Human long chain, very long chain and medium chain acyl-CoA dehydrogenases are specific for the S-enantiomer of 2- methylpentadecanoyl-CoA.

The acyl-CoA dehydrogenases are a family of mitochondrial flavoenzymes involved in fatty acid and branched chain amino-acid metabolism. Long chain acyl-CoA dehydrogenase (LCAD) and short/branched chain acyl-CoA dehydrogenase (SBCAD) have been shown to have activity towards 2-methyl branched chain acyl-CoA substrates of varying chain lengths. In humans, long chain 2-branched chain fatty acids such as pristanic acid are largely thought to be metabolized in peroxisomes through desaturation of their CoA esters by branched chain acyl-CoA oxidase, but LCAD is also capable of utilizing 2-methyldecanoyl- and 2-methylpalmitoyl-CoA as substrate [1]. Since the acyl-CoA oxidase reaction is specific for the S-enantiomer of the branched chain substrates, we investigated the stereo specificity of mitochondrial LCAD. Purified LCAD had a specific activity of 390 and 340 mU/mg of purified LCAD protein using palmitoyl-CoA and S-2-methylpentadecanoyl-CoA, respectively, as substrate. No activity was measurable with R-2-methylpentadecanoyl-CoA. Purified medium chain acyl-CoA dehydrogenase (MCAD) could also utilize S-2-methylpentadecanoyl-CoA as a substrate, but not R-2-methylpentadecanoyl-CoA. These results indicate that LCAD and MCAD are specific for the S-enantiomers of methylbranched chain substrates. Crude mitochondrial extracts showed no activity when dehydrogenating activity was measured with R/S-2-methylpalmitoyl-CoA or S-2-methylpentadecanoyl-CoA after inactivation of the extract with antibodies to very long chain acyl-CoA dehydrogenase and MCAD, suggesting that this substrate is not useful in identifyig clinical deficiencies of LCAD.

In vitro characterization and in vivo expression of human very-long chain acyl-CoA dehydrogenase.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a disorder of fatty acid beta-oxidation that can present at any age with cardiomyopathy, rhabdomyolysis, hepatic dysfunction, and/or nonketotic hypoglycemia. Through the expansion of newborn screening programs an increasing number of individuals with VLCAD deficiency are being identified prior to the onset of symptoms allowing early initiation of therapy. The development of a safe, durable, and effective VLCAD gene delivery system for use at the time of diagnosis could result in a significant improvement in the quality and duration of life for patients with VLCAD deficiency. To this end, we developed a construct containing the human VLCAD cDNA under the control of the strong CMV promoter (pCMV-hVLCAD). A novel rabbit polyclonal anti-VLCAD antibody was prepared using a 24 amino-acid peptide unique to the human VLCAD protein to study human VLCAD expression in immune competent mice. Antibody specificity was demonstrated in Western blots of human VLCAD deficient fibroblasts and in pCMV-hVLCAD transiently transfected VLCAD deficient fibroblasts. Transfected fibroblasts showed correction of the metabolic block as demonstrated by normalization of C14- and C16-acylcarnitine species in cell culture media and restoration of VLCAD activity in cells. Following tail vein injection of pCMV-hVLCAD into mice, we demonstrated expression of hVLCAD in liver. Altogether, these steps are important in the development of a durable gene therapy for VLCAD deficiency.

Immunochemical characterization of variant long-chain acyl-CoA dehydrogenase in cultured fibroblasts from nine patients with long-chain acyl-CoA dehydrogenase deficiency.

Long-chain acyl-CoA dehydrogenase (LCAD) deficiency is a disorder of mitochondrial fatty acid oxidation that is characterized by hypoglycemia, muscle weakness, and hepato- and cardiomegaly. To characterize variant LCAD, we first carried out preliminary experiments using pure enzyme preparations. Despite the significant sequence similarity of LCAD to medium-chain acyl-CoA dehydrogenase, the antibody raised against rat LCAD was monospecific for human and rat LCAD and did not cross-react with either human or rat medium-chain acyl-CoA dehydrogenase. Immunoblot analysis of variant LCAD in cultured fibroblasts from nine patients with LCAD deficiency revealed a single LCAD band in all nine LCAD-deficient cell lines. Each variant LCAD was comparable in molecular size and quantity to normal LCAD, suggesting that the LCAD mutation in each of these cell lines is likely to be a point mutation that produces a stable variant LCAD. The uniform nature of variant LCAD suggests that only a single, or at most a few, prevalent point mutations may be found in the majority of LCAD-deficient patients. If this is the case, it should be possible to devise a molecular diagnostic method for LCAD deficiency.