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

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

Human phytanoyl-CoA hydroxylase: resolution of the gene structure and the molecular basis of Refsum's disease.

Refsum's disease (RD) is an inherited neurological syndrome biochemically characterized by the accumulation of phytanic acid in plasma and tissues. Patients with RD are unable to degrade phytanic acid due to a deficient activity of phytanoyl-CoA hydroxyl-ase (PhyH), a peroxisomal enzyme catalysing the first step of phytanic acid alpha-oxidation. To enable mutation analysis of RD at the genome level, we have elucidated the genomic organization of the PHYH gene. The gene is approximately 21 kb and contains nine exons and eight introns. Mutation analysis of PHYH cDNA from 22 patients with RD revealed 14 different missense mutations, a 3 bp insertion, and a 1 bp deletion, which were all confirmed at the genome level. A 111 bp deletion identified in the PHYH cDNA of several patients with RD was due to either one of two different mutations in the same splice acceptor site, which result in skipping of exon 3. Six mutations, including a large in-frame deletion and five missense mutations, were expressed in the yeast Saccharomyces cerevisiae to study their effect on PhyH activity. The results showed that all these mutations lead to an enzymatically inactive PhyH protein.

Phytanoyl-CoA hydroxylase from rat liver. Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation.

Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway. Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes. In rat, however, the alpha-oxidation pathway has been reported to be mitochondrial. In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein. We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE. Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38. 6 kDa protein. The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2). Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa. This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2. This type of processing has been reported in several other peroxisomal proteins that contain a PTS2. Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat. The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid alpha-oxidation in rat and human.

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:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2.

Rhizomelic chondrodysplasia punctata (RCDP) is a genetic disorder which is clinically characterized by rhizomelic shortening of the upper extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation. Patients with RCDP can be subdivided into three subgroups based on biochemical analyses and complementation studies. The largest subgroup contains patients with mutations in the PEX7 gene encoding the PTS2 receptor. This results in multiple peroxisomal abnormalities which includes a deficiency of acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT), alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase), peroxisomal 3-ketoacyl-CoA thiolase and phytanoyl-CoA hydroxylase, although there are differences in the extent of the deficiencies observed. Patients in the two other subgroups have been reported to be either deficient in the activity of DHAPAT (RCDP type 2) or alkyl-DHAP synthase (RCDP type 3) while no other abnormalities could be observed. To examine whether the gene encoding DHAPAT is mutated in patients with RCDP type 2, we determined the N-terminal amino acid sequence of the enzyme isolated from human placenta. Using this sequence as a query, we identified a 2040 bp open reading frame (ORF) in the human database of expressed sequence tags. Expression of this ORF in the yeast Saccharomyces cerevisiae showed that we have identified the DHAPAT cDNA. The deduced amino acid sequence revealed no PTS2 consensus sequence. In contrast DHAPAT appears to contain a putative PTS1 at the extreme C-terminus. All RCDP type 2 patients analyzed were found to contain mutations in their DHAPAT cDNA. This demonstrates that RCDP type 2 is the result of mutations in DHAPAT.

Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a non-functional PTS2 receptor.

Rhizomelic chondrodysplasia punctata (RCDP) is an autosomal recessive disease characterized clinically by a disproportionately short stature primarily affecting the proximal parts of the extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation. Although some patients have single enzyme deficiencies, the majority of RCDP patients (86%) belong to a single complementation group (CG11, also known as complementation group I, Amsterdam nomenclature). Cells from CG11 show a tetrad of biochemical abnormalities: a deficiency of i) dihydroxyacetonephosphate acyltransferase, ii) alkyldihydroxyacetonephosphate synthase, iii) phytanic acid alpha-oxidation and iv) inability to import peroxisomal thiolase. These deficiencies indicate involvement of a component required for correct targeting of these peroxisomal proteins. Deficiencies in peroxisomal targeting are also found in Saccharomyces cerevisiae pex5 and pex7 mutants, which show differential protein import deficiencies corresponding to two peroxisomal targeting sequences (PTS1 and PTS2). These mutants lack their PTS1 and PTS2 receptors, respectively. Like S. cerevisiae pex cells, RCDP cells from CG11 cannot import a PTS2 reporter protein. Here we report the cloning of PEX7 encoding the human PTS2 receptor, based on its similarity to two yeast orthologues. All RCDP patients from CG11 with detectable PEX7 mRNA were found to contain mutations in PEX7. A mutation resulting in C-terminal truncation of PEX7 cosegregates with the disease and expression of PEX7 in RCDP fibroblasts from CG11 rescues the PTS2 protein import deficiency. These findings prove that mutations in PEX7 cause RCDP, CG11.