Reinvestigation of peroxisomal 3-ketoacyl-CoA thiolase deficiency: identification of the true defect at the level of d-bifunctional protein.

In this report, we reinvestigate the only patient ever reported with a deficiency of peroxisomal 3-ketoacyl-CoA thiolase (THIO). At the time when they were described, the abnormalities in this patient, which included accumulation of very-long-chain fatty acids and the bile-acid intermediate trihydroxycholestanoic acid, were believed to be the logical consequence of a deficiency of the peroxisomal beta-oxidation enzyme THIO. In light of the current knowledge of the peroxisomal beta-oxidation system, however, the reported biochemical aberrations can no longer be explained by a deficiency of this thiolase. In this study, we show that the true defect in this patient is at the level of d-bifunctional protein (DBP). Immunoblot analysis revealed the absence of DBP in postmortem brain of the patient, whereas THIO was normally present. In addition, we found that the patient had a homozygous deletion of part of exon 3 and intron 3 of the DBP gene, resulting in skipping of exon 3 at the cDNA level. Our findings imply that the group of single-peroxisomal beta-oxidation-enzyme deficiencies is limited to straight-chain acyl-CoA oxidase, DBP, and alpha-methylacyl-CoA racemase deficiency and that there is no longer evidence for the existence of THIO deficiency as a distinct clinical entity.

Peroxisomal fatty acid alpha- and beta-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases.

Peroxisomes are subcellular organelles with an indispensable role in cellular metabolism. The importance of peroxisomes for humans is stressed by the existence of a group of genetic diseases in humans in which there is an impairment in one or more peroxisomal functions. Most of these functions have to do with lipid metabolism including the alpha- and beta-oxidation of fatty acids. Here we describe the current state of knowledge about peroxisomal fatty acid alpha- and beta-oxidation with particular emphasis on the following: (1) the substrates beta-oxidized in peroxisomes; (2) the enzymology of the alpha- and beta-oxidation systems; (3) the permeability properties of the peroxisomal membrane and the role of the different transporters therein; (4) the interaction with other subcellular compartments, including the mitochondria, which are the ultimate site of NADH re-oxidation and full degradation of acetyl-CoA to CO(2) and water; and (5) the different disorders of peroxisomal alpha- and beta-oxidation.

Molecular basis of D-bifunctional protein deficiency.

Peroxisomal disorders appear with a frequency of 1:5000 in newborns. They are caused either by peroxisomal assembly defects or by deficiencies of single peroxisomal enzymes. The phenotypes vary widely: affected humans may die very early in life within a few days to several months as a result of the impairment in essential peroxisomal functions as, for example, in Zellweger syndrome, or they may show only minor disabilities as is in acatalasemia. The deficiency of D-bifunctional protein, an enzyme involved in peroxisomal beta-oxidation of certain fatty acids and the synthesis of bile acids, causes a very severe, Zellweger-like phenotype. A number of different mutations in the gene coding for the enzyme were found in humans causing the total or partial loss of its enzymatic function. This paper gives a review of cases and their molecular basis.

Lipid metabolism in peroxisomes: enzymology, functions and dysfunctions of the fatty acid alpha- and beta-oxidation systems in humans.

Peroxisomes are subcellular organelles present in virtually all eukaryotic cells catalysing a number of indispensable functions in cellular metabolism. The importance of peroxisomes in man is stressed by the existence of an expanding group of genetic diseases in which there is an impairment in one or more peroxisomal functions. One of the major functions of peroxisomes concerns their role in lipid metabolism, which includes: (i) fatty acid betaoxidation; (ii) ether phospholipid synthesis; (iii) fatty acid alpha-oxidation; and (iv) isoprenoid biosynthesis. In this paper, we review the current state of knowledge concerning the peroxisomal fatty acid alpha- and beta-oxidation systems with particular emphasis on the enzymes involved and the various disorders of fatty acid oxidation in peroxisomes. We also pay attention to the fact that some of the metabolites that accumulate as the result of a defect in peroxisomal alpha- and/or beta-oxidation are activators of members of the family of nuclear receptors, including peroxisome-proliferator-activated receptor alpha.

Characterization of the HSD17B4 gene: D-specific multifunctional protein 2/17beta-hydroxysteroid dehydrogenase IV.

The HSD17B4 gene codes for a 80 kDa multifunctional enzyme containing three distinct functional domains and is localized in peroxisomes. The N-terminal part exhibits 3-hydroxyacyl-CoA dehydrogenase and 17beta-hydroxysteroid dehydrogenase activity whereas the central part shows enoyl-CoA hydratase activity. The carboxy-terminal part of the protein has sterol-carrier-protein activity. The protein is widely expressed, however in several tissues like brain, uterus and lung its expression is limited to specific cells like Purkinje cells or luminal epithelium. The HSD17B4 gene consist of 24 exons and 23 introns with classical intron-exon junctions spanning more than 100 kbp. The importance of the HSD17B4 protein is stressed by the identification of patients with severe clinical abnormalities due to mutations in the HSD17B4 gene. We have now checked the consequences of one frequent mutation, G16 S, which results in inactivation of the enzyme due to loss of interaction with NAD+.

Enoyl-CoA hydratase deficiency: identification of a new type of D-bifunctional protein deficiency.

D-bifunctional protein is involved in the peroxisomal beta-oxidation of very long chain fatty acids, branched chain fatty acids and bile acid intermediates. In line with the central role of D-bifunctional protein in the beta-oxidation of these three types of fatty acids, all patients with D-bifunctional protein deficiency so far reported in the literature show elevated levels of very long chain fatty acids, branched chain fatty acids and bile acid inter-mediates. In contrast, we now report two novel patients with D-bifunctional protein deficiency who both have normal levels of bile acid intermediates. Complementation analysis and D-bifunctional protein activity measurements revealed that both patients had an isolated defect in the enoyl-CoA hydratase domain of D-bifunctional protein. Subsequent mutation analysis showed that both patients are homozygous for a missense mutation (N457Y), which is located in the enoyl-CoA hydratase coding part of the D-bifunctional protein gene. Expression of the mutant protein in the yeast Saccharomyces cerevisiae confirmed that the N457Y mutation is the disease-causing mutation. Immunoblot analysis of patient fibroblast homogenates showed that the protein levels of full-length D-bifunctional protein were strongly reduced while the enoyl-CoA hydratase component produced after processing within the peroxisome was undetectable, which indicates that the mutation leads to an unstable protein.

Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis.

In the past few years, many patients have been described who have a defect of unknown origin in the peroxisomal beta-oxidation pathway. Complementation analysis has been done by various groups to establish the extent of the genetic heterogeneity among the patients. These studies were based on the use of two established cell lines, one with a deficiency of acyl-CoA oxidase and one with a deficiency of l-bifunctional protein (l-BP), and they showed that most patients belong to the l-BP-deficient group. However, molecular analysis of the cDNA encoding l-BP in patients failed to show any mutations. The recent identification of a new d-specific bifunctional protein (d-BP) prompted us to reinvestigate the original patient with presumed l-BP deficiency. In a collaborative effort, we have now found that the true defect in this patient is at the level of the d-BP and not at the level of the l-BP. Our results suggest that most, if not all, patients whose condition has been diagnosed as l-BP are, in fact, d-BP deficient. We tested this hypothesis in nine patients whose condition was diagnosed as l-BP deficiency on the basis of complementation analysis and found clear-cut mutations in the d-BP cDNA from all patients.

D-hydroxyacyl-CoA dehydrogenase deficiency. Identification of a new peroxisomal disorder with implications for other disorders of beta-oxidation.

The second and third steps of peroxisomal beta-oxidation are catalysed by two multifunctional enzymes: D-bifunctional protein and L-bifunctional protein. Here we show that fibroblasts of a patient described as being deficient in the 3-hydroxyacyl-CoA dehydrogenase component of D-bifunctional protein and fibroblasts of a patient described as being deficient in L-bifunctional protein do not complement one another. Using a newly developed method to measure the activity of D-bifunctional protein in fibroblast homogenates, we found that the activity of the D-bifunctional protein was completely deficient in the patient with presumed L-bifunctional protein deficiency.

Sensitive analysis of serum 3alpha, 7alpha, 12alpha,24-tetrahydroxy- 5beta-cholestan-26-oic acid diastereomers using gas chromatography-mass spectrometry and its application in peroxisomal D-bifunctional protein deficiency.

The final steps in bile acid biosynthesis take place in peroxisomes and involve oxidative cleavage of the side chain of C27-5beta-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenodeoxycholic acid. The enoyl-CoA hydratase and beta-hydroxy acyl-CoA dehydrogenase reactions involved in the chain shortening of C27-5beta-cholestanoic acids are catalyzed by the recently identified peroxisomal d-bifunctional protein. Deficiencies of d-bifunctional protein lead, among others, to an accumulation of 3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholest-26-oic acid (varanic acid). The ability to resolve the four C24, C25 diastereomers of varanic acid has, so far, only been carried out on biliary bile acids using p -bromophenacyl derivatives. Here, we describe a sensitive gas chromatography-mass spectrometry (GC/MS) method that enables good separation of the four varanic acid diastereomers by use of 2R-butylester-trimethylsilylether derivatives. This method showed the specific accumulation of (24R,25R)-varanic acid in the serum of a patient with isolated deficiency of the d-3-hydroxy acyl-CoA dehydrogenase part of peroxisomal d-bifunctional protein, whereas this diastereomer was absent in a serum sample from a patient suffering from complete d-bifunctional protein deficiency. In samples from both patients an accumulation of (24S,25S)-varanic acid was observed, most likely due to the action of l-bifunctional protein on Delta24E-THCA-CoA. This GC/MS method is applicable to serum samples, obviating the use of bile fluid, and is a helpful tool in the subclassification of patients with peroxisomal d-bifunctional protein deficiency.

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.

Complementation analysis of fibroblasts from peroxisomal fatty acid oxidation deficient patients shows high frequency of bifunctional enzyme deficiency plus intragenic complementation: unequivocal evidence for differential defects in the same enzyme protein.

In the last few years many patients have been reported with a defect in peroxisomal fatty acid beta-oxidation of unknown origin. Using a combined approach based on direct activity measurements of straight-chain acyl-CoA oxidase and complementation analysis after somatic cell fusion of fibroblasts, we have now classified 13 patients into 4 distinct groups representing different gene defects. Remarkably, we found intragenic complementation in group 2 so that group 2 is in fact made up of 3 distinct subgroups. The underlying basis for this peculiar phenomenon probably has to do with the fact that bifunctional protein harbors two catalytic activities including enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. In group 2A enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase are defective whereas in group 2B and 2C either the hydratase or 3-hydroxyacyl-CoA dehydrogenase component of the bifunctional protein is deficient.