Elsevier

Biochimie

Volume 88, Issue 1, January 2006, Pages 113-116
Biochimie

The gene mutated in l-2-hydroxyglutaric aciduria encodes l-2-hydroxyglutarate dehydrogenase

https://doi.org/10.1016/j.biochi.2005.06.005 Get rights and content

Abstract

The biochemical defect in l-2-hydroxyglutaric aciduria is still unknown, but the mutated gene has recently been identified on chromosome 14q22. Transfection of human embryonic kidney (HEK) cells with a cDNA encoding the product of the human gene led to a > 15-fold increase in l-2-hydroxyglutarate dehydrogenase activity. The overexpressed enzyme had similar biochemical characteristics (including sensitivity to FAD and association with membranes) as the rat liver enzyme. Western blot analysis indicated that it is processed through the removal of a N-terminal ≈ 4 kDa fragment, in agreement with a mitochondrial localization. Transfection experiments indicated that the mutations (K81E, E176D, Δ-exon9) found in patients with l-2-hydroxyglutaric aciduria suppressed l-2-hydroxyglutarate dehydrogenase activity. Western blot analysis showed that the three mutated proteins were expressed to various degrees in HEK cells, but were abnormally processed. Taken together, these data indicate that l-2-hydroxyglutaric aciduria is due to a deficiency in l-2-hydroxyglutarate dehydrogenase.

Introduction

l-2-Hydroxyglutaric aciduria is a neurometabolic disorder characterized by the presence of elevated concentrations of l-2-hydroxyglutaric acid in plasma, cerebrospinal fluid and urine [1], [2]. The enzymatic defect leading to this disorder is still not known. l-2-hydroxyglutarate is metabolized to alpha-ketoglutarate by an FAD-dependent dehydrogenase linked to mitochondrial membranes, which most likely transfers its electrons to the respiratory chain [3], [4]. It is likely, although not yet proven, that deficiency of this enzyme is responsible for l-2-hydroxyglutaric aciduria. The gene mutated in this disorder has recently been identified through a homozygosity linkage approach [5] or through a functional candidate gene approach [4]. The protein encoded by this gene is homologous to FAD-dependent dehydrogenases, but its function has not yet been established. The purpose of the present work was to test if this gene encoded l-2-hydroxyglutarate dehydrogenase and investigate the effect of mutations found in patients on the biochemical function of the corresponding protein.

Section snippets

Preparation of plasmids

The open reading frame encoding the putative human l-2-hydroxyglutarate dehydrogenase (accession no NM_02488.1) was amplified by PCR using Pwo DNA polymerase, human liver cDNA as a template and the following primers: 5′-GGAATTCAGCGTGCGTAGGGGATG-3′ and 5′-GTACAACAAAGATTTGAATTATCTAGAATG-3′(Eurogentec, Ougrée, Belgium). In the latter primer, the natural stop codon is replaced by a XbaI restriction site. The PCR product was cloned into the EcoRV site of pBluescript II KS (Stratagene, La Jolla, CA,

Expression of the WT putative l-2-hydroxyglutarate dehydrogenase

Successful overexpression of the putative l-2-hydroxyglutarate dehydrogenase was obtained by transfecting human embryonic kidney (HEK) cells with linear, purified polyethyleneimine. As shown in Fig. 1, a faint band of ≈ 50 kDa and a stronger band of ≈ 45 kDa were revealed with antibodies directed against the His-tag present at the C-terminus of the recombinant protein. The putative l-2-hydroxyglutarate dehydrogenase, is predicted to be targeted to the mitochondria where its mitochondrial

Conclusion

Our data indicate that the gene mutated in l-2-hydroxyglutaric aciduria encodes l-2-hydroxyglutarate dehydrogenase and that the mutations found in patients with this disease abolish the activity of this enzyme. l-2-hydroxyglutarate dehydrogenase converts its substrate to alpha-ketoglutarate, a ubiquitous compound that is formed both in the Krebs cycle and by deamination of glutamate. The pathological symptoms found in l-2-hydroxyglutaric aciduria are, therefore, most likely the consequence of a

Acknowledgements

This work was supported by the Concerted research action program of the Communauté Française de Belgique; the Interuniversity Attraction Poles Program-Belgian Science Policy, and by the Belgian Scientific Fund for Medical Research (FRSM). RR is fellow of the Fonds pour l'Encouragement à la Recherche dans l’Industrie et dans l’Agriculture and MVDC, chercheur qualifié of the Belgian FNRS.

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