duta, Eva Richterova, and Jitka Sokolova for technical help. This study was supported by research program of the General University Hospital in Prague RVO-VFN 64165/2012. Institutional support was provided by research programs PRVOUK-P24/LF1/3 and UNCE 204011 of the Charles University in Prague with access to the LC-MS/MS made possible by the project OPPK No. CZ.2.16/3.1.00/24012.
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All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study. The study was approved by the Ethics Committee of General University Hospital and First Faculty of Medicine, Charles University, Prague (No. 949/10 S-IV); all patients gave their written informed consent.
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Communicated by: Jaak Jaeken
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Melenovská, P., Kopecká, J., Krijt, J. et al. Chaperone therapy for homocystinuria: the rescue of CBS mutations by heme arginate. J Inherit Metab Dis 38, 287–294 (2015). https://doi.org/10.1007/s10545-014-9781-9
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DOI: https://doi.org/10.1007/s10545-014-9781-9