Peroxisomal lignoceroyl-CoA ligase deficiency in childhood adrenoleukodystrophy and adrenomyeloneuropathy.

Abstract

We previously reported that in childhood adrenoleukodystrophy (C-ALD) and adrenomyeloneuropathy (AMN), the peroxisomal fl-oxidation system for very long chain (>C22) fatty acids is defective. To further define the defect in these two forms of X chromosome-linked ALD, we examined the oxidation of [1-14C]lignoceric acid (n-tetracosanoic acid, C24:0) and [1-'4C]lignoceroyl-CoA (substrates for the first and second steps of f8-oxidation, respectively). The oxidation rates of lignoceric acid in C-ALD and AMN were 43% and 36% of control values, respectively, whereas the oxidation rate of lignoceroyl-CoA was 109% (C-ALD) and 106% (AMN) of control values, respectively. On the other hand, the oxidation rates of palmitic acid (n-hexadecanoic acid) and palmitoyl-CoA in C-ALD and AMN were similar to the control values. These results suggest that lignoceroyl-CoA ligase activity may be impaired in C-ALD and AMN. To identify the specific enzymatic deficiency and its subcellular localization in C-ALD and AMN, we established a modified procedure for the subcellular fractionation of cultured skin fibroblasts. Determination of acyl-CoA ligase activities provided direct evidence that lignoceroyl-CoA ligase is deficient in peroxisomes while it is normal in mitochondria and microsomes. Moreover, the normal oxidation of lignoceroyl-CoA as compared with the deficient oxidation of lignoceric acid in isolated peroxisomes also supports the conclusion that peroxisomal lignoceroyl-CoA ligase is impaired in both C-ALD and AMN. Palmitoyl-CoA ligase activity was found to be normal in peroxisomes as well as in mitochondria and microsomes. This normal peroxisomal palmitoyl-CoA ligase activity as compared with the deficient activity of lignoceroyl-CoA ligase in C-ALD and AMN suggests the presence of two separate acyl-CoA ligases for palmitic and lignoceric acids in peroxisomes. These data clearly demonstrate that the pathognomonic accumulation of very long chain fatty acids in C-ALD and AMN is due to a deficiency of peroxisomal very long chain (lignoceric acid) acyl-CoA ligase.The peroxisomal disorders represent a newly characterized group of inherited diseases (1, 2). In the adrenoleukodystrophies (ALD), three forms are recognized: childhood ALD (C-ALD; X chromosome-linked), adult ALD [adrenomyeloneuropathy (AMN); X chromosome-linked], and neonatal ALD (autosomal recessive). C-ALD is the most common form (3, 4) and usually appears between the ages of 4 and 8 years. It is characterized by central nervous system demyelination and adrenal cortical insufficiency. Death occurs during the first or second decade. AMN occurs mainly in adults, progresses more slowly, and affects the adrenal cortex, spinal cord, and peripheral nerves (5). The occurrence of both C-ALD and AMN within the same kindred suggests that these forms of ALD are different clinical manifestations of the same mutation (4). The identification of an identical biochemical defect in both would confirm this assumption. The neonatal form of ALD is a severe disorder that is evident in...

Therapy of X-linked adrenoleukodystrophy

“…3 For reasons that are not yet understood this defect leads to the accumulation of saturated very long chain fatty acids (VLCFA) such as hexacosanoic acid (C26:0) in the adrenal gland and nervous system white matter 4 and other tissues and in plasma. 5 The VLCFA excess appears to be due to their impaired degradation, 5,6 a reaction that normally takes place in the peroxisome, 7 although recent studies indicate that the defects in fatty acid metabolism are complex and not yet fully understood. 8 Demonstration of excess of VLCFA in plasma is the most commonly used diagnostic assay.…”

The protein coded by the X‐adrenoleukodystrophy gene is a peroxisomal integral membrane protein

“…The deficient activity of lignoceroyl-CoA ligase as compared to the normal oxidation of lignoceroyl-CoA in purified peroxisomes supported this conclusion [14,17]. These observations suggested that the gene coding for lignoceroylCoA ligase may be a candidate for the X-ALD gene; however, the recent identification of the ALD gene by positional cloning [18][19][20] led to a different conclusion.…”

“…Thirty or more confluent flasks (75 cm*) of cultured skin fibroblasts from control and X-ALD patients were harvested, cell pellets were washed with homogenizing medium (0.25 M sucrose, 1 mM EDTA, 0.5pg/ml of antipain, 0.5,&ml of leupeptin, 1 &ml of aprotinin, 0.7 &ml pepstatin, 0.2 mM phenylmethylsulfonylfluoride, 0.1% ethanol in 3 mM imidazole buffer (pH 7.4)) at 4°C and the subcellular organelles were purified by differential and isopycnic density gradient centrifugation [14]. The gradient was collected from the bottom and analyzed by the marker enzyme activities of catalase for peroxisomes, NADPH cytochrome c reductase for endoplasmic reticulum and cytochrome c oxidase for mitochondria.…”

“…The subcellular organelles were prepared by isopycnic density gradient centrifugation in Nycodenz (Accurate Chemical and Scientific Corp., Westbury, NY) from cultured skin fibroblasts from controls and X-ALD patients as described previously [14]. Thirty or more confluent flasks (75 cm*) of cultured skin fibroblasts from control and X-ALD patients were harvested, cell pellets were washed with homogenizing medium (0.25 M sucrose, 1 mM EDTA, 0.5pg/ml of antipain, 0.5,&ml of leupeptin, 1 &ml of aprotinin, 0.7 &ml pepstatin, 0.2 mM phenylmethylsulfonylfluoride, 0.1% ethanol in 3 mM imidazole buffer (pH 7.4)) at 4°C and the subcellular organelles were purified by differential and isopycnic density gradient centrifugation [14].…”

“…The identification of peroxisomes as the site of oxidation of VLC fatty acids [10] suggested that X-ALD might be a peroxisomal disease. Direct demonstration of defective oxidation of VLC fatty acids in peroxisomes isolated from cultured X-ALD skin fibroblasts established that X-ALD is a disease associated with a defect in the peroxisomal fl-oxidation system [14].…”