Processing of mutant N-acetyl-α-glucosaminidase in mucopolysaccharidosis type IIIB fibroblasts cultured at low temperature.

BACKGROUND: The autosomal recessive, neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU), resulting in accumulation of heparan sulfate. The disease spectrum comprises a severe, rapidly progressing (RP) phenotype and a more attenuated, slowly progressing (SP) phenotype. Previous studies showed significantly higher NAGLU activity in skin fibroblasts of SP patients when cultured at 30°C which may be relevant for development of novel therapeutic strategies. Here we report on the processes involved in this phenomenon. METHODS: Fibroblasts from controls, one RP patient (homozygous for the p.R297* mutation) and three SP MPSIIIB patients (homozygous for the mutation p.S612G or p.R643C, or compound heterozygous for the mutations p.A72_G79dup8 and p.R565Q) were cultured at temperatures ranging from 37°C to 27°C and harvested at different time points to assess NAGLU activity, mRNA and protein levels, and NAGLU glycosylation. Intracellular localization of wild-type and mutant mCherry-tagged NAGLU was analyzed by immunofluorescence. RESULTS: In control fibroblasts NAGLU was present as a 85kDa precursor and a 82kDa mature form. In SP patients' fibroblasts cultured at 37°C, only the 85kDa form was detected. Culturing at lower temperatures resulted in higher NAGLU mRNA levels, increased levels of both precursor and mature NAGLU protein and improved processing. The formation of mature NAGLU corresponded with higher NAGLU activity levels. CONCLUSION: We show that the NAGLU protein consists of a precursor and a mature form and that in SP MPSIIIB patients' fibroblasts only the precursor protein is present at 37°C. Culturing at lower temperatures resulted in the formation of the mature, enzymatically active form, due to higher mRNA levels and improved processing.

Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene.

Refsum disease is an autosomal-recessively inherited disorder characterized clinically by a tetrad of abnormalities: retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia and elevated protein levels in the cerebrospinal fluid (CSF) without an increase in the number of cells in the CSF. All patients exhibit accumulation of an unusual branched-chain fatty acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), in blood and tissues. Biochemically, the disease is caused by the deficiency of phytanoyl-CoA hydroxylase (PhyH), a peroxisomal protein catalyzing the first step in the alpha-oxidation of phytanic acid. We have purified PhyH from rat-liver peroxisomes and determined the N-terminal amino-acid sequence, as well as an additional internal amino-acid sequence obtained after Lys-C digestion of the purified protein. A search of the EST database with these partial amino-acid sequences led to the identification of the full-length human cDNA sequence encoding PhyH: the open reading frame encodes a 41.2-kD protein of 338 amino acids, which contains a cleavable peroxisomal targeting signal type 2 (PTS2). Sequence analysis of PHYH fibroblast cDNA from five patients with Refsum disease revealed distinct mutations, including a one-nucleotide deletion, a 111-nucleotide deletion and a point mutation. This analysis confirms our finding that Refsum disease is caused by a deficiency of PhyH.

Cholesterol-deprivation increases mono-unsaturated very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is characterized by a striking and unpredictable variation in phenotypic expression. It ranges from a rapidly progressive and fatal cerebral demyelinating disease in childhood (CCALD), to the milder slowly progressive form in adulthood (AMN). X-ALD is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in reduced beta-oxidation of very long-chain fatty acids (VLCFA, >22 carbon atoms) in peroxisomes and elevated levels of VLCFA in plasma and tissues. Previously, it has been shown that culturing skin fibroblasts from X-ALD patients in lipoprotein-deficient medium results in reduced VLCFA levels and increased expression of the functionally redundant ALD-related protein (ALDRP). The aim of this study was to further resolve the interaction between cholesterol and VLCFA metabolism in X-ALD. Our data show that the reduction in 26:0 in X-ALD fibroblasts grown in lipoprotein-deficient culture medium (free of cholesterol) is offset by a significant increase in both the level and synthesis of 26:1. We also demonstrate that cholesterol-deprivation results in increased expression of stearoyl-CoA-desaturase (SCD) and increased desaturation of 18:0 to 18:1. Finally, there was no increase in [1-(14)C]-26:0 beta-oxidation. Taken together, we conclude that cholesterol-deprivation reduces saturated VLCFA, but increases mono-unsaturated VLCFA. These data may have implications for treatment of X-ALD patients with lovastatin.

High-Throughput Screen Fails to Identify Compounds That Enhance Residual Enzyme Activity of Mutant N-Acetyl-α-Glucosaminidase in Mucopolysaccharidosis Type IIIB.

BACKGROUND: In the severe neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB or Sanfilippo disease type B), deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU) results in accumulation of heparan sulfate. Patients present with a severe, rapidly progressing phenotype (RP) or a more attenuated, slowly progressing phenotype (SP). In a previous study, residual NAGLU activity in fibroblasts of SP patients could be increased by culturing at 30°C, probably as a result of improved protein folding and lysosomal targeting under these conditions. Chaperones are molecules which influence protein folding and could therefore have therapeutic potential in SP MPSIIIB patients. Here we studied the effects of 1,302 different compounds on residual NAGLU activity in SP MPSIIIB patient fibroblasts including 1,280 approved compounds from the Prestwick Chemical Library. METHODS: Skin fibroblasts of healthy controls, an SP MPSIIIB patient (homozygous for the temperature sensitive mutation p.S612G) and an RP MPSIIIB patient (homozygous for the p.R297* mutation and non-temperature sensitive), were used. A high-throughput assay for measurement of NAGLU activity was developed and validated, after which 1,302 different molecules were tested for their potential to increase NAGLU activity. RESULTS: None of the compounds tested were able to enhance NAGLU activity. CONCLUSIONS: This high-throughput screen failed to identify compounds that could enhance residual activity of mutant NAGLU in fibroblasts of SP MPSIIIB patients with temperature sensitive mutations. To therapeutically simulate the positive effect of lower temperatures on residual NAGLU activity, first more insight is needed into the mechanisms underlying this temperature dependent increase.

Rhizomelic chondrodysplasia punctata. Deficiency of 3-oxoacyl-coenzyme A thiolase in peroxisomes and impaired processing of the enzyme.

The rhizomelic form of chondrodysplasia punctata (RCDP) is a peroxisomal disorder characterized biochemically by an impairment of plasmalogen biosynthesis and phytanate catabolism. We have now found that the maturation of peroxisomal 3-oxoacyl-CoA thiolase is impaired in fibroblasts from RCDP patients. To establish the subcellular localization of the 3-oxoacyl-CoA thiolase precursor protein, cultured skin fibroblasts were fractionated on a continuous Nycodenz gradient. Only a small amount of 3-oxoacyl-CoA thiolase activity was present in the catalase-containing (peroxisomal) fractions of RCDP fibroblasts in comparison with control fibroblasts. Moreover, the amount of thiolase protein in immunoblots of the catalase-containing fractions was below the limit of detection. Finally, the beta-oxidation of [14C]palmitoyl-CoA was found to be reduced in these fractions. We conclude that the mutation in RCDP leads to a partial deficiency of 3-oxoacyl-CoA thiolase activity in the peroxisomes and, concomitantly, an impairment in the ability to convert the precursor of this protein to the mature form. The reduction of 3-oxoacyl-CoA thiolase activity results in a decrease in the rate of peroxisomal beta-oxidation of palmitoyl-CoA. However, the capacity of the peroxisomes to oxidize very-long-chain fatty acids must be sufficient to prevent excessive accumulation of these compounds in vivo.

Peroxisomal trans-2-enoyl-CoA reductase is involved in phytol degradation.

Phytol is a naturally occurring precursor of phytanic acid. The last step in the conversion of phytol to phytanoyl-CoA is the reduction of phytenoyl-CoA mediated by an, as yet, unidentified enzyme. A candidate for this reaction is a previously described peroxisomal trans-2-enoyl-CoA reductase (TER). To investigate this, human TER was expressed in E. coli as an MBP-fusion protein. The purified recombinant protein was shown to have high reductase activity towards trans-phytenoyl-CoA, but not towards the peroxisomal beta-oxidation intermediates C24:1-CoA and pristenoyl-CoA. In conclusion, our results show that human TER is responsible for the reduction of phytenoyl-CoA to phytanoyl-CoA in peroxisomes.

Purification of peroxisomal acyl-CoA: dihydroxyacetonephosphate acyltransferase from human placenta.

The peroxisomal enzyme acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT) was extracted from human placental membranes using CHAPS as a detergent in the presence of 1 M KCl. Prior to assay dipalmitoylphosphatidylcholine was added to the sample as eluted from the various columns in order to stabilize the protein for subsequent enzyme activity measurements at 37 degrees C. The enzyme was purified from the placental membrane using ocytl-Sepharose CL-4B chromatography, Hydroxyapatite HTP chromatography, CM-Sepharose CL-6B, PBE 94 chromatofocusing and TSK G3000 SW size exclusion chromatography. A final purification of more than 8000-fold with respect to the placental membranes was achieved with a final yield of about 5%. Upon chromatofocusing the peak of activity eluted at a pH of 5.1-5.3 indicating a low isoelectric point. A native M(r) of 60-80 kDa was calculated from HPLC size exclusion chromatography. SDS-PAGE of the final purified fraction showed one major band with a M(r) of 65 kDa. These results suggest that DHAPAT is a monomeric protein. A polyclonal antiserum raised against the purified fraction was prepared in rabbits. Immunoprecipitation experiments showed complete precipitation of DHAPAT activity in fractions prepared from human placenta, liver and skin fibroblasts. Immunoprecipitation was also used to determine the residual amount of DHAPAT protein in liver from a patient with the Zellweger syndrome. A value of about 10% was found, which closely corresponds to the residual amount of enzyme activity.

Identification and characterization of the mouse cDNA encoding acyl-CoA:dihydroxyacetone phosphate acyltransferase.

We used the amino acid sequence of human acyl-CoA:dihydroxyacetone phosphate acyltransferase (DHAPAT) as bait to screen the database of expressed sequence tags (dbEST) and identified several partial mouse cDNA clones showing high identity. Primers were selected based on the dbEST sequences and used for amplification of this transcript from cDNA prepared from mouse skin fibroblasts. The complete nucleotide sequence was then determined and revealed an open reading frame (ORF) of 2034 bp encoding a protein consisting of 678 amino acids with a calculated molecular mass of 76870. The deduced amino acid sequence showed high identity (80%) with that of human DHAPAT and also revealed a typical peroxisomal targeting signal type 1 (PTS1) at its extreme carboxy-terminus (alanine-lysine-leucine, AKL). Definitive evidence that this cDNA indeed codes for DHAPAT was obtained by heterologous expression in the yeast Saccharomyces cerevisiae. Northern blot analysis revealed high expression of DHAPAT especially in mouse heart, liver and testis.

Acyl-CoA:dihydroxyacetone phosphate acyltransferase in human skin fibroblasts: study of its properties using a new assay method.

In relation to the finding that human skin fibroblasts are capable of de novo either phospholipid biosynthesis, we have studied the properties of acyl-CoA:dihydroxyacetone phosphate acyltransferase in fibroblast homogenates using a new assay method. The results indicate that the acylation of dihydroxyacetone phosphate shows an optimum at pH 5.5 with a broad shoulder of activity up to pH 6.4 and a decline in activity up to pH 8.2. At pH 5.5 the acyltransferase accepts dihydroxyacetone phosphate, but not glycerol 3-phosphate as a substrate. Furthermore, the transferase activity was found to be membrane-bound and inactivated by Triton X-100 at concentrations above 0.025% (w/v). Similar properties have been described for the enzyme as present in rat-liver and guinea-pig liver peroxisomes. These data, together with the finding that acyl-CoA:dihydroxyacetone phosphate acyltransferase is deficient in cultured skin fibroblasts from patients without peroxisomes (Zellweger syndrome), suggest that in cultured skin fibroblasts the enzyme is primarily located in peroxisomes.

Production and characterisation of monoclonal antibodies against native and disassembled human catalase.

Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either monomer/dimer catalase or native, tetrameric enzyme. Three stable hybridoma clones were selected and the characteristics of the antibodies produced were investigated by ELISA, immunofluorescence, immunoprecipitation and immunoblotting experiments. One monoclonal antibody (17E10) was shown to interact with both native tetramer catalase and--to a lesser extent--with monomer/dimer catalase. Two monoclonal antibodies (10B12H9, 13A10) were found to react only with completely denatured catalase or with monomer/dimer catalase but not with native catalase.

Human liver L-alanine-glyoxylate aminotransferase: characteristics and activity in controls and hyperoxaluria type I patients using a simple spectrophotometric method.

We have studied the characteristics of human liver alanine-glyoxylate aminotransferase, which is deficient in hyperoxaluria type I, an inherited disorder of glyoxylate metabolism. The enzyme was optimally active at pH 8.0 showing apparent Km values for L-alanine and glyoxylate of 8.3 and 1.3 mmol/l, respectively. Activity was found to proceed linearly for up to 4 h. Measurements under these optimal conditions enabled the biochemical diagnosis of hyperoxaluria type I to be made via enzyme activity measurements in percutaneous needle biopsy specimens of liver tissue.

Carnitine biosynthesis. Purification of gamma-butyrobetaine hydroxylase from rat liver.

gamma-Butyrobetaine hydroxylase catalyse the last step in carnitine biosynthesis, the formation of L-carnitine from gamma-butyrobetaine, a reaction dependent on Fe2+, alpha-ketoglutarate, ascorbate and oxygen. Initial attempts to purify the protein from rat liver showed that gamma-butyrobetaine hydroxylase is unstable. We, therefore, determined the influence of various compounds on the stability of gamma-butyrobetaine hydroxylase at different storage temperatures. The enzyme activity was best conserved by storing the protein at 4 degrees C in the presence of 200 g/l glycerol and 10 mM DTT. We subsequently purified the enzyme from rat liver to apparent homogeneity by liquid chromatography.

Identification of fatty acid oxidation disorder patients with lowered acyl-CoA thioesterase activity in human skin fibroblasts.

BACKGROUND: Acyl-CoA thioesterases are enzymes that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A (CoASH). These enzymes have been identified in several cellular compartments and are thought to regulate intracellular levels of acyl-CoAs, free fatty acids and CoASH. However, to date no patients deficient in acyl-CoA thioesterases have been identified. DESIGN: Acyl-CoA thioesterase activity was measured in human skin fibroblasts. Western-blot analysis was used to determine Type-II acyl-CoA thioesterase protein levels in patients. RESULTS: Acyl-CoA thioesterase activity was found in human fibroblasts with all saturated acyl-CoAs from C4-CoA to C18-CoA, with highest activity detected with lauroyl-CoA and myristoyl-CoA (C12-CoA and C14-CoA). An antibody that recognizes the major isoforms of Type-II acyl-CoA thioesterases precipitated the majority of acyl-CoA thioesterase activity in fibroblasts, showing that the main thioesterase activity detected in fibroblasts is catalyzed by Type-II thioesterases. Measurement of acyl-CoA thioesterase activity from fibroblasts of 34 patients with putative fatty acid oxidation disorders resulted in the identification of three patients with lowered Type-II acyl-CoA thioesterase activity in fibroblasts. These patients also had lowered expression of Type-II acyl-CoA thioesterase protein in fibroblasts as judged by Western-blot analysis. However, mutation analysis failed to identify any mutation in the coding sequences for the mitochondrial acyl-CoA thioesterase II (MTE-II) or the cytosolic acyl-CoA thioesterase II (CTE-II). CONCLUSIONS: We have described three patients with lowered Type-II acyl-CoA thioesterase protein and activity in human skin fibroblasts, which is the first description of patients with a putative defect in acyl-CoA thioesterases.

Etherphospholipid biosynthesis and dihydroxyactetone-phosphate acyltransferase: resolution of the genomic organization of the human gnpat gene and its use in the identification of novel mutations.

Etherphospholipids are characterised by the occurrence of an alkyl- or alkenyl-group at the sn-1 position of the glycerol backbone. Peroxisomes play an essential role in the formation of etherphospholipids since the first two enzymes of the biosynthetic pathway are strictly peroxisomal. The function of plasmalogens is still an enigma but the recent identification of patients suffering from an isolated defect in either dihydroxyacetone phosphate acyltransferase (GNPAT) or alkyldihydroxyacetone phosphate synthase provides conclusive evidence that plasmalogens play an essential role for human survival and functioning. In this paper we report the complete genomic organisation of the GNPAT gene coding for the peroxisomal dihydroxyacetone phosphate acyltransferase. The gene is located on chromosome 1q42.12-43. It spans approximately 28 kb and consists of 16 exons and 15 introns. This information was used to analyse the GNPAT gene in 12 patients with GNPAT deficiency. All patients analysed were found to have mutations in their GNPAT gene. Of the 9 different mutations found, 2 were missense mutations, 2 small deletions, 1 insertion and 3 mutations were within splice donor/acceptor-sites. Another mutation created an alternative splice donor-site causing the partial deletion of an exon. The data obtained provide conclusive evidence for the major role of GNPAT in etherphospholipid biosynthesis.

Immunoblot analysis of peroxisomal proteins in liver and fibroblasts from patients.

Identification of a patient as suffering from a peroxisomal disorder usually starts by the finding of elevated very long-chain fatty acids in plasma and/or serum. This is followed by more detailed studies in blood, fibroblasts and tissues, including immunoblot analysis. Indeed, immunoblot analysis has become a valuable tool in the correct diagnosis and assignment of individual patients, except for X-linked adrenoleukodystrophy (X-ALD). We describe a simple immunoblotting procedure applicable to liver and fibroblast homo-genates using antibodies raised against catalase and the three beta-oxidation enzyme proteins acyl-CoA oxidase I, bifunctional protein and peroxisomal thiolase. The same procedure can also be used for chorionic villus biopsy specimens and has now become the method of choice for the prenatal diagnosis of Zellweger syndrome (and other disorders of peroxisome biogenesis) and rhizomelic chondrodysplasia punctata.

Molecular and biochemical characterization of rat gamma-trimethylaminobutyraldehyde dehydrogenase and evidence for the involvement of human aldehyde dehydrogenase 9 in carnitine biosynthesis.

The penultimate step in carnitine biosynthesis is mediated by gamma-trimethylaminobutyraldehyde dehydrogenase (EC 1.2.1.47), a cytosolic NAD(+)-dependent aldehyde dehydrogenase that converts gamma-trimethylaminobutyraldehyde into gamma-butyrobetaine. This enzyme was purified from rat liver, and two internal peptide fragments were sequenced by Edman degradation. The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA containing an open reading frame of 1485 base pairs encoding a polypeptide of 494 amino acids with a calculated molecular mass of 55 kDa. Expression of the coding sequence in Escherichia coli confirmed that the cDNA encodes gamma-trimethylaminobutyraldehyde dehydrogenase. The previously identified human aldehyde dehydrogenase 9 (EC 1.2.1.19) has 92% identity with rat trimethylaminobutyraldehyde dehydrogenase and has been reported to convert substrates that resemble gamma-trimethylaminobutyraldehyde. When aldehyde dehydrogenase 9 was expressed in E. coli, it exhibited high trimethylaminobutyraldehyde dehydrogenase activity. Furthermore, comparison of the enzymatic characteristics of the heterologously expressed human and rat dehydrogenases with those of purified rat liver trimethylaminobutyraldehyde dehydrogenase revealed that the three enzymes have highly similar substrate specificities. In addition, the highest V(max)/K(m) values were obtained with gamma-trimethylaminobutyraldehyde as substrate. This indicates that human aldehyde dehydrogenase 9 is the gamma-trimethylaminobutyraldehyde dehydrogenase, which functions in carnitine biosynthesis.

Carnitine biosynthesis: identification of the cDNA encoding human gamma-butyrobetaine hydroxylase.

gamma-Butyrobetaine hydroxylase (EC 1.14.11.1) is the last enzyme in the biosynthetic pathway of L-carnitine and catalyzes the formation of L-carnitine from gamma-butyrobetaine, a reaction dependent on alpha-ketoglutarate, Fe2+, and oxygen. We report the purification of the protein from rat liver to apparent homogeneity, which allowed N-terminal sequencing using Edman degradation. The obtained amino acid sequence was used to screen the expressed sequence tag database and led to the identification of a human cDNA containing an open reading frame of 1161 base pairs encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44.7 kDa. Heterologous expression of the open reading frame in the yeast Saccharomyces cerevisiae confirmed that the cDNA encodes the human gamma-butyrobetaine hydroxylase. Northern blot analysis showed gamma-butyrobetaine hydroxylase expression in kidney (high), liver (moderate), and brain (very low), while no expression could be detected in the other investigated tissues.

Molecular and Biochemical Characterization of Rat epsilon -N-Trimethyllysine Hydroxylase, the First Enzyme of Carnitine Biosynthesis.

epsilon-N-Trimethyllysine hydroxylase (EC ) is the first enzyme in the biosynthetic pathway of l-carnitine and catalyzes the formation of beta-hydroxy-N-epsilon-trimethyllysine from epsilon-N-trimethyllysine, a reaction dependent on alpha-ketoglutarate, Fe(2+), and oxygen. We purified the enzyme from rat kidney and sequenced two internal peptides by quadrupole-time-of-flight mass spectroscopy. The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA of 1218 base pairs encoding a polypeptide of 405 amino acids with a calculated molecular mass of 47.5 kDa. Using the rat sequence we also identified the homologous cDNAs from human and mouse. Heterologous expression of both the rat and human cDNAs in COS cells confirmed that they encode epsilon-N-trimethyllysine hydroxylase. Subcellular fractionation studies revealed that the rat enzyme is localized exclusively in mitochondria. Expression studies in yeast indicated that the rat enzyme is synthesized as a 47.5-kDa precursor and subsequently processed to a mature protein of 43 kDa, presumably upon import in mitochondria. The Michaelis-Menten constants of the purified rat enzyme for trimethyllysine, alpha-ketoglutarate, and Fe(2+) were 1.1 mm, 109 microm, and 54 microm, respectively. Both gel filtration and blue native polyacrylamide gel electrophoresis analysis showed that the native enzyme has a mass of approximately 87 kDa, indicating that in rat epsilon-N-trimethyllysine hydroxylase is a homodimer.

Demonstration of dimethylnonanoyl-CoA thioesterase activity in rat liver peroxisomes followed by purification and molecular cloning of the thioesterase involved.

Peroxisomes play an indispensable role in cellular fatty acid oxidation in higher eukaryotes by catalyzing the chain shortening of a distinct set of fatty acids and fatty acid derivatives including pristanic acid (2,6,10,14-tetramethylpentadecanoic acid). Earlier studies have shown that pristanic acid undergoes three cycles of beta-oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA (DMN-CoA) which is then transported to the mitochondria for full oxidation to CO(2) and H(2)O. In principle, this can be done via two different mechanisms in which DMN-CoA is either converted into the corresponding carnitine ester or hydrolyzed to 4,8-dimethylnonanoic acid plus CoASH. The latter pathway can only be operational if peroxisomes contain 4,8-dimethylnonanoyl-CoA thioesterase activity. In this paper we show that rat liver peroxisomes indeed contain 4,8-dimethylnonanoyl-CoA thioesterase activity. We have partially purified the enzyme involved from peroxisomes and identified the protein as the rat ortholog of a known human thioesterase using MALDI-TOF mass spectrometry in combination with the rat EST database. Heterologous expression studies in Escherichia coli established that the enzyme hydrolyzes not only DMN-CoA but also other branched-chain acyl-CoAs as well as straight-chain acyl-CoA-esters. Our data provide convincing evidence for the existence of the second pathway of acyl-CoA transport from peroxisomes to mitochondria by hydrolysis of the CoA-ester in peroxisomes followed by transport of the free acid to mitochondria, reactivation to its CoA-ester, and oxidation to CO(2) and H(2)O. (c)2002 Elsevier Science.

Abnormal myelin formation in rhizomelic chondrodysplasia punctata type 2 (DHAPAT-deficiency).

The case of a Yemeni girl with isolated peroxisomal acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT) deficiency is reported. She had rhizomelic chondrodysplasia punctata, microcephaly, failure to thrive, delayed motor and mental development, and spastic quadriplegia. Deficient de novo plasmalogen synthesis in her fibroblasts as a result of low DHAPAT activity was found, while her very-long-chain fatty acid profile, phytanic acid concentration, alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) activity, and peroxisomal 3-ketoacyl-CoA thiolase protein were normal. A mutation in her DHAPAT complementary DNA resulted in the substitution of an arginine residue in the protein at position 211 by a histidine (R211H). Magnetic resonance imaging showed abnormal white matter signal in the centrum semiovale involving the arcuate fibers, while the corpus callosum was normal. DHAPAT and alkyl-DHAP synthase initiate the synthesis of plasmalogens, which are major constituents of myelin phospholipids. The reported girl's abnormal formation of myelin is probably related to the inadequacy of plasmalogen biosynthesis, which is likely to be due to deficient DHAPAT activity.