Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling

Sara Violante (1, 2) , Lodewijk Ijlst (2) , Henk van Lenthe (2) , Isabel Tavares de Almeida (1) , Ronald J. Wanders (2) , Fátima V. Ventura (1)

1 Metabolism and Genetics Group
2 Laboratory Genetic Metabolic Disease

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid β-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. homogenates expressing human CPT2 were incubated with saturated and unsaturated C2-C26 acyl-CoAs and branched-chain aminoacid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8-C12) and long-chain (C14-C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain aminoacid oxidation intermediates. -2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that -2-C16:1-CoA may act as a competitive inhibitor of CPT2 ( of 18.8µM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that -2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of -2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.

Mots clés: mFAO; CPT1; CPT2; CACT; MTP; LCHAD; VLCAD; DMN; DMH; Acylcarnitines; Carnitine palmitoyltransferase 2 (CPT2); Long-chain acyl-CoA metabolites; Fatty acid oxidation (FAO); Mitochondrial Trifunctional Protein (MTP)

Sara Violante, Lodewijk Ijlst, Henk van Lenthe, Isabel Tavares de Almeida, Ronald J. Wanders, et al.. Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling. Biochimica et Biophysica Acta - Molecular Basis of Disease, 2010, 1802 (9), pp.728. ⟨10.1016/j.bbadis.2010.06.002⟩. ⟨hal-00608982⟩