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Peroxisomes, lipid metabolism, and peroxisomal disorders
Molecular Genetics and Metabolism 83 (2004) 16–27 www.elsevier.com/locate/ymgme
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Peroxisomes, lipid metabolism, and peroxisomal disorders R.J.A. Wanders¤ Laboratory for Genetic Metabolic Diseases, Department of Clinical Chemistry and Pediatrics, Academic Medical Center, University of Amsterdam, Emma Children’s Hospital, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Received 9 July 2004; accepted 30 August 2004
Abstract Peroxisomes catalyse a large variety of diVerent cellular functions of which most have to do with lipid metabolism. This paper deals with the role of peroxisomes in three key pathways of lipid metabolism, including: (1) etherphospholipid biosynthesis, (2) fatty acid -oxidation, and (3) fatty acid -oxidation. Apart from a brief description of the peroxisomal enzymes involved in each of these pathways, the interaction between peroxisomes and other subcellular organelles, notably microsomes and peroxisomes, will be discussed. Finally, the current state of knowledge with respect to the diVerent disorders of peroxisomal lipid metabolism will be described. 2004 Elsevier Inc. All rights reserved. Keywords: Etherphospholipid biosynthesis; Fatty acid -oxidation; Fatty acid -oxidation; Peroxisome biogenesis; Zellweger syndrome; Refsum disease
Introduction Peroxisomes play an essential role in human physiology as concluded from the devastating consequences of the lack of peroxisomes in patients with Zellweger syndrome. The peroxisomal disorders are usually divided into two groups, including: (1) the disorders of peroxisome biogenesis (PBDs) and (2) the single peroxisomal enzyme deWciencies. Zellweger syndrome is the prototype of the group of peroxisomal disorders and patients suVering from this syndrome display a range of abnormalities, including neurological, skeletal, hepatological, and ocular abnormalities. In Zellweger syndrome as well as in neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), which together constitute the Zellweger spectrum, peroxisome function is disrupted due to mutations in one of the many genes whose proper expression is required for peroxisome biogenesis. *
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1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2004.08.016
The genes involved in peroxisome biogenesis are called PEX genes and the proteins encoded by these genes are named peroxins [1]. At present, mutations in 14 diVerent PEX genes have been described to be associated with Zellweger spectrum disorders [2]. Studies on Zellweger syndrome and the other Zellweger spectrum disorders have contributed greatly to the elucidation of the essential role of peroxisomes in human physiology. Indeed, it was the crucial Wnding of Moser and co-workers [3] in 1982, which led to the recognition of the important role of peroxisomes in fatty acid -oxidation. Similarly, the discovery that plasmalogens were deWcient in Zellweger patients by Heymans et al. in 1983 [4], provided unequivocal evidence for the essential role of peroxisomes in etherphospholipid biosynthesis. Furthermore, the demonstration of markedly elevated phytanic acid levels in plasma from Zellweger patients pointed to the essential role of peroxisomes in fatty acid -oxidation. In this paper, we will describe the current state of knowledge about the metabolic role of peroxisomes in
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humans with particular attention to the role of peroxisomes in lipid metabolism. We will also brieXy describe the inborn errors of peroxisomal lipid metabolism.
Etherphospholipid biosynthesis Etherphospholipids constitute a special class of phospholipids characterised by an ether bond at the sn-1 position of the glycerol backbone rather than an ester bond as in diacylglycerophospholipids (Fig. 1). Two types of ether bonds occur in etherphospholipids: (1) an ether bond and (2) a vinyl-ether bond. The former is found in platelet activating factor (PAF) whereas the vinyl-ether bond is found in a class of etherphospholipids called plasmalogens. A schematic representation of the diVerent types of glycerophospholipids is shown in Fig. 1. In plasmalogens, the aliphatic moieties at the sn-1 position consist of C16:0 (palmitic acid), C18:0 (stearic acid), or C18:1 (oleic acid), whereas the sn-2 position is occupied by polyunsaturated fatty acids (PUFAs). The head group in etherphospholipids is usually of the ethanolamine or choline type. Brain myelin possesses the
Fig. 1. Structure of glycerophospholipids. Plasmalogens contain a vinyl ether bond at the sn-1 position of the glycerol backbone, compared to the ether bond present in plasmanyl-phospholipids (e.g., platelet activating factor) and the ester bond in diacylphospholipids. R1 and R2 represent long-chain fatty acids esteriWed at the sn-1 and sn-2 position, respectively. The head groups represent ethanolamine or choline in plasmalogens, choline in platelet activating factor, and ethanolamine, choline or serine in diacyl-phospholipids.
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highest content of ethanolamine plasmalogens (PE-plasmalogens) whereas heart muscle has a higher content of choline plasmalogens (PC plasmalogens). Moderate amounts of plasmalogens are found in kidney, skeletal muscle, spleen, and blood cells, whereas liver is known for its low plasmalogen content. Although the function of plasmalogens still remains obscure, the pathway of plasmalogen biosynthesis has been worked out in detail (Fig. 2). The Wrst step in the biosynthesis of plasmalogens involves the esteriWcation of dihydroxyacetonephosphate (DHAP) with a long-chain acyl-CoA ester and is carried out by dihydroxyacetonephosphate acyltransferase (DHAPAT). The characteristic etherbond at the sn-1 position of etherphospholipids is introduced by the replacement of the sn-1 fatty acid with a long-chain fatty alcohol. This reaction is catalysed by alkyldihydroxyacetonephosphate synthase (ADHAPS) with alkylDHAP as product. The fatty alcohol may be derived from dietary sources or by the reduction of longchain acyl-CoAs through the action of an acyl-CoA reductase (Fig. 3). The alkylDHAP produced in the alkylDHAP synthase reaction is subsequently converted into 1-alkylglycerol-3-phosphate (1-alkyl-G3P) as catalysed by the enzyme alkyl/acyldihydroxyacetonephosphate reductase (AADHAPR). Subsequently, 1-alkylG3P is converted into 1-alkyl-2-acyl-G3P by an alkyl/ acyl-glycerol-3-phosphate acyltransferase (AAG3P-AT). The phosphate group is subsequently removed by a
Fig. 2. Schematic representation of the steps involved in the biosynthesis of PC and PE plasmalogens. See text for further details.
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Fig. 3. Topology of the peroxisomal enzymes involved in the biosynthesis of plasmalogens. Symbols used: (1) DHAP-AT; (2) ADHAP-S; (3) acyl-CoA reductase (long-chain alcohol-forming); (4) glycerol-3phosphate dehydrogenase; (5) alkyl/acyl-DHAP reductase (AADHAP-R); (6) peroxisomal membrane protein of 34 kDa (PMP34); (7) very-long-chain acyl-CoA synthetase (VLACS). Adapted from Brites et.al. [9].
phosphohydrolase (PH) yielding 1-alkyl-2-acyl-sn-glycerol. Subsequently, cytidine diphosphate ethanolamine (CDP ethanolamine) is incorporated via the action of ethanolamine phosphotransferase to form 1-alkyl-2acyl-sn-glycerol-3-phosphoethanolamine (1-alkyl-2-acylGPE). Desaturation to 1-alk-11-enyl-2-acyl-GPE is carried out by a microsomal enzyme called delta-1-alkyldesaturase. 1-Alk-11-enyl-2-acyl-GPC (PC plasmalogens) is primarily formed from 1-alk-11-enyl-2-acyl-GPE via sn-2 and polar head group modiWcations (see Fig. 2). Dihydroxyacetonephosphate acyltransferase (DHAPAT) and alkyl-dihydroxyacetonephosphate synthase (ADHAP-S) are exclusively localised in peroxisomes [5]. Both enzymes have been puriWed from diVerent sources and the corresponding cDNAs and genes have been identiWed in diVerent species including Homo sapiens [6– 8]. It turns out that DHAPAT is a bona Wde PTS1 protein. Human DHAPAT for instance has the C-terminal tripeptide alanine/lysine/leucine [6,7]. On the other hand, human alkylDHAP synthase is a PTS2 protein [8]. Fig. 3 shows a schematic picture of the topology of the diVerent enzymes involved in the biosynthesis of plasmalogens and the role of peroxisomes and the endoplasmic reticulum therein (see Brites et al. [9] for review). Disorders of etherphospholipid biosynthesis Two single enzyme deWciencies in the etherphospholipid biosynthetic pathway have been identiWed so far.
These two include: (1) DHAPAT deWciency and (2) alkyl-DHAP synthase deWciency. DHAPAT deWciency was Wrst described in 1992 in a patient showing all the clinical signs and symptoms of rhizomelic chondrodysplasia punctata (RCDP) [10]. In its classical presentation RCDP is clinically characterised by a disproportionally short stature primarily aVecting the proximal parts of the extremities, typical facial appearance, congenital contractures, characteristic ocular involvement, dwarWsm, spasticity, and severe mental retardation. Radiologic studies show shortening, metaphyseal cupping, and disturbed ossiWcation of humeri and/or femora, together with epiphyseal and extra-epiphyseal calciWcations. Analysis of erythrocyte plasmalogens in this patient revealed a full deWciency. Subsequent studies in cultured skin Wbroblasts revealed an isolated deWciency of dihydroxyacetonephosphate acyltransferase, the Wrst enzyme in the etherphospholipid biosynthetic pathway [10]. In 1994 the Wrst case of isolated alkyl-DHAP synthase deWciency was described, again in a patient showing all the clinical signs and symptoms of RCDP [11]. In this patient erythrocyte plasmalogens were also completely deWcient and subsequent studies in Wbroblasts revealed the complete deWciency of alkyl-DHAP synthase with normal values for DHAPAT and other peroxisomal parameters. DHAPAT deWciency and alkylDHAP synthase deWciency have been described in very few patients only (see [12] for review). The molecular basis of both enzyme deWciencies has been resolved in recent years [7,13]. It should be noted that the majority of RCDP patients do not carry mutations in either the GNPAT or ADHAPS gene coding for DHAPAT and alkyl-DHAP synthase, respectively. In fact, most RCDP patients carry mutations in the PEX7 gene [14,15]. This gene codes for the so-called PTS2-receptor, which plays a crucial role in the correct targeting of a group of proteins to the peroxisome. Peroxisomal proteins are equipped with one of two diVerent peroxisomal targeting signals (PTS), called PTS1 and PTS2, which are recognised in the cytosol by either the PTS1- or PTS2- receptors, respectively, and then escorted to the peroxisomal membrane, followed by transport of the PTS1- and PTS2-proteins across the peroxisomal membrane and recycling of the receptors back into the cytosol [1]. In case of mutations in the PEX7 gene the PTS2 receptor will be dysfunctional, so that the correct targeting of peroxisomal proteins equipped with a PTS2 signal does not occur. AlkylDHAP synthase [8] and phytanoyl-CoA hydroxylase [16,17], the Wrst enzyme in the -oxidation of phytanic acid (see later) are PTS2 proteins, which explains their deWciency in RCDP patients with mutations in the PEX7 gene. Furthermore, the deWciency of alkyl-DHAP synthase explains the impairment in etherphospholipid biosynthesis as reXected in the deWciency of plasmalogens in erythrocytes and tissues.
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In addition to the deWciencies of alkyl-DHAP synthase and phytanoyl-CoA hydroxylase there are two other abnormalities in this form of RCDP, which include the partial deWciency of DHAPAT and the aberrant localization of peroxisomal thiolase in the cytosol in its abnormal precursor form (44 kDa rather than 41 kDa) [18]. The abnormality in peroxisomal thiolase is readily explained from the fact that this is a PTS2 protein too, whereas the partial deWciency of DHAPAT is due to the fact that alkyl-DHAP synthase and DHAPAT form a complex within the peroxisome interior [19]. DHAPAT is unstable in the absence of the alkyl-DHAP synthase protein thus explaining the partial deWciency of DHAPAT [20]. In our experience >90% of RCDP patients have mutations in the PEX7 gene [17]. We have been involved in the diagnosis of >120 patients with RCDP. If a patient presents with clinical signs and symptoms of RCDP, erythrocyte plasmalogen levels should be determined. Measurement of erythrocyte plasmalogens has proven to be extreme reliable because all patients studied by us to-date have shown deWcient plasmalogens in erythrocytes, independent of the type of RCDP. DeWnitive diagnosis to resolve the true underlying genetic defect in each patient requires detailed enzymatic studies in Wbroblasts, followed by molecular analysis of the genes coding for Pex7p, the PTS2 receptor (PEX7), DHAPAT (GNPAT), or alkylDHAP synthase (AGPS), respectively (see Wanders et al. [12] for details). Peroxisomal fatty acid -oxidation Although fatty acids can undergo oxidation via diVerent mechanisms, most fatty acids are degraded by means of -oxidation. Peroxisomes contain a fatty acid -oxidation machinery just like mitochondria. The mechanism of -oxidation in mitochondria and peroxisomes is identical and involves a set of four consecutive reactions: (1) dehydrogenation; (2) hydration (of the double bond); (3) dehydrogenation again; and (4) thiolytic cleavage. Through this 4-step pathway a 2-carbon unit is split from each fatty acid in the form of an acetyl-CoA unit, which can then be degraded in the citric acid (Krebs) cycle to produce CO2 and H2O. The peroxisomal and mitochondrial -oxidation systems have diVerent functions as they catalyse the -oxidation of diVerent fatty acids and fatty acid derivatives. Indeed, mitochondria catalyse the -oxidation of the bulk of fatty acids derived from the diet, including palmitate, oleate, linoleate, and linolenate, whereas peroxisomes play an important role by oxidising a diVerent set of fatty acids and fatty acids derivatives. These include: 1. very-long-chain fatty acids (VLCFA), notably hexacosanoic acid (C26:0),
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2. pristanic acid (2,6,10,14-tetramethylpentadecanoic acid), and 3. di- and trihydroxycholestanoic acid (DHCA and THCA). Very-long-chain fatty acids are derived from both dietary sources, but are also synthesised endogenously from shorter chain fatty acids, whereas pristanic acid cannot be synthesised endogenously, but is only derived from dietary sources, either directly or indirectly from phytanic acid, which is -oxidised to pristanic acid within peroxisomes. Finally, di- and trihydroxycholestanoic acid are intermediates in the formation of the primary bile acids, cholic acid and chenodeoxycholic acid, and are formed from cholesterol in the liver. Although generally marked as a biosynthetic reaction, biosynthesis of polyunsaturated fatty acids such as docosahexaenoic acid (C22:6-3) is actually catalysed by -oxidation in peroxisomes. Indeed, recent studies have established that the formation of C22:6-3 from linolenic acid (C18:3-3) involves the active participation of the peroxisomal fatty acid oxidation system [21,22]. It should be noted that the peroxisomal fatty acid oxidation system is only able to chain-shorten fatty acids and is not able to degrade fatty acids to completion. This is most convincingly demonstrated for pristanic acid, which undergoes three cycles of -oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA, which is then shuttled to the mitochondria in the form of its carnitine ester for full oxidation to CO2 and H2O. The same is probably true for very-long-chain fatty acids, which are chain-shortened to a medium-chain acyl-CoA ester, followed by transport to the mitochondria as carnitine ester to allow complete oxidation to CO2 and H2O again. Finally, with respect to chenodeoxycholoyl-CoA and choloyl-CoA as formed in peroxisomes, these are converted into the corresponding taurine or glycine conjugates within peroxisomes followed by export from peroxisomes to end up in bile. Fig. 4 depicts the enzymology of the peroxisomal fatty acid -oxidation system. Human peroxisomes contain two acyl-CoA oxidases, one speciWc for straightchain fatty acids like C26:0, and a second one catalysing the dehydrogenation of 2-methyl branched-chain fatty acids like pristanoyl-CoA and di- and trihydroxycholestanoyl-CoA. The enoyl-CoA esters of C26:0, pristanic acid, DHCA, and THCA are all handled by a single bifunctional enzyme, catalysing the second (hydration) and third (dehydrogenation) steps of peroxisomal -oxidation. This enzyme, with both enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, forms and dehydrogenates D-3-hydroxyacyl-CoA esters rather than L-3-hydroxyacyl-CoAs and is the single enzyme involved in the oxidation of C26:0, pristanic acid, as well as DHCA and THCA. DiVerent names have been given
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Fig. 4. Enzymology of the peroxisomal fatty acid -oxidation system. Human peroxisomes contain two acyl-CoA oxidases, one speciWc for straightchain fatty acids like C26:0, and a second one, catalysing the dehydrogenation of 2-methyl branched-chain fatty acids like pristanoyl-CoA and diand trihydroxycholestanoyl-CoA. The latter oxidase only accepts 2-methyl branched-chain fatty acyl-CoAs with the 2-methyl group in the (2S)-conWguration. The enoyl-CoA esters of C26:0, (2S)-pristanic acid and (25S)-DHCA and THCA are all handled by a single bifunctional enzyme harbouring both enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity. Finally, peroxisomes contain two thiolases of which one (pTH2/ SCPx) is involved in the oxidation of 2-methyl branched-chain fatty acids. Both pTH1 and pTH2/SCPx react with the 3-ketoacyl-CoA esters of straight-chain fatty acids which explains the involvement of both thiolases in C26:0 -oxidation.
to this enzyme including D-bifunctional protein (DBP), D-peroxisomal bifunctional enzyme (D-PBE), multifunctional enzyme II (MFE-II), and multifunctional protein 2 (MFP-2). Peroxisomes also contain a diVerent bifunctional protein, which forms and dehydrogenates L-3hydroxyacyl-CoA esters, but the function of this enzyme remains to be resolved in the future. Finally, human peroxisomes contain two peroxisomal thiolases, i.e., pTH1 and pTH2. Human pTH1 is
the human equivalent of the cloWbrate-inducible thiolase identiWed by Hashimoto and co-workers, whereas PTH2 is identical to the 58 kDa sterol carrier protein x (SCPx) with both thiolase activity as well as a sterol carrier protein domain. It is clear now that pTH2 (SCPx) plays an indispensable role in the oxidation of 2-methyl branched-chain fatty acids, whereas pTH1 and pTH2 are probably both involved in C26:0 -oxidation.
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The enzymes described above are necessary and suYcient for the -oxidation of straight-chain fatty acids like C26:0 as well as 2-methyl branched-chain fatty acids with the methyl group in the (2S)-conWguration. However, auxiliary enzymes are needed for the -oxidation of (2R)-methyl branched-chain FAs and unsaturated FAs. Oxidation of (2R)-methyl branched-chain FAs requires the active participation of an enzyme, capable of converting (2R)- into (2S)-branched-chain acyl-CoAs. Studies, notably by Schmitz et al. [23–25] have shown that peroxisomes contain 2-methylacyl-CoA racemase (AMACR) activity, which enzyme is able to convert (2R)-methylacyl-CoAs into the corresponding (2S)methyl-acyl-CoAs and vice versa. The importance of this enzyme for oxidation of 2-methyl branched-chain fatty acids is stressed by our Wndings in AMACR-deWcient patients in which (2R)-pristanic acid accumulates as well as di- and trihydroxycholestanoic acid [26–28]. The accumulation of the latter two species is explained by the fact that the 2-methyl group in DHCA and THCA, as produced from cholesterol, has the (R)-conWguration (see Fig. 4). Human peroxisomes also contain -3,-2-enoylCoA isomerase activity as well as 2,4-dienoylCoA reductase, and -3,5,-2,4-dienoylCoA isomerase activity, thereby allowing the oxidation of unsaturated fatty acids with double bonds at even or uneven positions [12]. Interaction between peroxisomes and mitochondria Since peroxisomes lack a respiratory chain and a citric acid (Krebs) cycle the products of -oxidation of fatty acids in peroxisomes have to be shuttled to mitochondria. With respect to the end products of peroxisomal -oxidation, such as acetyl-CoA, propionyl-CoA, and medium-chain acyl-CoA esters, it has been demonstrated that these acyl-CoAs are transported to the mitochondrion in the form of the corresponding carnitine esters. For this purpose peroxisomes are equipped with diVerent carnitine acyltransferases including carnitine acetyltransferase (CAT) and carnitine octanoyltransferase (COT), which allow formation of carnitine esters inside peroxisomes, followed by export across the peroxisomal membrane via an unidentiWed carrier system. Uptake of carnitine esters into mitochondria occurs via the mitochondrial carnitine/acylcarnitine transporter (CACT), followed by retroconversion of the acylcarnitines into the corresponding acyl-CoA esters via diVerent acyltransferases, present in mitochondria. An alternative mechanism by which the end-products of peroxisomal -oxidation may be transferred to mitochondria, is by cleavage of the acyl-CoA esters into the free acids plus CoASH via one of the many thioesterases identiWed in peroxisomes (see [3] for review) followed by transport of the free fatty acids to mitochondria [29].
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One aspect of peroxisomal -oxidation, which remains to be resolved deWnitively, is the way in which the NADH produced in the third step of -oxidation is reoxidised to NAD. Our studies in the yeast Saccharomyces cerevisiae [30] have shown the involvement of a NAD(H) redox shuttle in which the NADH produced is reoxidised to NAD by means of peroxisomal malate dehydrogenase (MDH), followed by the export of malate to the cytosol where the reverse occurs. This malate oxaloacetate shuttle resembles the mitochondrial glutamate/malate shuttle. The fact that mammalian peroxisomes do not contain peroxisomal malate dehydrogenase activity is evidence against the existence of a similar type of NAD(H) redox shuttle in mammals. According to Baumgart et al. [31] mammalian peroxisomes contain a diVerent type of NADH shuttle with lactate dehydrogenase playing the role of malate dehydrogenase. Transport of fatty acids and fatty acid derivatives across the peroxisomal membrane One of the truly unresolved issues of peroxisome metabolism is the transport of FAs across the peroxisomal membrane. In mitochondria FAs are carried across the mitochondrial innermembrane via the carnitine cycle in which FAs are Wrst activated to the corresponding coenzyme-A esters followed by substitution of the coenzyme-A unit by carnitine, as catalysed by the enzyme carnitine palmitoyltransferase 1 (CPT1). The acylcarnitines then enter the mitochondrion via a speciWc transporter, the carnitine/acylcarnitine transporter (CACT), which exchanges extramitochondrial acylcarnitine for intramitochondrial carnitine. Finally, the carnitine moiety in the acylcarnitine species is exchange for coenzyme-A to produce the acyl-CoA back again, a reaction mediated by carnitine palmitoyltransferase 2 (CPT2). Such a carnitine cycle is present in peroxisomes, but seems to work only in the export of acyl-CoA species from the interior of the peroxisome to the cytosol and subsequently to the mitochondrial space. How then are FAs transported across the peroxisomal membrane? The discovery that X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene coding for a protein (ALDP) which belongs to the family of ABCtransporters, led to the hypothesis that ALDP may actually catalyse the transport of FAs across the peroxisomal membrane. ALDP is a so-called half-ABC-transporter with six transmembrane spanning regions. Interestingly, human peroxisomes contain three additional half-ABCtransporters including ALDR, PMP70, and PMP69. Since these are all half-ABC-transporters, the most likely scenario is that these half-transporters form homo- and/ or heterodimers within the peroxisomal membrane. The yeast S. cerevisiae contains two peroxisomal halfABC-transporters, called Pxa1p and Pxa2p. Mutants in which either the PXA1 or PXA2 gene has been
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disrupted, show a similar phenotype, characterised by lack of growth on oleate-containing medium as caused by the reduced ability to oxidize oleic acid. Oxidation of a medium-chain fatty acid like octanoate (C8:0) is fully normal in these mutants, suggesting the existence of two routes of fatty acid import into peroxisomes. Based on experiments in which the peroxisomal acyl-CoA synthetase (Faa2p) was redirected to the cytosol by removing the PTS1 targeting signal, Hettema et al. [32] concluded that the Pxa1p/Pxa2p dimer catalyses the transport of acyl-CoA esters across the peroxisomal membrane. DeWnitive proof of this postulate awaits reconstitution of the Pxa1p/Pxa2p dimer in artiWcial liposomes. Such a system would also be required to test whether ALDP either as homo- or heterodimer transports the CoA esters of very-long-chain fatty acids or not. It should be noted that others favour a diVerent role for ALDP. Indeed, McGuinness et al. [33] have proposed that ALDP is involved in shuttling the end products of peroxisomal -oxidation to the mitochondria. Disorders of peroxisomal -oxidation X-linked adrenoleukodystrophy (X-ALD) is the most common single peroxisomal disorder with a minimum incidence of 1:21,000 males [34] in the USA to 1:15,000 males in France (Dr. P. Aubourg, Paris, France, personal communication, cited in Kemp et al. [35]). The phenotypic expression of X-ALD varies wildly. At present, at least six phenotypic variants of X-ALD can be distinguished [36]. This classiWcation is somewhat arbitrary and is based on the age of onset and the organs principally involved. The two most frequent phenotypes, together accounting for >80% of all cases, are childhood cerebral ALD (CCALD) and adrenomyeloneuropathy (AMN). Onset of CCALD is between 3 and 10 years of age with progressive behavioural, cognitive, and neurologic deterioration, often leading to total disability within 3 years. The cerebral phenotype is not only observed in childhood but may also present later in life in adolescence (adolescent cerebral ALD) or even adulthood (adult cerebral ALD). There is a marked diVerence between the cerebral phenotypes and AMN since cerebral phenotypes show an inXammatory reaction in the cerebral white matter, which resembles, but can be distinguished from, what is observed in multiple sclerosis. In contrast to CCALD, the inXammatory response is absent or mild in AMN, which has a much later age of onset (28 § 9 years) and a much slower rate of progression. Nevertheless, approximately 40% of AMN patients do develop some cerebral involvement associated with a more rapid downhill progression. It is important to emphasize that women, heterozygous for ALD, may develop AMN-like symptoms in middle age or later. Current information holds that this is more frequent than previously anticipated. Cerebral involvement and
adrenocortical insuYciency are rare, however, in heterozygous women. The biochemical hallmark of X-ALD is the accumulation of VLCFAs, notably C24:0 and C26:0, in plasma and tissues. Analysis of VLCFAs in plasma has turned out to be an extremely reliable diagnostic method with few, if any, false negatives, at least in hemizygotes. If an abnormal VLCFA proWle is found in a particular patient, molecular analysis of the ABCD1 gene, which codes for the ALD protein, should be done. At present, >650 diVerent mutations in the ABCD1 gene have been identiWed (see www.X-ALD.nl). Interestingly, in the majority of X-linked ALD patients, the ALD protein is markedly deWcient as concluded from immunoXuorescence microscopy analysis [37,38]. The accumulation of VLCFAs in X-linked ALD patients is the result of the impaired oxidation of these fatty acids in peroxisomes. As discussed above, it has to be established what the association is between mutations in the ABCD1 gene, the consequent loss of function of ALDP and the impaired oxidation of VLCFAs in peroxisomes. Acyl-CoA oxidase deWciency was Wrst described in 1988 by Poll-The et al. [39] in two siblings with neonatal onset hypotonia, delayed motor development, sensory deafness, and retinopathy, with extinguished electroretinograms, but no craniofacial dysmorphia. Since this initial description only few patients have been described since then (see [12] for review). In patients with acyl-CoA oxidase deWciency plasma very-long-chain fatty acids are markedly elevated whereas other parameters of the peroxisomal -oxidation system, including pristanic acid and di- and trihydroxycholestanoic acid are completely normal. The acyl-CoA oxidase gene has been identiWed and the molecular basis of acyl-CoA oxidase deWciency has been resolved in a few cases (see [12] for review). Much more frequent among the disorders of peroxisomal -oxidation is D-bifunctional protein deWciency. The clinical presentation of D-bifunctional protein deWcient patients resembles that of the peroxisome biogenesis disorders (PBDs) in many respects. Indeed, virtually all patients with D-bifunctional protein deWciency identiWed so far (>60) show severe clinical abnormalities including hypotonia, craniofacial dysmorphia, neonatal seizures, hepatomegaly, and developmental delay. Most patients with D-BP deWciency die in the Wrst year of life. A remarkable observation is that patients with D-BP deWciency often show disordered neuronal migration. In retrospect the Wrst case of D-bifunctional protein deWciency was described by Watkins et al. [40] in 1989 in a male patient with severe hypotonia, and neonatal seizures. An electroencephalogram revealed multifocal spikes. No developmental progress was observed. Interestingly, there was no dysmorphia in this patient and no hepatomegaly. Fontanelles were large with open metopic sutures. Visual evoked responses and brainstem auditory
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evoked responses were grossly abnormal. A brain biopsy at 6 weeks revealed polymicrogyria. The patient died at 5 months of age after a clinical course marked by absent developmental progress and therapy refractory seizures. The true defect in this patient at the level of D-bifunctional protein was only resolved in 1999 by van Grunsven et al. [41]. In this patient D-bifunctional protein was completely missing as assessed by immunoblot analysis, which explains the combined loss of both the enoyl-CoA hydratase as well as 3-hydroxyacyl-CoA dehydrogenase activity of D-BP. In addition to complete D-bifunctional protein deWciency also referred to as D-BP deWciency type 1, there are two additional forms of D-bifunctional protein deWciency in which either the enoyl-CoA hydratase (see van Grunsven et al. [42]) or the 3-hydroxyacylCoA dehydrogenase (van Grunsven et al. [43]) component of D-bifunctional protein is functionally inactive, respectively. For more detailed information the reader is referred to Wanders et al. [12]. 2-Methyl-acyl-CoA racemase (AMACR) deWciency is the fourth and last disorder of peroxisomal -oxidation. Although only a few patients with this defect have been described in literature so far, the clinical picture of AMACR deWciency is usually that of a slowly progressive adult-onset sensory motor neuropathy. Indeed, two of the three initial patients described by Ferdinandusse et al. [26] suVered from adult-onset sensory motor neuropathy. One patient also had pigmentary retinopathy, suggestive of Refsum disease, whereas the other patient had upper motor neuron signs in the legs, suggesting adrenomyeloneuropathy. The third patient was a child without neuropathy but also aVected by Nieman-Pick type C. Interestingly, a new patient with AMACR deWciency was recently described with a completely diVerent clinical presentation, dominated by liver dysfunction. Apparently, AMACR deWciency can have widely diVerent clinical presentations with some patients showing a slowly progressive neuropathy whereas others present with fulminant liver disease. In literature, a case of presumed peroxisomal thiolase deWciency has been described in a patient showing all signs and symptoms of Zellweger syndrome [44]. Since there were several inconsistencies in this patient, Ferdinandusse et al. [45] decided to reinvestigate this patient, which disclosed that the true defect in this patient is not at the level of peroxisomal thiolase but in D-bifunctional protein. As a consequence, peroxisomal thiolase deWciency can no longer be considered a distinct peroxisomal disorder [45]. Fatty acid -oxidation Ever since the discovery by Klenk and Kahlke in the early 1960s [46] that phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) accumulates in Refsum disease patients, the mechanism of oxidation of phytanic acid
23
has been studied. Since phytanic acid is a 3-methyl branched-chain fatty acid, phytanic acid cannot undergo -oxidation. Studies notably by Steinberg and co-workers in the late 1960s (see [47] for review) revealed that phytanic acid Wrst undergoes -oxidation whereby the terminal carboxyl-group is removed as CO2. The other product of this -oxidation reaction is pristanic acid, a 2methyl branched-chain fatty acid, which can undergo normal -oxidation like any 2-methyl fatty acid. Mitochondria were long believed to be the site of phytanic acid -oxidation. Prompted by the Wnding that phytanic acid does not only accumulate in Refsum disease patients but also in patients suVering from diVerent peroxisomal biogenesis defects [48], the issue of the subcellular localization of phytanic acid -oxidation was reinvestigated, which led to the conclusion that peroxisomes are the exclusive site of phytanic acid -oxidation [47]. The mechanism of phytanic acid -oxidation has long remained mysterious but has been resolved in recent years. A major break-through in this respect was the discovery by Mihalik et al. in 1995 [49] of the enzyme phytanoyl-CoA hydroxylase, which turned out to be the key enzyme in fatty acid -oxidation. Phytanoyl-CoA hydroxylase belongs to the family of 2-oxoglutarate dependent hydroxylases in which the formation of 2hydroxyphytanoyl-CoA is driven by 2-oxoglutarate, which is decarboxylated to succinate. The identiWcation of phytanoyl-CoA hydroxylase by Mihalik et al. [49] was soon followed by the discovery of phytanoyl-CoA hydroxylase as the enzyme deWcient in Refsum disease [50]. This Wnding not only ended the longstanding quest for the primary defect in Refsum’s disease but at the same time established that -oxidation of phytanic acid does not involve phytanic acid, as long believed, but phytanoyl-CoA with the hydroxylase as the key enzyme. Studies by diVerent groups have shown that phytanoylCoA hydroxylase is a true peroxisomal enzyme in all mammalian species, including humans, rats, and mice. All phytanoyl-CoA hydroxylases identiWed to-date contain bona Wde peroxisomal targeting signals of the PTS2 type. The product of the hydroxylase reaction is 2-hydroxyphytanoyl-CoA, which is split into formyl-CoA and pristanal within peroxisomes, a reaction catalysed by the enzyme 2-hydroxyphytanoyl-CoA lyase. This enzyme was puriWed by Foulon et al. [51] and subsequently cloned. The lyase has been found to contain a true peroxisomal targeting signal of the PTS1 type [51]. Interestingly, the 2hydroxyphytanoyl-CoA lyase is a thiamine pyrophosphate dependent enzyme. The subsequent step in the pathway, i.e., the conversion of pristanal to pristanic acid, remains ill-deWned. It was originally suggested that ALDH10, an aldehyde dehydrogenase localised in the endoplasmic reticulum membrane would catalyse this step, although recent observations suggest otherwise [52].
24
R.J.A. Wanders / Molecular Genetics and Metabolism 83 (2004) 16–27
The fact that peroxisomes contain abundant pristanal dehydrogenase activity suggests that the conversion of pristanal to pristanic acid occurs in peroxisomes via an aldehyde dehydrogenase yet to be identiWed. Finally, pristanic acid needs to be activated to its coenzyme-A ester. Interestingly, Steinberg et al. [53] identiWed a distinct acyl-CoA synthetase in peroxisomes, which faces the peroxisomal lumen and which is reactive with pristanic acid. This enzyme, called VLACS could catalyse the intraperoxisomal conversion of pristanic acid into pristanoyl-CoA. The pristanoyl-CoA thus generated within peroxisomes can then undergo -oxidation as established by Verhoeven et al. [54]. As discussed before, pristanoyl-CoA undergoes three cycles of -oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA, which is then subsequently shuttled to mitochondria in its carnitine ester form to undergo full oxidation to CO2 and H2O (see Fig. 5). Disorders of fatty acid -oxidation Refsum’s disease is the only true disorder of fatty acid -oxidation and was Wrst delineated as a distinct clinical entity by Sigvald Refsum in the 1940s. Indeed, in 1945 Refsum described a clinical condition, characterised by cerebellar ataxia, atypical retinitis pigmentosa, anosmia, and elevated cerebrospinal Xuid (CSF) protein, and other organ malformations, and speculated that it was inherited. The subsequent identiWcation of two Norwegian families with similar signs and symptoms led him to conclude that this was a new syndrome transmitted by autosomal recessive inheritance. A major discovery was the Wnding of Klenk and Kahlke in the 1960s [46] of the accumulation of phytanic acid in plasma and tissues of Refsum disease patients thus generating a biochemical marker for the disease. According to Refsum, the tetrad of retinitis pigmentosa, cerebellar ataxia, peripheral polyneuropathy, and high CSF protein without pleocytosis is diagnostic for Refsum’s disease. Other frequent Wndings are olefactory impairment, impaired vestibular, and cochlear function, pupillary abnormalities, shortened distal phalanges, and broad, short Wngernails, lens opacities, ichthyosis, renal failure, and cardiac arrhythmias. Refsum patients usually present in late childhood with progressive deterioration of night vision, the occurrence of a progressive retinitis pigmentosa, and anosmia, later in time followed by deafness, ataxia, polyneuropathy, ichthyosis, and cardiac arrhythmias. Short metacarpals and metatarsals are also found in some 30% of Refsum patients. Furthermore, psychiatric disturbances have been reported in a subset of patients. It is quite clear now that only few patients develop the full spectrum of clinical signs and symptoms of Refsum’s disease. Indeed, a study of 16 Refsum’s disease patients with full clinical information for a period up to 60 years showed that all 16 patients developed retinitis pigmentosa, 15 out of 16 developed
Fig. 5. Peroxisomes and the -oxidation of phytanic acid. Phytanic acid is either activated outside peroxisomes, followed by transport of phytanoyl-CoA across the peroxisomal membrane or Wrst transported as free phytanic acid to be activated inside peroxisomes. Subsequently, phytanoyl-CoA is converted into 2-hydroxyphytanoyl-CoA by the enzyme phytanoyl-CoA hydroxylase, which is coupled to the decarboxylation of 2-oxoglutarate to succinate plus CO2. 2-Hydroxyphytanoyl-CoA is then cleaved into pristanal plus formyl-CoA, which is probably converted inside peroxisomes to formate and free coenzymeA, followed by subsequent degradation of formate. The pristanal formed in the lyase-reaction is converted to pristanic acid inside peroxisomes via an ill-deWned aldehyde dehydrogenase. Finally, pristanic acid is converted into pristanoyl-CoA, probably by the enzyme verylong-chain acyl-CoA synthetase (VLACS) which is a peripheral peroxisomal membrane protein with its catalytic site facing the interior of the peroxisome. Finally, pristanoyl-CoA undergoes three cycles of oxidation inside peroxisomes to produce 4,8-dimethylnonanoyl-CoA, which is then converted into the corresponding carnitine ester, which is then shuttled to the mitochondrion for full oxidation to CO2 and H2O.
anosmia, whereas neuropathy, deafness, and ataxia was only found in 11, 10, and 8 patients, respectively [55]. Finally, ichthyosis was only present in 4 of the 16 patients studied.
R.J.A. Wanders / Molecular Genetics and Metabolism 83 (2004) 16–27
The mainstay of management of Refsum’s disease is restriction of dietary phytanic acid. The average human daily dietary intake of phytanic acid in Western countries amounts to 50–100 mg. The usual goal is to maintain phytanic acid intake in Refsum’s disease patients below 10 mg daily. This means restriction of common foods such as butter, cheeses, lamb, beef, and certain Wsh. In patients, who respond with a fall in plasma phytanic acid levels there may be an arrest in the progression of the peripheral neuropathy, improved muscle strength, regression of ichthyosis, and correction of non-speciWc abnormalities of the electrocardiogram. A major problem is that the elimination of total body phytanic acid usually takes 1–2 years. For this reason plasma exchange has been used to rapidly lower plasma phytanic acid levels but only in acutely ill patients. The fact that it is diYcult and often impossible to normalize plasma phytanic acid levels in Refsum disease patients, has inspired us to look for alternative strategies to remove phytanic acid. One possibility is to look for other pathways, which may be used to degrade phytanic acid. Such a pathway is the -oxidation system, which allows degradation of fatty acids starting from the end. We have recently shown that human liver has the capacity to form -hydroxyphytanic acid from phytanic acid in a cytochrome P450-mediated reaction as we found earlier for rat liver microsomes [56]. We are currently studying whether we can induce the capacity to oxidize phytanic acid by known inducers of diVerent cytochrome P450s in an eVort to generate a new therapeutic option for Refsum disease patients by induction of the capacity to -oxidize phytanic acid.
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Acknowledgments [13]
The author gratefully acknowledges Mrs. Maddy Festen for expert preparation of the manuscript and Mr. Jos Ruiter for the artwork. The author’s studies were Wnancially supported by the Dutch Foundations for ScientiWc Research (NOW-MW; EU Project, No. QLG1CT-2001-01277 Mouse models of peroxisomal diseases (MMPD); EU Project, No. QLG3-CT-2002-00696 Refsum’s disease: Diagnosis, Pathology, and Treatment (RDDPT); NWO, Project No. 901-03-159; NWO, Project No. 916.46.109; NWO Project Number 901-03-097; NWO Project No. 99008; PBF Project No. 97-0216; PBF Project No. 99-0220; Royal Dutch Academy of Sciences).
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R.J.A. Wanders / Molecular Genetics and Metabolism 83 (2004) 16–27 [50] G.A. Jansen, R.J.A. Wanders, P.A. Watkins, S.J. Mihalik, Phytanoyl-Coenzyme A hydroxylase deWciency—the enzyme defect in Refsum’s disease, N. Engl. J. Med. 337 (1997) 133–134. [51] V. Foulon, V.D. Antonenkov, K. Croes, E. Waelkens, G.P. Mannaerts, P.P. Van Veldhoven, M. Casteels, PuriWcation, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during alpha-oxidation of 3-methyl-branched fatty acids, Proc. Natl. Acad. Sci. USA 96 (1999) 10039–10044. [52] G.A. Jansen, D.M. vanden Brink, R. Ofman, O. Draghici, G. Dacremont, R.J.A. Wanders, IdentiWcation of pristanal dehydrogenase activity in peroxisomes: conclusive evidence that the complete phytanic acid alpha-oxidation pathway is localized in peroxisomes, Biochem. Biophys. Res. Commun. 283 (2001) 674–679.
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[53] S.J. Steinberg, S.J. Wang, D.G. Kim, S.J. Mihalik, P.A. Watkins, Human very-long-chain acyl-CoA synthetase: cloning, topography, and relevance to branched-chain fatty acid metabolism, Biochem. Biophys. Res. Commun. 257 (1999) 615–621. [54] N.M. Verhoeven, D.S. Roe, R.M. Kok, R.J.A. Wanders, C. Jakobs, C. Roe, Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in cultured Wbroblasts, J. Lipid Res. 39 (1998) 66–74. [55] A.S. Wierzbicki, M.D. Lloyd, C.J. SchoWeld, M.D. Feher, F.B. Gibberd, Refsum’s disease: a peroxisomal disorder aVecting phytanic acid alpha-oxidation, J. Neurochem. 80 (2002) 727– 735. [56] J.C. Komen, M. Duran, R.J. Wanders, omicron-Hydroxylation of phytanic acid in rat liver microsomes: implications for Refsum disease, J. Lipid Res. 45 (2004) 1341–1346.
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Peroxisomes, lipid metabolism, and peroxisomal disorders R.J.A. Wanders¤ Laboratory for Genetic Metabolic Diseases, Department of Clinical Chemistry and Pediatrics, Academic Medical Center, University of Amsterdam, Emma Children’s Hospital, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Received 9 July 2004; accepted 30 August 2004
Abstract Peroxisomes catalyse a large variety of diVerent cellular functions of which most have to do with lipid metabolism. This paper deals with the role of peroxisomes in three key pathways of lipid metabolism, including: (1) etherphospholipid biosynthesis, (2) fatty acid -oxidation, and (3) fatty acid -oxidation. Apart from a brief description of the peroxisomal enzymes involved in each of these pathways, the interaction between peroxisomes and other subcellular organelles, notably microsomes and peroxisomes, will be discussed. Finally, the current state of knowledge with respect to the diVerent disorders of peroxisomal lipid metabolism will be described. 2004 Elsevier Inc. All rights reserved. Keywords: Etherphospholipid biosynthesis; Fatty acid -oxidation; Fatty acid -oxidation; Peroxisome biogenesis; Zellweger syndrome; Refsum disease
Introduction Peroxisomes play an essential role in human physiology as concluded from the devastating consequences of the lack of peroxisomes in patients with Zellweger syndrome. The peroxisomal disorders are usually divided into two groups, including: (1) the disorders of peroxisome biogenesis (PBDs) and (2) the single peroxisomal enzyme deWciencies. Zellweger syndrome is the prototype of the group of peroxisomal disorders and patients suVering from this syndrome display a range of abnormalities, including neurological, skeletal, hepatological, and ocular abnormalities. In Zellweger syndrome as well as in neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), which together constitute the Zellweger spectrum, peroxisome function is disrupted due to mutations in one of the many genes whose proper expression is required for peroxisome biogenesis. *
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1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2004.08.016
The genes involved in peroxisome biogenesis are called PEX genes and the proteins encoded by these genes are named peroxins [1]. At present, mutations in 14 diVerent PEX genes have been described to be associated with Zellweger spectrum disorders [2]. Studies on Zellweger syndrome and the other Zellweger spectrum disorders have contributed greatly to the elucidation of the essential role of peroxisomes in human physiology. Indeed, it was the crucial Wnding of Moser and co-workers [3] in 1982, which led to the recognition of the important role of peroxisomes in fatty acid -oxidation. Similarly, the discovery that plasmalogens were deWcient in Zellweger patients by Heymans et al. in 1983 [4], provided unequivocal evidence for the essential role of peroxisomes in etherphospholipid biosynthesis. Furthermore, the demonstration of markedly elevated phytanic acid levels in plasma from Zellweger patients pointed to the essential role of peroxisomes in fatty acid -oxidation. In this paper, we will describe the current state of knowledge about the metabolic role of peroxisomes in
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humans with particular attention to the role of peroxisomes in lipid metabolism. We will also brieXy describe the inborn errors of peroxisomal lipid metabolism.
Etherphospholipid biosynthesis Etherphospholipids constitute a special class of phospholipids characterised by an ether bond at the sn-1 position of the glycerol backbone rather than an ester bond as in diacylglycerophospholipids (Fig. 1). Two types of ether bonds occur in etherphospholipids: (1) an ether bond and (2) a vinyl-ether bond. The former is found in platelet activating factor (PAF) whereas the vinyl-ether bond is found in a class of etherphospholipids called plasmalogens. A schematic representation of the diVerent types of glycerophospholipids is shown in Fig. 1. In plasmalogens, the aliphatic moieties at the sn-1 position consist of C16:0 (palmitic acid), C18:0 (stearic acid), or C18:1 (oleic acid), whereas the sn-2 position is occupied by polyunsaturated fatty acids (PUFAs). The head group in etherphospholipids is usually of the ethanolamine or choline type. Brain myelin possesses the
Fig. 1. Structure of glycerophospholipids. Plasmalogens contain a vinyl ether bond at the sn-1 position of the glycerol backbone, compared to the ether bond present in plasmanyl-phospholipids (e.g., platelet activating factor) and the ester bond in diacylphospholipids. R1 and R2 represent long-chain fatty acids esteriWed at the sn-1 and sn-2 position, respectively. The head groups represent ethanolamine or choline in plasmalogens, choline in platelet activating factor, and ethanolamine, choline or serine in diacyl-phospholipids.
17
highest content of ethanolamine plasmalogens (PE-plasmalogens) whereas heart muscle has a higher content of choline plasmalogens (PC plasmalogens). Moderate amounts of plasmalogens are found in kidney, skeletal muscle, spleen, and blood cells, whereas liver is known for its low plasmalogen content. Although the function of plasmalogens still remains obscure, the pathway of plasmalogen biosynthesis has been worked out in detail (Fig. 2). The Wrst step in the biosynthesis of plasmalogens involves the esteriWcation of dihydroxyacetonephosphate (DHAP) with a long-chain acyl-CoA ester and is carried out by dihydroxyacetonephosphate acyltransferase (DHAPAT). The characteristic etherbond at the sn-1 position of etherphospholipids is introduced by the replacement of the sn-1 fatty acid with a long-chain fatty alcohol. This reaction is catalysed by alkyldihydroxyacetonephosphate synthase (ADHAPS) with alkylDHAP as product. The fatty alcohol may be derived from dietary sources or by the reduction of longchain acyl-CoAs through the action of an acyl-CoA reductase (Fig. 3). The alkylDHAP produced in the alkylDHAP synthase reaction is subsequently converted into 1-alkylglycerol-3-phosphate (1-alkyl-G3P) as catalysed by the enzyme alkyl/acyldihydroxyacetonephosphate reductase (AADHAPR). Subsequently, 1-alkylG3P is converted into 1-alkyl-2-acyl-G3P by an alkyl/ acyl-glycerol-3-phosphate acyltransferase (AAG3P-AT). The phosphate group is subsequently removed by a
Fig. 2. Schematic representation of the steps involved in the biosynthesis of PC and PE plasmalogens. See text for further details.
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Fig. 3. Topology of the peroxisomal enzymes involved in the biosynthesis of plasmalogens. Symbols used: (1) DHAP-AT; (2) ADHAP-S; (3) acyl-CoA reductase (long-chain alcohol-forming); (4) glycerol-3phosphate dehydrogenase; (5) alkyl/acyl-DHAP reductase (AADHAP-R); (6) peroxisomal membrane protein of 34 kDa (PMP34); (7) very-long-chain acyl-CoA synthetase (VLACS). Adapted from Brites et.al. [9].
phosphohydrolase (PH) yielding 1-alkyl-2-acyl-sn-glycerol. Subsequently, cytidine diphosphate ethanolamine (CDP ethanolamine) is incorporated via the action of ethanolamine phosphotransferase to form 1-alkyl-2acyl-sn-glycerol-3-phosphoethanolamine (1-alkyl-2-acylGPE). Desaturation to 1-alk-11-enyl-2-acyl-GPE is carried out by a microsomal enzyme called delta-1-alkyldesaturase. 1-Alk-11-enyl-2-acyl-GPC (PC plasmalogens) is primarily formed from 1-alk-11-enyl-2-acyl-GPE via sn-2 and polar head group modiWcations (see Fig. 2). Dihydroxyacetonephosphate acyltransferase (DHAPAT) and alkyl-dihydroxyacetonephosphate synthase (ADHAP-S) are exclusively localised in peroxisomes [5]. Both enzymes have been puriWed from diVerent sources and the corresponding cDNAs and genes have been identiWed in diVerent species including Homo sapiens [6– 8]. It turns out that DHAPAT is a bona Wde PTS1 protein. Human DHAPAT for instance has the C-terminal tripeptide alanine/lysine/leucine [6,7]. On the other hand, human alkylDHAP synthase is a PTS2 protein [8]. Fig. 3 shows a schematic picture of the topology of the diVerent enzymes involved in the biosynthesis of plasmalogens and the role of peroxisomes and the endoplasmic reticulum therein (see Brites et al. [9] for review). Disorders of etherphospholipid biosynthesis Two single enzyme deWciencies in the etherphospholipid biosynthetic pathway have been identiWed so far.
These two include: (1) DHAPAT deWciency and (2) alkyl-DHAP synthase deWciency. DHAPAT deWciency was Wrst described in 1992 in a patient showing all the clinical signs and symptoms of rhizomelic chondrodysplasia punctata (RCDP) [10]. In its classical presentation RCDP is clinically characterised by a disproportionally short stature primarily aVecting the proximal parts of the extremities, typical facial appearance, congenital contractures, characteristic ocular involvement, dwarWsm, spasticity, and severe mental retardation. Radiologic studies show shortening, metaphyseal cupping, and disturbed ossiWcation of humeri and/or femora, together with epiphyseal and extra-epiphyseal calciWcations. Analysis of erythrocyte plasmalogens in this patient revealed a full deWciency. Subsequent studies in cultured skin Wbroblasts revealed an isolated deWciency of dihydroxyacetonephosphate acyltransferase, the Wrst enzyme in the etherphospholipid biosynthetic pathway [10]. In 1994 the Wrst case of isolated alkyl-DHAP synthase deWciency was described, again in a patient showing all the clinical signs and symptoms of RCDP [11]. In this patient erythrocyte plasmalogens were also completely deWcient and subsequent studies in Wbroblasts revealed the complete deWciency of alkyl-DHAP synthase with normal values for DHAPAT and other peroxisomal parameters. DHAPAT deWciency and alkylDHAP synthase deWciency have been described in very few patients only (see [12] for review). The molecular basis of both enzyme deWciencies has been resolved in recent years [7,13]. It should be noted that the majority of RCDP patients do not carry mutations in either the GNPAT or ADHAPS gene coding for DHAPAT and alkyl-DHAP synthase, respectively. In fact, most RCDP patients carry mutations in the PEX7 gene [14,15]. This gene codes for the so-called PTS2-receptor, which plays a crucial role in the correct targeting of a group of proteins to the peroxisome. Peroxisomal proteins are equipped with one of two diVerent peroxisomal targeting signals (PTS), called PTS1 and PTS2, which are recognised in the cytosol by either the PTS1- or PTS2- receptors, respectively, and then escorted to the peroxisomal membrane, followed by transport of the PTS1- and PTS2-proteins across the peroxisomal membrane and recycling of the receptors back into the cytosol [1]. In case of mutations in the PEX7 gene the PTS2 receptor will be dysfunctional, so that the correct targeting of peroxisomal proteins equipped with a PTS2 signal does not occur. AlkylDHAP synthase [8] and phytanoyl-CoA hydroxylase [16,17], the Wrst enzyme in the -oxidation of phytanic acid (see later) are PTS2 proteins, which explains their deWciency in RCDP patients with mutations in the PEX7 gene. Furthermore, the deWciency of alkyl-DHAP synthase explains the impairment in etherphospholipid biosynthesis as reXected in the deWciency of plasmalogens in erythrocytes and tissues.
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In addition to the deWciencies of alkyl-DHAP synthase and phytanoyl-CoA hydroxylase there are two other abnormalities in this form of RCDP, which include the partial deWciency of DHAPAT and the aberrant localization of peroxisomal thiolase in the cytosol in its abnormal precursor form (44 kDa rather than 41 kDa) [18]. The abnormality in peroxisomal thiolase is readily explained from the fact that this is a PTS2 protein too, whereas the partial deWciency of DHAPAT is due to the fact that alkyl-DHAP synthase and DHAPAT form a complex within the peroxisome interior [19]. DHAPAT is unstable in the absence of the alkyl-DHAP synthase protein thus explaining the partial deWciency of DHAPAT [20]. In our experience >90% of RCDP patients have mutations in the PEX7 gene [17]. We have been involved in the diagnosis of >120 patients with RCDP. If a patient presents with clinical signs and symptoms of RCDP, erythrocyte plasmalogen levels should be determined. Measurement of erythrocyte plasmalogens has proven to be extreme reliable because all patients studied by us to-date have shown deWcient plasmalogens in erythrocytes, independent of the type of RCDP. DeWnitive diagnosis to resolve the true underlying genetic defect in each patient requires detailed enzymatic studies in Wbroblasts, followed by molecular analysis of the genes coding for Pex7p, the PTS2 receptor (PEX7), DHAPAT (GNPAT), or alkylDHAP synthase (AGPS), respectively (see Wanders et al. [12] for details). Peroxisomal fatty acid -oxidation Although fatty acids can undergo oxidation via diVerent mechanisms, most fatty acids are degraded by means of -oxidation. Peroxisomes contain a fatty acid -oxidation machinery just like mitochondria. The mechanism of -oxidation in mitochondria and peroxisomes is identical and involves a set of four consecutive reactions: (1) dehydrogenation; (2) hydration (of the double bond); (3) dehydrogenation again; and (4) thiolytic cleavage. Through this 4-step pathway a 2-carbon unit is split from each fatty acid in the form of an acetyl-CoA unit, which can then be degraded in the citric acid (Krebs) cycle to produce CO2 and H2O. The peroxisomal and mitochondrial -oxidation systems have diVerent functions as they catalyse the -oxidation of diVerent fatty acids and fatty acid derivatives. Indeed, mitochondria catalyse the -oxidation of the bulk of fatty acids derived from the diet, including palmitate, oleate, linoleate, and linolenate, whereas peroxisomes play an important role by oxidising a diVerent set of fatty acids and fatty acids derivatives. These include: 1. very-long-chain fatty acids (VLCFA), notably hexacosanoic acid (C26:0),
19
2. pristanic acid (2,6,10,14-tetramethylpentadecanoic acid), and 3. di- and trihydroxycholestanoic acid (DHCA and THCA). Very-long-chain fatty acids are derived from both dietary sources, but are also synthesised endogenously from shorter chain fatty acids, whereas pristanic acid cannot be synthesised endogenously, but is only derived from dietary sources, either directly or indirectly from phytanic acid, which is -oxidised to pristanic acid within peroxisomes. Finally, di- and trihydroxycholestanoic acid are intermediates in the formation of the primary bile acids, cholic acid and chenodeoxycholic acid, and are formed from cholesterol in the liver. Although generally marked as a biosynthetic reaction, biosynthesis of polyunsaturated fatty acids such as docosahexaenoic acid (C22:6-3) is actually catalysed by -oxidation in peroxisomes. Indeed, recent studies have established that the formation of C22:6-3 from linolenic acid (C18:3-3) involves the active participation of the peroxisomal fatty acid oxidation system [21,22]. It should be noted that the peroxisomal fatty acid oxidation system is only able to chain-shorten fatty acids and is not able to degrade fatty acids to completion. This is most convincingly demonstrated for pristanic acid, which undergoes three cycles of -oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA, which is then shuttled to the mitochondria in the form of its carnitine ester for full oxidation to CO2 and H2O. The same is probably true for very-long-chain fatty acids, which are chain-shortened to a medium-chain acyl-CoA ester, followed by transport to the mitochondria as carnitine ester to allow complete oxidation to CO2 and H2O again. Finally, with respect to chenodeoxycholoyl-CoA and choloyl-CoA as formed in peroxisomes, these are converted into the corresponding taurine or glycine conjugates within peroxisomes followed by export from peroxisomes to end up in bile. Fig. 4 depicts the enzymology of the peroxisomal fatty acid -oxidation system. Human peroxisomes contain two acyl-CoA oxidases, one speciWc for straightchain fatty acids like C26:0, and a second one catalysing the dehydrogenation of 2-methyl branched-chain fatty acids like pristanoyl-CoA and di- and trihydroxycholestanoyl-CoA. The enoyl-CoA esters of C26:0, pristanic acid, DHCA, and THCA are all handled by a single bifunctional enzyme, catalysing the second (hydration) and third (dehydrogenation) steps of peroxisomal -oxidation. This enzyme, with both enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, forms and dehydrogenates D-3-hydroxyacyl-CoA esters rather than L-3-hydroxyacyl-CoAs and is the single enzyme involved in the oxidation of C26:0, pristanic acid, as well as DHCA and THCA. DiVerent names have been given
20
R.J.A. Wanders / Molecular Genetics and Metabolism 83 (2004) 16–27
Fig. 4. Enzymology of the peroxisomal fatty acid -oxidation system. Human peroxisomes contain two acyl-CoA oxidases, one speciWc for straightchain fatty acids like C26:0, and a second one, catalysing the dehydrogenation of 2-methyl branched-chain fatty acids like pristanoyl-CoA and diand trihydroxycholestanoyl-CoA. The latter oxidase only accepts 2-methyl branched-chain fatty acyl-CoAs with the 2-methyl group in the (2S)-conWguration. The enoyl-CoA esters of C26:0, (2S)-pristanic acid and (25S)-DHCA and THCA are all handled by a single bifunctional enzyme harbouring both enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity. Finally, peroxisomes contain two thiolases of which one (pTH2/ SCPx) is involved in the oxidation of 2-methyl branched-chain fatty acids. Both pTH1 and pTH2/SCPx react with the 3-ketoacyl-CoA esters of straight-chain fatty acids which explains the involvement of both thiolases in C26:0 -oxidation.
to this enzyme including D-bifunctional protein (DBP), D-peroxisomal bifunctional enzyme (D-PBE), multifunctional enzyme II (MFE-II), and multifunctional protein 2 (MFP-2). Peroxisomes also contain a diVerent bifunctional protein, which forms and dehydrogenates L-3hydroxyacyl-CoA esters, but the function of this enzyme remains to be resolved in the future. Finally, human peroxisomes contain two peroxisomal thiolases, i.e., pTH1 and pTH2. Human pTH1 is
the human equivalent of the cloWbrate-inducible thiolase identiWed by Hashimoto and co-workers, whereas PTH2 is identical to the 58 kDa sterol carrier protein x (SCPx) with both thiolase activity as well as a sterol carrier protein domain. It is clear now that pTH2 (SCPx) plays an indispensable role in the oxidation of 2-methyl branched-chain fatty acids, whereas pTH1 and pTH2 are probably both involved in C26:0 -oxidation.
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The enzymes described above are necessary and suYcient for the -oxidation of straight-chain fatty acids like C26:0 as well as 2-methyl branched-chain fatty acids with the methyl group in the (2S)-conWguration. However, auxiliary enzymes are needed for the -oxidation of (2R)-methyl branched-chain FAs and unsaturated FAs. Oxidation of (2R)-methyl branched-chain FAs requires the active participation of an enzyme, capable of converting (2R)- into (2S)-branched-chain acyl-CoAs. Studies, notably by Schmitz et al. [23–25] have shown that peroxisomes contain 2-methylacyl-CoA racemase (AMACR) activity, which enzyme is able to convert (2R)-methylacyl-CoAs into the corresponding (2S)methyl-acyl-CoAs and vice versa. The importance of this enzyme for oxidation of 2-methyl branched-chain fatty acids is stressed by our Wndings in AMACR-deWcient patients in which (2R)-pristanic acid accumulates as well as di- and trihydroxycholestanoic acid [26–28]. The accumulation of the latter two species is explained by the fact that the 2-methyl group in DHCA and THCA, as produced from cholesterol, has the (R)-conWguration (see Fig. 4). Human peroxisomes also contain -3,-2-enoylCoA isomerase activity as well as 2,4-dienoylCoA reductase, and -3,5,-2,4-dienoylCoA isomerase activity, thereby allowing the oxidation of unsaturated fatty acids with double bonds at even or uneven positions [12]. Interaction between peroxisomes and mitochondria Since peroxisomes lack a respiratory chain and a citric acid (Krebs) cycle the products of -oxidation of fatty acids in peroxisomes have to be shuttled to mitochondria. With respect to the end products of peroxisomal -oxidation, such as acetyl-CoA, propionyl-CoA, and medium-chain acyl-CoA esters, it has been demonstrated that these acyl-CoAs are transported to the mitochondrion in the form of the corresponding carnitine esters. For this purpose peroxisomes are equipped with diVerent carnitine acyltransferases including carnitine acetyltransferase (CAT) and carnitine octanoyltransferase (COT), which allow formation of carnitine esters inside peroxisomes, followed by export across the peroxisomal membrane via an unidentiWed carrier system. Uptake of carnitine esters into mitochondria occurs via the mitochondrial carnitine/acylcarnitine transporter (CACT), followed by retroconversion of the acylcarnitines into the corresponding acyl-CoA esters via diVerent acyltransferases, present in mitochondria. An alternative mechanism by which the end-products of peroxisomal -oxidation may be transferred to mitochondria, is by cleavage of the acyl-CoA esters into the free acids plus CoASH via one of the many thioesterases identiWed in peroxisomes (see [3] for review) followed by transport of the free fatty acids to mitochondria [29].
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One aspect of peroxisomal -oxidation, which remains to be resolved deWnitively, is the way in which the NADH produced in the third step of -oxidation is reoxidised to NAD. Our studies in the yeast Saccharomyces cerevisiae [30] have shown the involvement of a NAD(H) redox shuttle in which the NADH produced is reoxidised to NAD by means of peroxisomal malate dehydrogenase (MDH), followed by the export of malate to the cytosol where the reverse occurs. This malate oxaloacetate shuttle resembles the mitochondrial glutamate/malate shuttle. The fact that mammalian peroxisomes do not contain peroxisomal malate dehydrogenase activity is evidence against the existence of a similar type of NAD(H) redox shuttle in mammals. According to Baumgart et al. [31] mammalian peroxisomes contain a diVerent type of NADH shuttle with lactate dehydrogenase playing the role of malate dehydrogenase. Transport of fatty acids and fatty acid derivatives across the peroxisomal membrane One of the truly unresolved issues of peroxisome metabolism is the transport of FAs across the peroxisomal membrane. In mitochondria FAs are carried across the mitochondrial innermembrane via the carnitine cycle in which FAs are Wrst activated to the corresponding coenzyme-A esters followed by substitution of the coenzyme-A unit by carnitine, as catalysed by the enzyme carnitine palmitoyltransferase 1 (CPT1). The acylcarnitines then enter the mitochondrion via a speciWc transporter, the carnitine/acylcarnitine transporter (CACT), which exchanges extramitochondrial acylcarnitine for intramitochondrial carnitine. Finally, the carnitine moiety in the acylcarnitine species is exchange for coenzyme-A to produce the acyl-CoA back again, a reaction mediated by carnitine palmitoyltransferase 2 (CPT2). Such a carnitine cycle is present in peroxisomes, but seems to work only in the export of acyl-CoA species from the interior of the peroxisome to the cytosol and subsequently to the mitochondrial space. How then are FAs transported across the peroxisomal membrane? The discovery that X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene coding for a protein (ALDP) which belongs to the family of ABCtransporters, led to the hypothesis that ALDP may actually catalyse the transport of FAs across the peroxisomal membrane. ALDP is a so-called half-ABC-transporter with six transmembrane spanning regions. Interestingly, human peroxisomes contain three additional half-ABCtransporters including ALDR, PMP70, and PMP69. Since these are all half-ABC-transporters, the most likely scenario is that these half-transporters form homo- and/ or heterodimers within the peroxisomal membrane. The yeast S. cerevisiae contains two peroxisomal halfABC-transporters, called Pxa1p and Pxa2p. Mutants in which either the PXA1 or PXA2 gene has been
22
R.J.A. Wanders / Molecular Genetics and Metabolism 83 (2004) 16–27
disrupted, show a similar phenotype, characterised by lack of growth on oleate-containing medium as caused by the reduced ability to oxidize oleic acid. Oxidation of a medium-chain fatty acid like octanoate (C8:0) is fully normal in these mutants, suggesting the existence of two routes of fatty acid import into peroxisomes. Based on experiments in which the peroxisomal acyl-CoA synthetase (Faa2p) was redirected to the cytosol by removing the PTS1 targeting signal, Hettema et al. [32] concluded that the Pxa1p/Pxa2p dimer catalyses the transport of acyl-CoA esters across the peroxisomal membrane. DeWnitive proof of this postulate awaits reconstitution of the Pxa1p/Pxa2p dimer in artiWcial liposomes. Such a system would also be required to test whether ALDP either as homo- or heterodimer transports the CoA esters of very-long-chain fatty acids or not. It should be noted that others favour a diVerent role for ALDP. Indeed, McGuinness et al. [33] have proposed that ALDP is involved in shuttling the end products of peroxisomal -oxidation to the mitochondria. Disorders of peroxisomal -oxidation X-linked adrenoleukodystrophy (X-ALD) is the most common single peroxisomal disorder with a minimum incidence of 1:21,000 males [34] in the USA to 1:15,000 males in France (Dr. P. Aubourg, Paris, France, personal communication, cited in Kemp et al. [35]). The phenotypic expression of X-ALD varies wildly. At present, at least six phenotypic variants of X-ALD can be distinguished [36]. This classiWcation is somewhat arbitrary and is based on the age of onset and the organs principally involved. The two most frequent phenotypes, together accounting for >80% of all cases, are childhood cerebral ALD (CCALD) and adrenomyeloneuropathy (AMN). Onset of CCALD is between 3 and 10 years of age with progressive behavioural, cognitive, and neurologic deterioration, often leading to total disability within 3 years. The cerebral phenotype is not only observed in childhood but may also present later in life in adolescence (adolescent cerebral ALD) or even adulthood (adult cerebral ALD). There is a marked diVerence between the cerebral phenotypes and AMN since cerebral phenotypes show an inXammatory reaction in the cerebral white matter, which resembles, but can be distinguished from, what is observed in multiple sclerosis. In contrast to CCALD, the inXammatory response is absent or mild in AMN, which has a much later age of onset (28 § 9 years) and a much slower rate of progression. Nevertheless, approximately 40% of AMN patients do develop some cerebral involvement associated with a more rapid downhill progression. It is important to emphasize that women, heterozygous for ALD, may develop AMN-like symptoms in middle age or later. Current information holds that this is more frequent than previously anticipated. Cerebral involvement and
adrenocortical insuYciency are rare, however, in heterozygous women. The biochemical hallmark of X-ALD is the accumulation of VLCFAs, notably C24:0 and C26:0, in plasma and tissues. Analysis of VLCFAs in plasma has turned out to be an extremely reliable diagnostic method with few, if any, false negatives, at least in hemizygotes. If an abnormal VLCFA proWle is found in a particular patient, molecular analysis of the ABCD1 gene, which codes for the ALD protein, should be done. At present, >650 diVerent mutations in the ABCD1 gene have been identiWed (see www.X-ALD.nl). Interestingly, in the majority of X-linked ALD patients, the ALD protein is markedly deWcient as concluded from immunoXuorescence microscopy analysis [37,38]. The accumulation of VLCFAs in X-linked ALD patients is the result of the impaired oxidation of these fatty acids in peroxisomes. As discussed above, it has to be established what the association is between mutations in the ABCD1 gene, the consequent loss of function of ALDP and the impaired oxidation of VLCFAs in peroxisomes. Acyl-CoA oxidase deWciency was Wrst described in 1988 by Poll-The et al. [39] in two siblings with neonatal onset hypotonia, delayed motor development, sensory deafness, and retinopathy, with extinguished electroretinograms, but no craniofacial dysmorphia. Since this initial description only few patients have been described since then (see [12] for review). In patients with acyl-CoA oxidase deWciency plasma very-long-chain fatty acids are markedly elevated whereas other parameters of the peroxisomal -oxidation system, including pristanic acid and di- and trihydroxycholestanoic acid are completely normal. The acyl-CoA oxidase gene has been identiWed and the molecular basis of acyl-CoA oxidase deWciency has been resolved in a few cases (see [12] for review). Much more frequent among the disorders of peroxisomal -oxidation is D-bifunctional protein deWciency. The clinical presentation of D-bifunctional protein deWcient patients resembles that of the peroxisome biogenesis disorders (PBDs) in many respects. Indeed, virtually all patients with D-bifunctional protein deWciency identiWed so far (>60) show severe clinical abnormalities including hypotonia, craniofacial dysmorphia, neonatal seizures, hepatomegaly, and developmental delay. Most patients with D-BP deWciency die in the Wrst year of life. A remarkable observation is that patients with D-BP deWciency often show disordered neuronal migration. In retrospect the Wrst case of D-bifunctional protein deWciency was described by Watkins et al. [40] in 1989 in a male patient with severe hypotonia, and neonatal seizures. An electroencephalogram revealed multifocal spikes. No developmental progress was observed. Interestingly, there was no dysmorphia in this patient and no hepatomegaly. Fontanelles were large with open metopic sutures. Visual evoked responses and brainstem auditory
R.J.A. Wanders / Molecular Genetics and Metabolism 83 (2004) 16–27
evoked responses were grossly abnormal. A brain biopsy at 6 weeks revealed polymicrogyria. The patient died at 5 months of age after a clinical course marked by absent developmental progress and therapy refractory seizures. The true defect in this patient at the level of D-bifunctional protein was only resolved in 1999 by van Grunsven et al. [41]. In this patient D-bifunctional protein was completely missing as assessed by immunoblot analysis, which explains the combined loss of both the enoyl-CoA hydratase as well as 3-hydroxyacyl-CoA dehydrogenase activity of D-BP. In addition to complete D-bifunctional protein deWciency also referred to as D-BP deWciency type 1, there are two additional forms of D-bifunctional protein deWciency in which either the enoyl-CoA hydratase (see van Grunsven et al. [42]) or the 3-hydroxyacylCoA dehydrogenase (van Grunsven et al. [43]) component of D-bifunctional protein is functionally inactive, respectively. For more detailed information the reader is referred to Wanders et al. [12]. 2-Methyl-acyl-CoA racemase (AMACR) deWciency is the fourth and last disorder of peroxisomal -oxidation. Although only a few patients with this defect have been described in literature so far, the clinical picture of AMACR deWciency is usually that of a slowly progressive adult-onset sensory motor neuropathy. Indeed, two of the three initial patients described by Ferdinandusse et al. [26] suVered from adult-onset sensory motor neuropathy. One patient also had pigmentary retinopathy, suggestive of Refsum disease, whereas the other patient had upper motor neuron signs in the legs, suggesting adrenomyeloneuropathy. The third patient was a child without neuropathy but also aVected by Nieman-Pick type C. Interestingly, a new patient with AMACR deWciency was recently described with a completely diVerent clinical presentation, dominated by liver dysfunction. Apparently, AMACR deWciency can have widely diVerent clinical presentations with some patients showing a slowly progressive neuropathy whereas others present with fulminant liver disease. In literature, a case of presumed peroxisomal thiolase deWciency has been described in a patient showing all signs and symptoms of Zellweger syndrome [44]. Since there were several inconsistencies in this patient, Ferdinandusse et al. [45] decided to reinvestigate this patient, which disclosed that the true defect in this patient is not at the level of peroxisomal thiolase but in D-bifunctional protein. As a consequence, peroxisomal thiolase deWciency can no longer be considered a distinct peroxisomal disorder [45]. Fatty acid -oxidation Ever since the discovery by Klenk and Kahlke in the early 1960s [46] that phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) accumulates in Refsum disease patients, the mechanism of oxidation of phytanic acid
23
has been studied. Since phytanic acid is a 3-methyl branched-chain fatty acid, phytanic acid cannot undergo -oxidation. Studies notably by Steinberg and co-workers in the late 1960s (see [47] for review) revealed that phytanic acid Wrst undergoes -oxidation whereby the terminal carboxyl-group is removed as CO2. The other product of this -oxidation reaction is pristanic acid, a 2methyl branched-chain fatty acid, which can undergo normal -oxidation like any 2-methyl fatty acid. Mitochondria were long believed to be the site of phytanic acid -oxidation. Prompted by the Wnding that phytanic acid does not only accumulate in Refsum disease patients but also in patients suVering from diVerent peroxisomal biogenesis defects [48], the issue of the subcellular localization of phytanic acid -oxidation was reinvestigated, which led to the conclusion that peroxisomes are the exclusive site of phytanic acid -oxidation [47]. The mechanism of phytanic acid -oxidation has long remained mysterious but has been resolved in recent years. A major break-through in this respect was the discovery by Mihalik et al. in 1995 [49] of the enzyme phytanoyl-CoA hydroxylase, which turned out to be the key enzyme in fatty acid -oxidation. Phytanoyl-CoA hydroxylase belongs to the family of 2-oxoglutarate dependent hydroxylases in which the formation of 2hydroxyphytanoyl-CoA is driven by 2-oxoglutarate, which is decarboxylated to succinate. The identiWcation of phytanoyl-CoA hydroxylase by Mihalik et al. [49] was soon followed by the discovery of phytanoyl-CoA hydroxylase as the enzyme deWcient in Refsum disease [50]. This Wnding not only ended the longstanding quest for the primary defect in Refsum’s disease but at the same time established that -oxidation of phytanic acid does not involve phytanic acid, as long believed, but phytanoyl-CoA with the hydroxylase as the key enzyme. Studies by diVerent groups have shown that phytanoylCoA hydroxylase is a true peroxisomal enzyme in all mammalian species, including humans, rats, and mice. All phytanoyl-CoA hydroxylases identiWed to-date contain bona Wde peroxisomal targeting signals of the PTS2 type. The product of the hydroxylase reaction is 2-hydroxyphytanoyl-CoA, which is split into formyl-CoA and pristanal within peroxisomes, a reaction catalysed by the enzyme 2-hydroxyphytanoyl-CoA lyase. This enzyme was puriWed by Foulon et al. [51] and subsequently cloned. The lyase has been found to contain a true peroxisomal targeting signal of the PTS1 type [51]. Interestingly, the 2hydroxyphytanoyl-CoA lyase is a thiamine pyrophosphate dependent enzyme. The subsequent step in the pathway, i.e., the conversion of pristanal to pristanic acid, remains ill-deWned. It was originally suggested that ALDH10, an aldehyde dehydrogenase localised in the endoplasmic reticulum membrane would catalyse this step, although recent observations suggest otherwise [52].
24
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The fact that peroxisomes contain abundant pristanal dehydrogenase activity suggests that the conversion of pristanal to pristanic acid occurs in peroxisomes via an aldehyde dehydrogenase yet to be identiWed. Finally, pristanic acid needs to be activated to its coenzyme-A ester. Interestingly, Steinberg et al. [53] identiWed a distinct acyl-CoA synthetase in peroxisomes, which faces the peroxisomal lumen and which is reactive with pristanic acid. This enzyme, called VLACS could catalyse the intraperoxisomal conversion of pristanic acid into pristanoyl-CoA. The pristanoyl-CoA thus generated within peroxisomes can then undergo -oxidation as established by Verhoeven et al. [54]. As discussed before, pristanoyl-CoA undergoes three cycles of -oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA, which is then subsequently shuttled to mitochondria in its carnitine ester form to undergo full oxidation to CO2 and H2O (see Fig. 5). Disorders of fatty acid -oxidation Refsum’s disease is the only true disorder of fatty acid -oxidation and was Wrst delineated as a distinct clinical entity by Sigvald Refsum in the 1940s. Indeed, in 1945 Refsum described a clinical condition, characterised by cerebellar ataxia, atypical retinitis pigmentosa, anosmia, and elevated cerebrospinal Xuid (CSF) protein, and other organ malformations, and speculated that it was inherited. The subsequent identiWcation of two Norwegian families with similar signs and symptoms led him to conclude that this was a new syndrome transmitted by autosomal recessive inheritance. A major discovery was the Wnding of Klenk and Kahlke in the 1960s [46] of the accumulation of phytanic acid in plasma and tissues of Refsum disease patients thus generating a biochemical marker for the disease. According to Refsum, the tetrad of retinitis pigmentosa, cerebellar ataxia, peripheral polyneuropathy, and high CSF protein without pleocytosis is diagnostic for Refsum’s disease. Other frequent Wndings are olefactory impairment, impaired vestibular, and cochlear function, pupillary abnormalities, shortened distal phalanges, and broad, short Wngernails, lens opacities, ichthyosis, renal failure, and cardiac arrhythmias. Refsum patients usually present in late childhood with progressive deterioration of night vision, the occurrence of a progressive retinitis pigmentosa, and anosmia, later in time followed by deafness, ataxia, polyneuropathy, ichthyosis, and cardiac arrhythmias. Short metacarpals and metatarsals are also found in some 30% of Refsum patients. Furthermore, psychiatric disturbances have been reported in a subset of patients. It is quite clear now that only few patients develop the full spectrum of clinical signs and symptoms of Refsum’s disease. Indeed, a study of 16 Refsum’s disease patients with full clinical information for a period up to 60 years showed that all 16 patients developed retinitis pigmentosa, 15 out of 16 developed
Fig. 5. Peroxisomes and the -oxidation of phytanic acid. Phytanic acid is either activated outside peroxisomes, followed by transport of phytanoyl-CoA across the peroxisomal membrane or Wrst transported as free phytanic acid to be activated inside peroxisomes. Subsequently, phytanoyl-CoA is converted into 2-hydroxyphytanoyl-CoA by the enzyme phytanoyl-CoA hydroxylase, which is coupled to the decarboxylation of 2-oxoglutarate to succinate plus CO2. 2-Hydroxyphytanoyl-CoA is then cleaved into pristanal plus formyl-CoA, which is probably converted inside peroxisomes to formate and free coenzymeA, followed by subsequent degradation of formate. The pristanal formed in the lyase-reaction is converted to pristanic acid inside peroxisomes via an ill-deWned aldehyde dehydrogenase. Finally, pristanic acid is converted into pristanoyl-CoA, probably by the enzyme verylong-chain acyl-CoA synthetase (VLACS) which is a peripheral peroxisomal membrane protein with its catalytic site facing the interior of the peroxisome. Finally, pristanoyl-CoA undergoes three cycles of oxidation inside peroxisomes to produce 4,8-dimethylnonanoyl-CoA, which is then converted into the corresponding carnitine ester, which is then shuttled to the mitochondrion for full oxidation to CO2 and H2O.
anosmia, whereas neuropathy, deafness, and ataxia was only found in 11, 10, and 8 patients, respectively [55]. Finally, ichthyosis was only present in 4 of the 16 patients studied.
R.J.A. Wanders / Molecular Genetics and Metabolism 83 (2004) 16–27
The mainstay of management of Refsum’s disease is restriction of dietary phytanic acid. The average human daily dietary intake of phytanic acid in Western countries amounts to 50–100 mg. The usual goal is to maintain phytanic acid intake in Refsum’s disease patients below 10 mg daily. This means restriction of common foods such as butter, cheeses, lamb, beef, and certain Wsh. In patients, who respond with a fall in plasma phytanic acid levels there may be an arrest in the progression of the peripheral neuropathy, improved muscle strength, regression of ichthyosis, and correction of non-speciWc abnormalities of the electrocardiogram. A major problem is that the elimination of total body phytanic acid usually takes 1–2 years. For this reason plasma exchange has been used to rapidly lower plasma phytanic acid levels but only in acutely ill patients. The fact that it is diYcult and often impossible to normalize plasma phytanic acid levels in Refsum disease patients, has inspired us to look for alternative strategies to remove phytanic acid. One possibility is to look for other pathways, which may be used to degrade phytanic acid. Such a pathway is the -oxidation system, which allows degradation of fatty acids starting from the end. We have recently shown that human liver has the capacity to form -hydroxyphytanic acid from phytanic acid in a cytochrome P450-mediated reaction as we found earlier for rat liver microsomes [56]. We are currently studying whether we can induce the capacity to oxidize phytanic acid by known inducers of diVerent cytochrome P450s in an eVort to generate a new therapeutic option for Refsum disease patients by induction of the capacity to -oxidize phytanic acid.
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Acknowledgments [13]
The author gratefully acknowledges Mrs. Maddy Festen for expert preparation of the manuscript and Mr. Jos Ruiter for the artwork. The author’s studies were Wnancially supported by the Dutch Foundations for ScientiWc Research (NOW-MW; EU Project, No. QLG1CT-2001-01277 Mouse models of peroxisomal diseases (MMPD); EU Project, No. QLG3-CT-2002-00696 Refsum’s disease: Diagnosis, Pathology, and Treatment (RDDPT); NWO, Project No. 901-03-159; NWO, Project No. 916.46.109; NWO Project Number 901-03-097; NWO Project No. 99008; PBF Project No. 97-0216; PBF Project No. 99-0220; Royal Dutch Academy of Sciences).
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