Phytanoyl-CoA hydroxylase deficiency. Enzymological and molecular basis of classical Refsum disease.
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Cloning, Molecular↗
Biomedical subjects
R J Wanders
Publications and source records attributed to R J Wanders.
Analysis of pristanic acid beta-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry.
In this paper we report the development of highly sensitive, selective, and accurate stable isotope dilution gas chromatography negative chemical ionization mass spectrometry (GC-NCI-MS) methods for quantification of peroxisomal beta-oxidation intermediates of pristanic acid in human plasma: 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid. The carboxylic groups of the intermediates were converted into pentafluorobenzyl esters, whereas hydroxyl groups were acetylated and ketogroups were methoximized. Hereafter, the samples were subjected to clean-up by high performance liquid chromatography. Analyses were performed by selected monitoring of the carboxylate anions of the derivatives. Control values of all three metabolites were established (2,3-pristenic acid: 2-48 nm, 3-hydroxypristanic acid: 0.02-0.81 nm, 3-ketopristanic acid: 0.07-1.45 nm). A correlation between the concentrations of pristanic acid and its intermediates in plasma was found. The diagnostic value of the methods is illustrated by measurements of the intermediates in plasma from patients with peroxisomal disorders. It is shown that in generalized peroxisomal disorders, the absolute concentrations of 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid were comparable to those in the controls, whereas relative to the pristanic acid concentrations these intermediates were significantly decreased. In bifunctional protein deficiency, elevated levels of 2,3-pristenic acid and 3-hydroxypristanic acid were found. 3-Ketopristanic acid, although within the normal range, was relatively low when compared to the high pristanic acid levels in these patients.-Verhoeven, N. M., D. S. M. Schor, E. A. Struys, E. E. W. Jansen, H. J. ten Brink, R. J. A. Wanders, and C. Jakobs. Analysis of pristanic acid beta-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry.
Diagnosis, Differential↗
The structure and organization of the human carnitine/acylcarnitine translocase (CACT1) gene2.
The carnitine/acylcarnitine translocase (CACT) transports acylcarnitines into mitochondria in exchange for free carnitine and it is, therefore, essential for the fatty acid beta-oxidation pathway. We have determined the exon-intron structure of the human CACT gene, which is responsible for a genetic disorder of fatty acid oxidation called CACT deficiency. The gene spans about 16.5 kb and consists of nine exons with the translation start site in exon 1. All the splice acceptor and donor sites conform to the AG/GT rules. All the introns except one are located at the level of the sequences coding for the extramembranous loops of CACT. We have designed a series of intronic oligonucleotide primers for amplifying each of the CACT exons together with their flanking intronic sequences, in segments well suited to detect mutations that would affect splicing of mRNA as well as the coding sequence itself.
Carnitine Acyltransferases↗
Plasmalogen phospholipids are involved in HDL-mediated cholesterol efflux: insights from investigations with plasmalogen-deficient cells.
Plasmalogens are ether-glycerophospholipids that exist in all mammalian cells, but their physiological function remains thus far an enigma. It has been previously suggested that the association of high-density lipoprotein (HDL) with cellular phospholipid is a pre-requisite for the process of HDL-mediated cholesterol efflux (HDL-MCE). To investigate our hypothesis that plasmalogens might play a role in HDL-MCE, we used a model composed of plasmalogen-deficient cells including RAW mutant macrophages and fibroblasts from patients with rhizomelic chondrodysplasia punctata type II. In mutant macrophages, HDL-MCE was reduced by 57% compared to control macrophages, after 16 hours. A similar phenomenon was observed in plasmalogen-deficient patients fibroblasts. Incubation of plasmalogen-deficient fibroblasts with 1-0-hexadecyl-sn-glycerol, which restored plasmalogen levels to that of control cells, resulted in a 35% increase in HDL-MCE, compared to a 10% increment in controls. The novel finding that HDL-MCE is reduced in plasmalogen-deficient cells and increases following plasmalogen restoration leads us to suggest that plasmalogen has an important function in the mediation of cellular cholesterol efflux.
Animals↗
Carnitine biosynthesis: identification of the cDNA encoding human gamma-butyrobetaine hydroxylase.
gamma-Butyrobetaine hydroxylase (EC 1.14.11.1) is the last enzyme in the biosynthetic pathway of L-carnitine and catalyzes the formation of L-carnitine from gamma-butyrobetaine, a reaction dependent on alpha-ketoglutarate, Fe2+, and oxygen. We report the purification of the protein from rat liver to apparent homogeneity, which allowed N-terminal sequencing using Edman degradation. The obtained amino acid sequence was used to screen the expressed sequence tag database and led to the identification of a human cDNA containing an open reading frame of 1161 base pairs encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44.7 kDa. Heterologous expression of the open reading frame in the yeast Saccharomyces cerevisiae confirmed that the cDNA encodes the human gamma-butyrobetaine hydroxylase. Northern blot analysis showed gamma-butyrobetaine hydroxylase expression in kidney (high), liver (moderate), and brain (very low), while no expression could be detected in the other investigated tissues.
Amino Acid Sequence↗
Mild trifunctional protein deficiency is associated with progressive neuropathy and myopathy and suggests a novel genotype-phenotype correlation.
Human mitochondrial trifunctional protein (TFP) is a heterooctamer of four alpha- and four beta-subunits that catalyzes three steps in the beta-oxidation spiral of long-chain fatty acids. TFP deficiency causes a Reye-like syndrome, cardiomyopathy, or sudden, unexpected death. We delineated the molecular basis for TFP deficiency in two patients with a unique phenotype characterized by chronic progressive polyneuropathy and myopathy without hepatic or cardiac involvement. Single-stranded conformation variance and nucleotide sequencing identified all patient mutations in exon 9 of the alpha-subunit. One patient is homozygous for the T845A mutation that substitutes aspartic acid for valine at residue 246. The second patient is a compound heterozygote for the T914A that substitutes asparagine for isoleucine at residue 269 and a C871T that creates a premature termination at residue 255. Allele-specific oligonucleotide hybridization studies revealed undetectable levels of the mRNA corresponding to the mutant allele carrying the termination codon. This study suggests a novel genotype-phenotype correlation in TFP deficiency; that is, mutations in exon 9 of the alpha-subunit, which encodes a linker domain between the NH2-terminal hydratase and the COOH-terminal 3-hydroxyacyl-CoA dehydrogenase, result in a unique neuromuscular phenotype.
Adolescent↗
Newly identified Chinese hamster ovary cell mutants are defective in biogenesis of peroxisomal membrane vesicles (Peroxisomal ghosts), representing a novel complementation group in mammals.
We isolated peroxisome biogenesis-defective mutants from Chinese hamster ovary cells by the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method. Seven cell mutants, ZP116, ZP119, ZP160, ZP161, ZP162, ZP164, and ZP165, of 11 P9OH/UV-resistant cell clones showed cytosolic localization of catalase, a peroxisomal matrix enzyme, apparently indicating a defect of peroxisome biogenesis. By transfection of PEX cDNAs and cell fusion analysis, mutants ZP119 and ZP165 were found to belong to a novel complementation group (CG), distinct from earlier mutants. CG analysis by cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome indicated that ZP119 and ZP165 were in the same CG as the most recently identified human CG-J. The peroxisomal matrix proteins examined, including PTS1 proteins as well as a PTS2 protein, 3-ketoacyl-CoA thiolase, were also found in the cytosol in ZP119 and ZP165. Furthermore, these mutants showed typical peroxisome assembly-defective phenotype such as severe loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. Most strikingly, peroxisomal reminiscent vesicular structures, so-called peroxisomal ghosts noted in all CGs of earlier Chinese hamster ovary cell mutants as well as in eight CGs of patients' fibroblasts, were not discernible in ZP119 and ZP165, despite normal synthesis of peroxisomal membrane proteins. Accordingly, ZP119 and ZP165 are the first cell mutants defective in import of both soluble and membrane proteins, representing the 14th peroxisome-deficient CG in mammals, including humans.
ATPases Associated with Diverse Cellular Activitie↗
Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry.
A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography-mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.
Deuterium↗
Molecular basis of hepatic carnitine palmitoyltransferase I deficiency.
Mitochondrial fatty acid beta-oxidation is important for energy production, which is stressed by the different defects found in this pathway. Most of the enzyme deficiencies causing these defects are well characterized at both the protein and genomic levels. One exception is carnitine palmitoyltransferase I (CPT I) deficiency, of which until now no mutations have been reported although the defect is enzymatically well characterized. CPT I is the key enzyme in the carnitine-dependent transport across the mitochondrial inner membrane and its deficiency results in a decreased rate of fatty acid beta-oxidation. Here we report the first delineation of the molecular basis of hepatic CPT I deficiency in a new case. cDNA analysis revealed that this patient was homozygous for a missense mutation (D454G). The effect of the identified mutation was investigated by heterologous expression in yeast. The expressed mutant CPT IA displayed only 2% of the activity of the expressed wild-type CPT IA, indicating that the D454G mutation is the disease-causing mutation. Furthermore, in patient's fibroblasts the CPT IA protein was markedly reduced on immunoblot, suggesting that the mutation renders the protein unstable.
Amino Acid Sequence↗
2,6-Dimethylheptanoyl-CoA is a specific substrate for long-chain acyl-CoA dehydrogenase (LCAD): evidence for a major role of LCAD in branched-chain fatty acid oxidation.
Oxidation of straight-chain fatty acids in mitochondria involves the complicated interaction between a large variety of different enzymes. So far four different mitochondrial straight-chain acyl-CoA dehydrogenases have been identified. The physiological function of three of the four acyl-CoA dehydrogenases has been resolved in recent years especially from studies on patients suffering from certain inborn errors of mitochondrial fatty acid beta-oxidation. The physiological role of long-chain acyl-CoA dehydrogenase (LCAD) has remained obscure, however. The results described in this paper provide strong evidence suggesting that LCAD plays a central role in branched-chain fatty acid metabolism since it turns out to be the major acyl-CoA dehydrogenase reacting with 2,6-dimethylheptanoyl-CoA, a metabolite of pristanic acid, which itself is the alpha-oxidation product of phytanic acid.
Acyl-CoA Dehydrogenase, Long-Chain↗
Differential deficiency of mevalonate kinase and phosphomevalonate kinase in patients with distinct defects in peroxisome biogenesis: evidence for a major role of peroxisomes in cholesterol biosynthesis.
Peroxisomes catalyze a number of essential metabolic functions especially related to lipid metabolism. There is increasing evidence suggesting that peroxisomes are also involved in the synthesis of isoprenoids via the mevalonate pathway at least in rat liver. In order to obtain independent evidence for a role of peroxisomes in isoprenoid synthesis in man, we have measured the activity of two key enzymes of the mevalonate pathway in patients suffering from certain defined defects in peroxisome biogenesis. We now report that mevalonate kinase is not only deficient in livers from Zellweger patients in which peroxisome biogenesis is defective, but also in livers from rhizomelic chondrodysplasia punctata (RCDP) Type 1 patients. In the latter group of patients there is a selective defect in peroxisome biogenesis due to a genetic defect in the PTS2-receptor, a mobile receptor-protein guiding peroxisomal proteins with a certain peroxisomal targeting signal (PTS2) to the peroxisome. Phosphomevalonate kinase was found to be strongly deficient in Zellweger patients thus suggesting that this enzyme is also peroxisomal. Taken together, our data indicate that in human liver mevalonate kinase and phosphomevalonate kinase are truly peroxisomal enzymes which strongly suggests that peroxisomes play a major role in cholesterol biosynthesis.
Amino Acid Sequence↗
Involvement of microsomal fatty aldehyde dehydrogenase in the alpha-oxidation of phytanic acid.
We investigated the role of microsomal fatty aldehyde dehydrogenase (FALDH) in the conversion of pristanal into pristanic acid. Cultured skin fibroblasts from controls and patients with Sjögren-Larsson syndrome (SLS) who are genetically deficient in FALDH activity were incubated with [2,3-(3)H]phytanic acid. The release of aqueous-soluble radioactivity by the SLS cells was decreased to 25% of normal, consistent with an intact formation of pristanal but a deficiency of further oxidation. SLS cells also accumulated four-fold more radioactivity in N-alkyl-phosphatidyl ethanolamine, which arises from incorporation of free aldehyde into phosphatidyl ethanolamine. Recombinant human FALDH expressed in Chinese hamster ovary cells readily oxidized pristanal and cultured fibroblasts from SLS patients showed a severe deficiency in FALDH activity (13% of normal) when pristanal was used as substrate. Nevertheless, SLS patients did not accumulate phytanic acid in their plasma. We conclude that FALDH is involved in the oxidation of pristanal to pristanic acid and that this reaction is deficient in patients with SLS.
Aldehyde Oxidoreductases↗
Carnitine palmitoyltransferase II specificity towards beta-oxidation intermediates--evidence for a reverse carnitine cycle in mitochondria.
Using isolated rat liver mitochondria, in the absence or presence of malonyl-CoA (an inhibitor of carnitine palmitoyltransferase I), we have found that carnitine palmitoyltransferase II (CPT II) is active with palmitoyl-CoA as well as with its beta-oxidation intermediates. A partially purified CPT II fraction from rat liver mitochondria was shown to be able to convert 3-hydroxypalmitoyl-CoA to 3-hydroxypalmitoylcarnitine, which could be identified by fast-atom-bombardment mass spectrometry. This apparent broad specificity of CPT II was further evaluated by kinetic studies using purified CPT II. It was found that CPT II readily accepts 3-oxopalmitoyl-CoA, palmitoyl-CoA, 3-hydroxypalmitoyl-CoA and 2,3-unsaturated palmitoyl-CoA as substrates with decreasing order of affinity. The apparent Vmax values found for the first three compounds were of the same order of magnitude; the 2,3-unsaturated acyl-CoA was the poorest substrate. Kinetic studies with purified CPT II showed 3-hydroxypalmitoyl-CoA to have the lowest K0.5 value (20 +/- 6 microM) of all the CoA esters studied; the highest K0.5 value (65 +/- 17 microM) was found for the 3-oxo intermediate. These findings support the hypothesis that CPT II is involved in the export of toxic long-chain acyl-CoA esters from the mitochondria by first converting them into the corresponding carnitine esters, followed by transport out of the mitochondria and subsequently out of the cell.
Animals↗
Alkyl-dihydroxyacetonephosphate synthase. Fate in peroxisome biogenesis disorders and identification of the point mutation underlying a single enzyme deficiency.
Peroxisomes play an indispensible role in ether lipid biosynthesis as evidenced by the deficiency of ether phospholipids in fibroblasts and tissues from patients suffering from a number of peroxisomal disorders. Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme playing a key role in the biosynthesis of ether phospholipids, contains the peroxisomal targeting signal type 2 in a N-terminal cleavable presequence. Using a polyclonal antiserum raised against alkyl-dihydroxyacetonephosphate synthase, levels of this enzyme were examined in fibroblast cell lines from patients affected by peroxisomal disorders. Strongly reduced levels were found in fibroblasts of Zellweger syndrome and rhizomelic chondrodysplasia punctata patients, indicating that the enzyme is not stable in the cytoplasm as a result of defective import into peroxisomes. In a neonatal adrenoleukodystrophy patient with an isolated import deficiency of proteins carrying the peroxisomal targeting signal type 1, the precursor form of alkyl-dihydroxyacetonephosphate synthase was detected at a level comparable to that of the mature form in control fibroblasts, in line with an intraperoxisomal localization. A patient with an isolated deficiency in alkyl-dihydroxyacetonephosphate (DHAP) synthase activity had normal levels of this protein. Analysis at the cDNA level revealed a missense mutation leading to a R419H substitution in the enzyme of this patient. Expression of a recombinant protein carrying this mutation in Escherichia coli yielded an inactive enzyme, whereas a comparable control recombinant enzyme was active, providing further proof that this substitution is responsible for the inactivity of the enzyme and the phenotype. In line with this result is the observation that wild-type alkyl-DHAP synthase activity can be inactivated by the arginine-modifying agent phenylglyoxal. The enzyme is efficiently protected against this inactivation when the substrate palmitoyl-DHAP is present at a saturating concentration. The gene encoding human alkyl-dihydroxyacetonephosphate synthase was mapped on chromosome 2q31.
Alkyl and Aryl Transferases↗
Pristanic acid beta-oxidation in peroxisomal disorders: studies in cultured human fibroblasts.
To investigate the individual steps of peroxisomal beta-oxidation, human fibroblasts from controls and patients affected by different peroxisomal disorders were incubated for 96 h with pristanic acid. Hereafter, 2,3-pristenic acid and 3-hydroxypristanic acid in the incubation medium were quantified by stable isotope dilution gas chromatography mass spectrometry (GC-MS). In control fibroblasts, both intermediates were formed and excreted into the medium in significant amounts. In cells from patients affected with different types of generalized peroxisomal disorders, the formation of both intermediates was absent or low, depending on the clinical severity of the disorder. In fibroblasts from patients affected with bifunctional protein deficiency, the concentrations of 2,3-pristenic acid and 3-hydroxypristanic acid in the medium were higher than in control cell lines.
Cell Line↗
Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function.
Gene targeting in mice was used to investigate the unknown function of Scp2, encoding sterol carrier protein-2 (SCP2; a peroxisomal lipid carrier) and sterol carrier protein-x (SCPx; a fusion protein between SCP2 and a peroxisomal thiolase). Complete deficiency of SCP2 and SCPx was associated with marked alterations in gene expression, peroxisome proliferation, hypolipidemia, impaired body weight control, and neuropathy. Along with these abnormalities, catabolism of methyl-branched fatty acyl CoAs was impaired. The defect became evident from up to 10-fold accumulation of the tetramethyl-branched fatty acid phytanic acid in Scp2(-/-) mice. Further characterization supported that the gene disruption led to inefficient import of phytanoyl-CoA into peroxisomes and to defective thiolytic cleavage of 3-ketopristanoyl-CoA. These results corresponded to high-affinity binding of phytanoyl-CoA to the recombinant rat SCP2 protein, as well as high 3-ketopristanoyl-CoA thiolase activity of the recombinant rat SCPx protein.
Acetyl-CoA C-Acetyltransferase↗
Characterization of phytanoyl-Coenzyme A hydroxylase in human liver and activity measurements in patients with peroxisomal disorders.
Phytanoyl-Coenzyme A hydroxylase is a newly recognized peroxisomal enzyme which catalyses the first step in the alpha-oxidation of phytanoyl-Coenzyme A. Since measurement of this enzyme activity in human liver homogenate is of great importance especially in relation to inherited diseases in which this enzyme activity is deficient, we have studied its characteristics in human liver. The results described in this paper show that optimal activity measurements require preformed phytanoyl-Coenzyme A plus 2-oxoglutarate, Fe2+ and ascorbate. The conditions developed can be used to determine phytanoyl-Coenzyme A hydroxylase activity in human liver homogenates which is of utmost importance not only for the diagnosis of patients, but also for the purification of the enzyme from various sources.
Enzyme Inhibitors↗
Peroxisomal D-hydroxyacyl-CoA dehydrogenase deficiency: resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency.
Peroxisomes play an essential role in a number of different metabolic pathways, including the beta-oxidation of a distinct set of fatty acids and fatty acid derivatives. The importance of the peroxisomal beta-oxidation system in humans is made apparent by the existence of a group of inherited diseases in which peroxisomal beta-oxidation is impaired. This includes X-linked adrenoleukodystrophy and other disorders with a defined defect. On the other hand, many patients have been described with a defect in peroxisomal beta-oxidation of unknown etiology. Resolution of the defects in these patients requires the elucidation of the enzymatic organization of the peroxisomal beta-oxidation system. Importantly, a new peroxisomal beta-oxidation enzyme was recently described called D-bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity primarily reacting with alpha-methyl fatty acids like pristanic acid and di- and trihydroxycholestanoic acid. In this patient we describe the first case of D-bifunctional protein deficiency as resolved by enzyme activity measurements and mutation analysis. The mutation found (Gly16Ser) is in the dehydrogenase coding part of the gene in an important loop of the Rossman fold forming the NAD+-binding site. The results show that the newly identified D-bifunctional protein plays an essential role in the peroxisomal beta-oxidation pathway that cannot be compensated for by the L-specific bifunctional protein.
17-Hydroxysteroid Dehydrogenases↗