Search

To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions. In an initial series of biopanning experiments, four different cDNA clones were obtained. These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1). A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms. Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells. The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1. That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein. These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR. In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins.

Pex5p is the receptor for the peroxisomal targeting signal 1 (PTS1) that consists of a C-terminal tripeptide (consensus (S/A/C)(K/R/H)(L/M)). Hexadecapeptides recognized by Pex5p from Homo sapiens and Saccharomyces cerevisiae were identified by screening a two-hybrid peptide library, and the targeting ability of the peptides was demonstrated using the green fluorescent protein as reporter. The PTS1 receptors recognized in a species-specific manner a broad range of C-terminal tripeptides, and these are reported herein. In addition, residues upstream of the tripeptide influenced the strength of the interaction in the two-hybrid system as well as in an in vitro competition assay. In peptides interacting with the human protein, hydrophobic residues were found with high frequency especially at positions -2 and -5, whereas peptides interacting with S. cerevisiae Pex5p were more hydrophilic and frequently contained arginine at position -2. In instances where the terminal tripeptide deviated from the consensus, upstream residues exerted a greater influence on the ability of the hexadecapeptides to bind Pex5p.

The discovery of many fatal human disorders resulting from impaired peroxisomal protein import makes the functional characterization of human peroxins critical. As part of our attempt to identify novel human genes and gene products involved in the import of peroxisomal proteins, we raised antisera against peroxisomal membrane proteins. One such antiserum inhibited peroxisomal protein import in semipermeabilized mammalian cells. This "import inhibiting" antiserum, ab-MF3, specifically recognized a 57-kDa protein. Immunoblot analysis of rat liver subcellular fractions demonstrated that this protein was present exclusively in peroxisomal membranes. Functional analysis revealed that this 57-kDa molecule bound the PTS1 receptor, Pex5p, in ligand blots, suggesting it is a docking site on the peroxisomal membrane. Previous studies have identified two yeast proteins, Pex14p and Pex13p, as Pex5p-binding proteins. To facilitate the biochemical analysis of peroxisomal membrane docking proteins, we cloned and expressed the previously unidentified human Pex14p, as well as a human Pex13p that is 39 aa longer than previously reported. Recombinant Pex14p was specifically recognized by the "import inhibiting" ab-MF3 and bound Pex5p and the Src homology 3 (SH3) domain of Pex13p in ligand blots. These studies demonstrate that the ab-MF3-immunoreactive, 57-kDa peroxisomal membrane protein is Pex14p. Furthermore, this peroxin interacts with Pex5p and Pex13p(SH3) and is directly required for peroxisomal protein import.

Peroxisomes in human liver contain two distinct acyl-CoA oxidases with different substrate specificities: (i) palmitoyl-CoA oxidase, oxidizing very long straight-chain fatty acids and eicosanoids, and (ii) a branched-chain acyl-CoA oxidase (hBRCACox), involved in the degradation of long branched fatty acids and bile acid intermediates. The accumulation of branched fatty acids and bile acid intermediates leads to severe mental retardation and death of the diseased children. In this study, we report the molecular characterization of the hBRCACox, a prerequisite for studying mutations in patients with a single enzyme deficiency. The composite cDNA sequence of hBRCACox, derived from overlapping clones isolated via immunoscreening and hybridization of human liver cDNA expression libraries, consisted of 2225 bases and contained an open reading frame of 2046 bases, encoding a protein of 681 amino acids with a calculated molecular mass of 76,739 Da. The C-terminal tripeptide of the protein is SKL, a known peroxisome targeting signal. Sequence comparison with the other acyl-CoA oxidases and evolutionary analysis revealed that, despite its broader substrate specificity, the hBRCACox is the human homolog of rat trihydroxycoprostanoyl-CoA oxidase (rTHCCox) and that separate gene duplication events led to the occurrence in mammals of acyl-CoA oxidases with different substrate specificities. Northern blot analysis demonstrated that--in contrast to the rTHCCox gene--the hBRCACox gene is transcribed also in extrahepatic tissues such as heart, kidney, skeletal muscle, and pancreas. The highest levels of the 2.6-kb mRNA were found in heart, followed by liver. The enzyme is encoded by a single-copy gene, which was assigned to chromosome 3p14.3 by fluorescent in situ hybridization. It was absent from livers of Zellweger patients as shown by immunoblot analysis and immunocytochemistry.

The composite trihydroxycoprostanoyl-CoA oxidase cDNA sequence, derived from overlapping clones isolated via screening of two different rat liver expression libraries, consisted of 2509 bases and contained an open reading frame of 2046 bases, encoding a protein of 681 amino acids with a calculated molecular mass of 76711 Da. The reading frame and identity of the trihydroxycoprostanoyl-CoA oxidase cDNA were confirmed by the location of various tryptic peptides, obtained from the purified enzyme, in the deduced amino acid sequence. The C-terminus (His-Lys-Met) of trihydroxycoprostanoyl-CoA oxidase did not seem to interact with the C-terminal peroxisomal targeting signal 1 (PTS1) import receptor, although the tripeptide fits the rule of conserved PTS1 variants for targeting of proteins to glycosomes of Trypanosomatidae. At the protein level, trihydroxycoprostanoyl-CoA oxidase showed 45% identical amino acids with rat palmitoyl-CoA oxidase, whereas the identity with pristanoyl-CoA oxidase was much lower (22%). Northern analysis of multiple rat tissues revealed a signal (approx. 2.6 kb) only in liver and (although much weaker) in kidney. Dot-blot analysis of total liver RNA revealed that the mRNA for trihydroxy-coprostanoyl-CoA oxidase is not induced after treatment of rats with structurally unrelated peroxisome proliferators and indicates that highly similar mRNAs are present in other mammals, including man. Immunocytochemistry showed a decrease in trihydroxycoprostanoyl-CoA oxidase protein in individual liver peroxisomes ('diluting-out effect') after treatment of rats with bezafibrate, whereas the palmitoyl-CoA oxidase labelling was significantly increased.

A procedure to visualize proteins containing a C-terminal peroxisomal targeting signal (PTS1) in complex protein mixtures was developed using a bacterially expressed, biotinylated form of the human PTS1-receptor. The binding of this fusion product to purified PTS1-containing proteins that were separated by SDS-PAGE and blotted onto nitrocellulose was detected by means of streptavidin-alkaline phosphatase and shown to be both saturable and specific. When applied to total tissue extracts, in addition to PTS1-containing proteins various endogenous biotinylated proteins were visualized. Therefore, a two-step staining procedure was optimized whereby the endogenous biotinylated proteins were shielded with a blue precipitate, followed by incubation with the biotinylated receptor and detection of the resulting PTS1-receptor/PTS1-protein complexes with a phosphatase reaction coupled to the formation of a red-colored precipitate. This relatively inexpensive, simple, and fast technique enabled us to visualize a variety of PTS1-containing proteins. In addition, the information presented in this study can be used to facilitate the identification and characterization of receptor-ligand interaction in general and to eliminate interference by endogenous biotinylated proteins intrinsic to the streptavidin-biotin detection system.

To identify proteins interacting with the C-terminal peroxisomal targeting signal (PTS1), we screened a human liver cDNA library by means of a Saccharomyces cerevisiae genetic system, known as the two-hybrid system. We isolated a cDNA encoding a protein that specifically bound the PTS1 topogenic signal in the intact yeast cell but also in vitro after bacterial expression and purification. Sequence analysis of the full-length cDNA revealed the presence of an open reading frame encoding a 70-kDa polypeptide that belongs to the tetratricopeptide repeat family and that is homologous to the PAS8 and PAS10 gene products, which are required for the formation of normal peroxisomes in yeast. Subcellular fractionation of human liver and immunofluorescence studies on HepG2 cells demonstrated that this PTS1-binding protein is present exclusively in peroxisomes and that the PTS1-binding domain is located to the cytosolic side of the peroxisomal membrane. All available evidence indicates that the PTS1-binding protein is part of the peroxisomal protein import machinery and most probably is the long sought after human PTS1 import receptor.

In an attempt to identify putative peroxisomal import receptors, we investigated the cross-linking of a radioiodinated peptide consisting of the 13 last amino acids of acyl-CoA oxidase and comprising the carboxy-terminal SKL-peroxisomal targeting motive, to proteins present in different subcellular fractions from rat liver. The radiolabeled peptide could be cross-linked to an 80 kDa protein present in the cytosol but not to proteins present in other subcellular fractions including highly purified peroxisomes. Binding was reversible, saturable and dependent on the presence of Mg2+ and ATP or GTP but hydrolysis of the nucleotides was not required. Binding was abolished by pretreatment of the cytosol--but not of the peptide--with N-ethylmaleimide. Binding was not specific for peptides containing the carboxy-terminal SKL-motive, since binding was competed for by the SKL-peptide from which the SKL-motive had been deleted, by the SKL-peptide with reversed sequence and by the SV40 T-antigen nuclear localisation signal peptide, but not by other peptides tested. The 80 kDa binding protein cross-reacted with a monoclonal antibody against hsp90. Purification and internal peptide sequencing of the binding protein revealed its identity as prolyl-endopeptidase. In retrospect, we realized that the SKL-peptide and all competing peptides contained a proline residue, which was not present in the non-competing peptides. In recent experiments in yeast McNew et al. (McNew, J.A., Sykes, K. and Goodman, J.M. (1993) Mol. Biol. Cell 4, 223-232) cross-linked a peroxisomal targeting peptide to a 20 kDa cytosolic protein that was identified as proline isomerase despite the fact that the peptide did not contain proline. The experiments by McNew et al. in yeast and our experiments in the rat suggest that the (peroxisomal) targeting sequence cross-linking approach may not be suited for the identification of (peroxisomal) import receptors.

Rat liver peroxisomes contain three acyl-CoA oxidases: palmitoyl-CoA oxidase, which oxidizes the CoA esters of straight chain fatty acids and prostaglandins; pristanoyl-CoA oxidase, which oxidizes the CoA esters of 2-methyl-branched fatty acids (e.g. pristanic acid); and trihydroxycoprostanoyl-CoA oxidase, which oxidizes the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (Van Veldhoven, P. P., Vanhove, G., Asselberghs, S., Eyssen, H. J., and Mannaerts, G. P. (1992) J. Biol. Chem. 267, 20065-20074). In the present report we demonstrate that human liver peroxisomes contain only two acyl-CoA oxidases: palmitoyl-CoA oxidase, which oxidizes the CoA esters of straight chain fatty acids and prostaglandins, and a novel branched chain acyl-CoA oxidase, which oxidizes the CoA esters of 2-methyl-branched fatty acids as well as those of the bile acid intermediates (which also possess a 2-methyl substitution in their side chains). The branched chain acyl-CoA oxidase was purified to near homogeneity by means of column chromatography. It appeared to be a 70-kDa monomeric protein that did not cross-react with antisera raised against rat palmitoyl-CoA oxidase and pristanoyl-CoA oxidase. No indication was found for the presence of a separate trihydroxycoprostanoyl-CoA oxidase in human liver. The branched chain acyl-CoA oxidase was present also in human kidney, suggesting that it is expressed in other extrahepatic tissues as well. Our results explain a number of clinical-chemical observations made in certain cases of peroxisomal beta-oxidation disorders.

Highly purified peroxisomal membranes stripped from their peripheral membrane proteins and only minimally contaminated with other membranes, contained three GTP-binding proteins of 29, 27 and 25 kDa, respectively. Bound radioactive GTP was displaced by unlabelled GTP, GTP analogs and GDP but not by GMP or other nucleotides. GTP binding was markedly decreased by trypsin treatment of intact purified peroxisomes; it increased 2-3-fold after pretreatment of the animals with a peroxisome proliferator. We conclude that the peroxisomal membrane contains small GTP-binding proteins that are exposed to the cytosol and that are firmly anchored in the membrane. We speculate that these proteins are involved in peroxisome multiplication by fission or budding during peroxisome biogenesis and proliferation.

Background: Peroxisome biogenesis disorders are a clinically and genetically heterogeneous group of very severe autosomal recessive disorders caused by impaired peroxisome biogenesis. The prototype of this group of disorders is the cerebro-hepato-renal syndrome of Zellweger. Methods and results: Here we report a patient with Zellweger syndrome, who presented at the age of 3 months with icterus, dystrophy, axial hypotonia, facial dysmorphy, posterior embryotoxon, and hepatomegaly. Abnormal findings of metabolic screening tests included hyperbilirubinaemia, hypoketotic dicarboxylic aciduria, increased C26.0 and decreased C22.0 plasma levels, and strongly reduced plasmalogen concentrations. In fibroblasts, both peroxisomal α- and β-oxidation were impaired. Liver histology revealed bile duct paucity, cholestasis, arterial hyperplasia, very small branches of the vena portae, and parenchymatic destruction. Immunocytochemical analysis of cultured fibroblasts demonstrated that the cells contain peroxisomal remnants lacking apparent matrix protein content and PEX1 4, a central membrane component of the peroxisomal matrix protein import machinery. Transfection of fibroblasts with a plasmid coding for wild-type PEX14 restored peroxisomal matrix protein import, indicating that the primary genetic defect affecting the patient is indeed linked to PEX14. Mutational analysis of this gene revealed a genomic deletion leading to the deletion of exon 3 from the coding DNA (c.85-?̱170+?del) and a concomitant change of the reading frame (p. [Ile29̱Lys56del;Gly57GlyfsX2]). Conclusions: This report represents the second PEX14-deficiency associated with Zellweger syndrome and the first documentation of a PEX14-deficient patient with detailed clinical follow-up and biochemical, morphological, and radiological data. [ABSTRACT FROM AUTHOR]