Nobuyuki Shimozawa

Mutation in PEX16 Is Causal in the Peroxisome-Deficient Zellweger Syndrome of Complementation Group D

Peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome (ZS), are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as by a malfunction of peroxisomes, among which>10 genotypes have been identified. We have isolated a human PEX16 cDNA (HsPEX16) by performing an expressed-sequence-tag homology search on a human DNA database, by using yeast PEX16 from Yarrowia lipolytica and then screening the human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex16p) made up of 336 amino acids. Among 13 peroxisome-deficiency complementation groups (CGs), HsPEX16 expression morphologically and biochemically restored peroxisome biogenesis only in fibroblasts from a CG-D patient with ZS in Japan (the same group as CG-IX in the United States). Pex16p was localized to peroxisomes through expression study of epitope-tagged Pex16p. One patient (PBDD-01) possessed a homozygous, inactivating nonsense mutation, C-->T at position 526 in a codon (CGA) for 176Arg, that resulted in a termination codon (TGA). This implies that the C-terminal half is required for the biological function of Pex16p. PBDD-01-derived PEX16 cDNA was defective in peroxisome-restoring activity when expressed in the patients fibroblasts. These results demonstrate that mutation in PEX16 is the genetic cause of CG-D PBDs.

Newly Identified Chinese Hamster Ovary Cell Mutants Are Defective in Biogenesis of Peroxisomal Membrane Vesicles (Peroxisomal Ghosts), Representing a Novel Complementation Group in Mammals

We isolated peroxisome biogenesis-defective mutants from Chinese hamster ovary cells by the 9-(1′-pyrene)nonanol/ultraviolet (P9OH/UV) method. Seven cell mutants, ZP116, ZP119, ZP160, ZP161, ZP162, ZP164, and ZP165, of 11 P9OH/UV-resistant cell clones showed cytosolic localization of catalase, a peroxisomal matrix enzyme, apparently indicating a defect of peroxisome biogenesis. By transfection of PEX cDNAs and cell fusion analysis, mutants ZP119 and ZP165 were found to belong to a novel complementation group (CG), distinct from earlier mutants. CG analysis by cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome indicated that ZP119 and ZP165 were in the same CG as the most recently identified human CG-J. The peroxisomal matrix proteins examined, including PTS1 proteins as well as a PTS2 protein, 3-ketoacyl-CoA thiolase, were also found in the cytosol in ZP119 and ZP165. Furthermore, these mutants showed typical peroxisome assembly-defective phenotype such as severe loss of resistance to 12-(1′-pyrene)dodecanoic acid/UV treatment. Most strikingly, peroxisomal reminiscent vesicular structures, so-called peroxisomal ghosts noted in all CGs of earlier Chinese hamster ovary cell mutants as well as in eight CGs of patients’ fibroblasts, were not discernible in ZP119 and ZP165, despite normal synthesis of peroxisomal membrane proteins. Accordingly, ZP119 and ZP165 are the first cell mutants defective in import of both soluble and membrane proteins, representing the 14th peroxisome-deficient CG in mammals, including humans.

Adrenoleukodystrophy protein enhances association of very long-chain acyl-coenzyme A synthetase with the peroxisome

Objective: To clarify the function of adrenoleukodystrophy protein (ALDP) using our ALDP-deficient mice established by gene targeting. Background: X-linked adrenoleukodystrophy (ALD) is characterized biochemically by the accumulation of very long-chain fatty acids (VLCFA) in tissues and body fluids, and is caused by impairment of peroxisomal β-oxidation. In ALD, very long-chain acyl-coenzyme A synthetase (VLACS), which is necessary for peroxisomal β-oxidation, does not function. Methods: The ALDP-deficient mice and C57BL/6J mice were used. VLACS or ALDP were transiently expressed by lipofection in murine fibroblasts, and VLCFA β-oxidation was assayed. Liver peroxisomes were purified by sequential centrifugations and a Nycodenz gradient centrifugation. The peroxisomal localization of VLACS was compared between the mutant and control mice using a Western blot analysis. Results: Impairment of VLCFA β-oxidation in ALDP-deficient fibroblasts was not corrected by the additional expression of VLACS alone but was by the coexpression of VLACS and ALDP. Although the tissue-specific expression of VLACS was similar in ALDP-deficient and normal mice, peroxisomal VLACS was clearly lower in ALDP-deficient than in normal mice. Conclusions: ALDP plays a role in the peroxisomal localization of VLACS, and VLACS does not function unless localized in the peroxisome.

Genomic structure and identification of 11 novel mutations of the PEX6 (peroxisome assembly factor-2) gene in patients with peroxisome biogenesis disorders

The PEX6 (peroxisome assembly factor‐2, PAF‐2) gene encodes a member of the AAA protein (ATPases associated with diverse cellular activities) family and restores peroxisome assembly in fibroblasts from peroxisome biogenesis disorder patients belonging to complementation group C (group 4 in the United States). We have now clarified the genomic DNA structure of human PEX6 and identified mutations in patients from various ethnic groups. The human PEX6 gene consists of 17 exons and 16 introns, spanning about 14kb. The largest exon, exon 1, has at least 952 bp nucleotides. Eleven novel mutations (18 alleles) were identified by direct sequencing of the PEX6 cDNA from 10 patients. All these mutations have been confirmed in the corresponding genomic DNA. There was no common mutation, but an exon skip was identified in two unrelated Japanese patients. Most of the mutations led to premature termination or large deletions of the PEX6 protein and resulted in the most severe peroxisome biogenesis disorder phenotype of Zellweger syndrome. A patient with an atypical Zellweger syndrome had a missense mutation that was shown to disrupt the cells ability to form peroxisomes. This mutation analysis will aid in understanding the functions of the PEX6 protein in peroxisomal biogenesis. Hum Mutat 13:487–496, 1999.

Accumulation of glycolipids in mutant Chinese hamster ovary cells (Z65) with defective peroxisomal assembly and comparison of the metabolic rate of glycosphingolipids between Z65 cells and wild-type CHO-K1 cells

The influence of peroxisomal dysfunction on glycosphingolipid metabolism was investigated using mutant Chinese hamster ovary (CHO) cells (Z65) with defective assembly of the peroxisomal membranes. In accordance with previous observations, the concentration of very long chain fatty acid (C24:0) was shown to be higher in Z65 cells than in control cells. We then compared the composition of glycolipids in Z65 cells with that in CHO-K1 cells, which are wild-type Chinese hamster ovary cells with intact peroxisomes, and found significantly increased concentrations of ceramide monohexoside (CMH) and ganglioside GM3 in Z65 cells. However, there were no differences in the concentrations of glycerophospholipids, triglycerides, free fatty acids and cholesterol between Z65 and CHO-K1 cells. Further, to investigate the metabolic rate of the major lipids, Z65 and CHO-K1 cells were pulse-labeled with [3-14C]serine. [3-14C]Serine was incorporated into phosphatidylserine, phosphatidylethanolamine and sphingomyelin more quickly in CHO-K1 than in Z65 cells. However, after 48 h, the radioactivity incorporated into those lipids, including CMH, was greater in Z65 cells than in CHO-K1 cells. Thus, the altered metabolism of glycosphingolipids, probably due to peroxisomal dysfunction, was thought to be responsible for the change in glycosphingolipid composition in Z65 cells.

A NOVEL NONSENSE MUTATION OF THE PEX7 GENE IN A PATIENT WITH RHIZOMELIC CHONDRODYSPLASIA PUNCTATA

AbstractMutations in the PEX7 gene encoding a peroxisome targeting signal 2 (PTS2) were identified in two patients with rhizomelic chondrodysplasia punctata (RCDP). A 7-year-old girl, the first Japanese individual to be diagnosed biochemically as a case of RCDP, had a novel nonsense mutation, R232ter, in the PEX7 gene, which had been inherited from her consanguineous parents. Another patient, a Chilean boy with RCDP, had compound heterozygous mutations of PEX7, L292ter and A218V, both of which have been documented. R232ter, which deletes all of the last two WP40 repeats in the PEX7 gene, is sufficient to inactivate functions of the PEX7 gene.

Amino acid and nucleotide sequences of human peroxisomal enoyl-CoA hydratase : 3-hydroxyacyl-CoA dehydrogenase cDNA

Deficiency of enoyl-CoA hydratase : 3-hydroxyacyl-CoA dehydrogenase (peroxisomal bifunctional enzyme), one of the enzymes of the peroxisomal β-oxidation system, leads to clinical manifestations resembling Zellweger syndrome with hypotonia, psychomotor delay, hepatomegaly, typical facial appearance and accumulation of very long-chain fatty acids. The nucleotide sequence of the human peroxisomal enoyl-CoA hydratase : 3-hydroxyacyl-CoA dehydrogenase cDNA has been reported by Hoefler and colleagues; however, we have found some amino acid differences from our originally isolated cDNA. Contrary to the findings described in a previous paper, we report here the cDNA sequence of human peroxisomal enoyl-CoA hydratase : 3-hydroxyacyl-CoA dehydrogenase in which there are 9 authenticated amino acid alterations.

Prenatal diagnosis of peroxisome biogenesis disorders by means of immunofluorescence staining of cultured chorionic villous cells.

To the Editor: Peroxisome biogenesis disorders (PBD) are fatal autosomal recessive diseases characterized by severe brain and hepatic dysfunction, facial dysmorphism, multiple metabolic dysfunction, and absence of peroxisomes. Zellweger syndrome (ZS) is the most severe phenotype and patients usually die within a year after birth. Neonatal adrenoleukodystrophy (NALD) is less severe and infantile Refsum disease is the mildest phenotype (1). Prenatal diagnosis of PBD is usually made by the findings including deficient enzyme activities of peroxisomal b-oxidation, plasmalogen biosynthesis, and absence of peroxisomes in cultured amniocytes (2, 3). Chorionic villi can be also utilized for the biochemical analyses (2, 4) and morphological investigation of peroxisomes (5). However, indirect immunofluorescence staining of peroxisomes in cultured villous cells has not been reported. Here we present the prenatal diagnosis of PBD by means of immunofluorescence staining using cultured villous fibroblasts. Chorionic villi were obtained by suction from three subjects who elected to take prenatal diagnosis for PBD (two ZS and one NALD) in their 10th week of gestation under the informed consent of the parents and the permission of the Investigational Review Board of the University. Villi were thoroughly washed with sterile saline, minced with scissors, put into culture dishes, then culture medium (MEM supplemented with 10% FCS) was added 15 min later and incubated in a CO2 incubator for about 2 weeks. Villous fibroblasts growing around villous tissues were harvested with trypsin and seeded again onto sterile fluorescence-free cover slips in culture dishes. The following day, these cells were fixed with 4% paraformaldehyde and stained by indirect immunofluorescence staining using antibody against human erythrocyte catalase and FITC-conjugated goat F(ab%)2 antirabbit IgG (TAGO, Burlingame, CA) (6). Fig. 1 shows the results of immunofluorescence staining. Peroxisomes were visualized in villous cells from cases 1 and 2 (left), suggesting that the fetuses were normal. Peroxisomes were also detected in amniocytes (right). Biochemical studies of amniocytes supported the diagnosis (data not shown). The newborn babies were healthy. On the other hand, peroxisomes were not detected in villous cells from case 3. Biochemical and morphological studies of amniocytes also suggested that the fetus was affected. Pregnancy was terminated according to the parents’ will. Autopsy findings of the fetus were compatible with a diagnosis of ZS.