Peroxisome Biogenesis Disorders Patients Research Articles

Molecular Mechanism of Detectable Catalase-Containing Particles, Peroxisomes, in Fibroblasts from a PEX2-Defective Patient

Patients with peroxisome biogenesis disorders (PBD) can be identified by detection of peroxisomes in their fibroblasts, by means of immunocytochemical staining using an anti-catalase antibody. We report here data on three PBD patients with newly identified mutations (del550C and del642G) in the PEX2 gene which encodes a 35-kDa peroxisomal membrane protein containing two membrane-spanning and a C-terminal cysteine-rich region. Some of the fibroblasts from the patient with the del642G mutation contained numerous catalase-containing particles, whereas no fibroblasts containing such particles were found in the patient with the del550C mutation. We confirmed that the del642G mutation caused a partial defect in peroxisome synthesis and import by expression of the mutated PEX2 into PEX2-defective CHO mutant cells. We propose that the two putative membrane-spanning segments in Pex2p are important domains for peroxisome assembly and import and that a defect in one of these domains severely affects PBD patients. Furthermore, a defect in the C-terminal portion of Pex2p exposed to the cytosol containing a RING finger motif caused the mild phenotype, residual enzyme activities, and mosaic detectable peroxisomes in fibroblasts from the patient.

L-Pipecolic acid oxidase, a human enzyme essential for the degradation of L-pipecolic acid, is most similar to the monomeric sarcosine oxidases

L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, we cloned the associated human gene and expressed its protein product. The cDNA was cloned with the use of a reverse genetics approach based on the amino acid sequence obtained from purified L-pipecolic acid oxidase from monkey. The complete cDNA, obtained by conventional library screening and 5' rapid amplification of cDNA ends, encompassed an open reading frame of 1170 bases, translating to a 390-residue protein. The translated protein terminated with the sequence AHL, a peroxisomal targeting signal 1. Indirect immunofluorescence studies showed that the protein product was expressed in human fibroblasts in a punctate pattern that co-localized with the peroxisomal enzyme catalase. A BLAST search with the amino acid sequence showed 31% identity and 53% similarity with Bacillus sp. NS-129 monomeric sarcosine oxidase, as well as similarity to all sarcosine oxidases and dehydrogenases. No similarity was found to the peroxisomal D-amino acid oxidases. The recombinant enzyme oxidized both L-pipecolic acid and sarcosine. However, PBD patients who lack the enzyme activity accumulate only L-pipecolic acid, suggesting that in humans in vivo, this enzyme is involved mainly in the degradation of L-pipecolic acid.

Phenotype-genotype relationships inPEX10-deficient peroxisome biogenesis disorder patients

The peroxisome biogenesis disorders (PBD) are characterized by neural, hepatic, and renal deficiencies, severe mental retardation, and are often lethal. These disorders are genetically and phenotypically heterogeneous and are caused by defective peroxisomal protein import and decreased peroxisomal metabolic function. Mutations in PEX10 have been identified in patients from complementation group 7 (CG7) of the PBDs and we report here an analysis of the genotypes and phenotypes of PEX10-deficient patients. All four PEX10-deficient Zellweger Syndrome (ZS) patients were found to have nonsense, frameshift, or splice site mutations that remove large portions of the PEX10 coding region. In contrast, a more mildly affected PEX10-deficient neonatal adrenoleukodystrophy patient expressed a PEX10 allele with a missense mutation, H290Q, affecting the C-terminal zinc-binding domain of the PEX10 product. These results support the hypothesis that severe, loss-of-function mutations in PEX genes cause more severe clinical phenotypes, whereas mildly affected PBD patients have PEX gene mutations that retain residual function. To quantitate the effects of the PEX10 mutations identified here and elsewhere we employed a functional complementation assay. Surprisingly, we observed that nonsense and frameshift mutations predicted to delete the C-terminal 2/3 (R125X) or 1/3 (c.704insA) of the protein displayed nearly normal PEX10 activity. Even more surprising, we found that the unexpectedly high PEX10 activity displayed by these cDNAs could be eliminated by removing or mutating segments of the PEX10 cDNA downstream of the mutations. Although these results demonstrate serious flaws in the PEX10 functional complementation assay, they do suggest that the C-terminal zinc-binding domain is critical for PEX10 function.

Metabolic control of peroxisome abundance.

Zellweger syndrome and related disorders represent a group of lethal, genetically heterogeneous diseases. These peroxisome biogenesis disorders (PBDs) are characterized by defective peroxisomal matrix protein import and comprise at least 10 complementation groups. The genes defective in seven of these groups and more than 90% of PBD patients are now known. Here we examine the distribution of peroxisomal membrane proteins in fibroblasts from PBD patients representing the seven complementation groups for which the mutant gene is known. Peroxisomes were detected in all PBD cells, indicating that the ability to form a minimal peroxisomal structure is not blocked in these mutants. We also observed that peroxisome abundance was reduced fivefold in PBD cells that are defective in the PEX1, PEX5, PEX12, PEX6, PEX10, and PEX2 genes. These cell lines all display a defect in the import of proteins with the type-1 peroxisomal targeting signal (PTS1). In contrast, peroxisome abundance was unaffected in cells that are mutated in PEX7 and are defective only in the import of proteins with the type-2 peroxisomal targeting signal. Interestingly, a fivefold reduction in peroxisome abundance was also observed for cells lacking either of two PTS1-targeted peroxisomal beta-oxidation enzymes, acyl-CoA oxidase and 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase. These results indicate that reduced peroxisome abundance in PBD cells may be caused by their inability to import these PTS1-containing enzymes. Furthermore, the fact that peroxisome abundance is influenced by peroxisomal 105-oxidation activities suggests that there may be metabolic control of peroxisome abundance.

Mutations in PEX1 in peroxisome biogenesis disorders: G843D and a mild clinical phenotype.

Peroxisomes are single, membrane-bound organelles present in nearly all eukaryotic cells (Lazarow and Moser 1995). The polypeptide composition of the peroxisomal membrane is distinct from that of other organelles. The peroxisome matrix has more than 50 enzymes involved in numerous metabolic pathways. Biogenesis of peroxisomes appears to proceed by import of newly synthesized proteins from the cytoplasm into existing peroxisomes, which enlarge and divide. Most peroxisomal proteins carry a peroxisomal targeting signal, PTS1 or PTS2 for matrix enzymes and mPTS for membrane proteins (Dyer et al 1996; Gould et al 1989; Swinkels et al 1991). The peroxisome biogenesis disorders (PBD) are a group of lethal autosomal-recessive diseases with overlapping phenotypes including Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease (Lazarow and Moser 1995). Failure to assemble normal peroxisomes, with the concomitant loss of multiple peroxisomal enzyme activities, is the cellular hallmark. Somatic cell fusion studies indicate that there are at least 10 complementation groups for PBDs, with complementation group (CG) 1 accounting for more than half of all PBD patients. Recently, PEXI encoding a member of the AAA protein family of ATPases was found to be the gene responsible for CG 1 (Portsteffen et al 1997; Reuber et al 1997). We report a common PEXI gene mutation, G843D, in relation to the PBD phenotype in a series of unrelated German patients in CG 1.

Genomic Structure ofPEX13,a Candidate Peroxisome Biogenesis Disorder Gene

The peroxisome biogenesis disorders (PBDs) are a set of lethal genetic diseases characterized by peroxisomal metabolic deficiencies, multisystem abnormalities, mental retardation, and premature death. These disorders are genetically heterogeneous and are caused by mutations in genes, termedPEXgenes, required for import of proteins into the peroxisomal matrix. We have previously reported the identification of humanPEX13,the gene encoding the docking factor for the PTS1 receptor, or PEX5 protein. As such, mutations inPEX13would be expected to abrogate peroxisomal protein import and result in PBD phenotypes. We report here the structure of the humanPEX13gene.PEX13spans approximately 11 kb on chromosome 2 and contains four exons, one more than previously thought. The corrected PEX13 cDNA is predicted to encode a protein product with a molecular mass of 44,312 Da. We examined the ability ofPEX13expression to rescue the peroxisomal protein import defects of fibroblast cells representing all known PBD complementation groups. No complementation was observed, suggesting that this gene is not mutated in any set of existing patients. However, given that complementation group assignments have been determined for only a subset of PBD patients, it is possible thatPEX13-deficient patients may exist at a low frequency within our existing PBD patient population or within ethnic groups underrepresented in our patient pool.

Phenotype-Genotype Relationships in Complementation Group 3 of the Peroxisome-Biogenesis Disorders

The peroxisome-biogenesis disorders (PBDs) are a set of often lethal genetic diseases characterized by mental retardation and defective peroxisomal matrix protein import. Mutations in PEX12 are known to underlie the disease in two patients from complementation group 3 of the PBDs. Here we show that all patients from this group carry mutations on both alleles of PEX12. A comparison between PEX12 genotypes and the clinical and cellular phenotypes of the corresponding PBD patients suggests a relatively straightforward relationship between genotype and phenotype in this group of the PBDs, such that the loss of PEX12 function leads to more-severe cellular and clinical phenotypes. However, one patient who presented relatively mild clinical and cellular phenotypes was a compound heterozygote for two seemingly severe mutations on each PEX12 allele. PEX12 mRNA present in the patient's cells was derived from only one allele, the one that carried a 2-bp deletion early in the PEX12 coding region, c.26,27Delta. The deduced protein product of this mRNA would contain only the first eight amino acids of the protein, and yet this mutant PEX12 cDNA displayed significant PEX12 activity in a functional complementation assay. Surprisingly, the PEX12/c.26, 27Delta cDNA directed the synthesis of a 29-kD PEX12 protein in vitro, a result that is consistent with translation initiation at a downstream AUG codon. Transfection studies confirmed the expression of similarly sized PEX12 proteins from the PEX12/c.26,27Delta allele. Thus, it appears that translation initiation at internal AUG codons may modulate disease phenotypes and should be considered whenever unexpectedly mild phenotypes result from severe mutations early in the coding region.

Identification of PEX10, the Gene Defective in Complementation Group 7 of the Peroxisome-Biogenesis Disorders

The peroxisome-biogenesis disorders (PBDs) are a group of genetically heterogeneous, lethal diseases that are characterized by neuronal, hepatic, and renal abnormalities; severe mental retardation; and, in their most severe form, death within the 1st year of life. Cells from all PBD patients exhibit decreased import of one or more classes of peroxisome matrix proteins, a phenotype shared by yeast pex mutants. We identified the human orthologue of yeast PEX10 and observed that its expression rescues peroxisomal matrix-protein import in PBD patients' fibroblasts from complementation group 7 (CG7). In addition, we detected mutations on both copies of PEX10 in two unrelated CG7 patients. A Zellweger syndrome patient, PBD100, was homozygous for a splice donor-site mutation that results in exon skipping and loss of 407 bp from the PEX10 open reading frame. A more mildly affected neonatal adrenoleukodystrophy patient was a compound heterozygote for a missense mutation in the PEX10 zinc-binding domain, H290Q, and for a nonsense mutation, R125ter. Although all three mutations attenuate PEX10 activity, the two alleles detected in the mildly affected patient, PBD052, encode partially functional PEX10 proteins. PEX10-deficient PBD100 cells contain many peroxisomes and import peroxisomal membrane proteins but do not import peroxisomal matrix proteins, indicating that loss of PEX10 has its most pronounced effect on peroxisomal matrix-protein import.

Mutations in PEX1 are the most common cause of peroxisome biogenesis disorders.

The peroxisome biogenesis disorders (PBDs) are a group of lethal autosomal-recessive diseases caused by defects in peroxisomal matrix protein import, with the concomitant loss of multiple peroxisomal enzyme activities. Ten complementation groups (CGs) have been identified for the PBDs, with CG1 accounting for 51% of all PBD patients. We identified the human orthologue of yeast PEX1, a gene required for peroxisomal matrix protein import. Expression of human PEX1 restored peroxisomal protein import in fibroblasts from 30 CG1 patients, and PEX1 mutations were detected in multiple CG1 probands. A common PEX1 allele, G843D, is present in approximately half of CG1 patients and has a deleterious effect on PEX1 activity. Phenotypic analysis of PEX1-deficient cells revealed severe defects in peroxisomal matrix protein import and destabilization of PEX5, the receptor for the type-1 peroxisomal targetting signal, even though peroxisomes were present in these cells and capable of importing peroxisomal membrane proteins. These data demonstrate an important role for PEX1 in peroxisome biogenesis and suggest that mutations in this gene are the most common cause of the PBDs.

Isolation of the human PEX12 gene, mutated in group 3 of the peroxisome biogenesis disorders.

The peroxisome biogenesis disorders (PBDs) are a group of genetically heterogeneous diseases lethal in early infancy. Although the clinical features of PBD patients may vary, cells from all PBD patients exhibit a defect in the import of one or more classes of peroxisomal matrix proteins. This cellular phenotype is shared by yeast pex mutants, and human orthologues of yeast PEX genes have been shown to be defective in some groups of PBD patients. We identified a putative human orthologue of ScPEX12 by screening the database of expressed sequence tags for cDNAs capable of encoding a protein similar to yeast Pex12p. Although its sequence similarity to yeast Pex12 proteins was limited, PEX12 shared the same subcellular distribution as yeast Pex12p and localized to the peroxisome membrane. PEX12 expression restored peroxisomal protein import in fibroblasts from PBD patients of complement group 3 (CG3) and frameshift mutations in PEX12 were detected in two unrelated CG3 patients. These data demonstrate that mutations in PEX12 are responsible for CG3 of the PBD and that PEX12 plays an essential role in peroxisomal matrix protein import.