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N Shimozawa

Publications and source records attributed to N Shimozawa.

At least 55 records · Page 3Linked to original sources
PubMed1999

Isolation and characterization of novel peroxisome biogenesis-defective Chinese hamster ovary cell mutants using green fluorescent protein.

We developed an improved method for isolation of peroxisome biogenesis-defective somatic animal cell mutants, using a combination of green fluorescent protein (GFP) expression and the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) selection method. We used TKaG1 and TKaG2 cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNAs each encoding rat Pex2p as well as GFP tagged at the C-terminus with peroxisome targeting signal type 1 (PTS1) or N-terminally PTS2-tagged GFP. P9OH/UV-resistant cell colonies were examined for intracellular location of GFP on unfixed cells, by fluorescence microscopy. Seven each of the mutant cell clones isolated from TKaG1 and TKaG2 showed cytosolic GFP-PTS1 and PTS2-GFP, respectively, indicating the defect in peroxisome assembly. By transfection of PEX2, PEX5, PEX6, and PEX12 cDNAs and cell fusion analysis between the CHO cell mutants, five different complementation groups (CGs) were identified. Two mutant clones, ZPG207 and ZPG208, belonged to novel CGs. Further CG analysis using fibroblasts from patients with peroxisome biogenesis disorders, including rhizomelic chondrodysplasia punctata (RCDP), revealed that ZPG208 belonged to none of human CGs. ZPG207 was classified into the same CG as RCDP. Taken together, ZPG208 is in a newly identified, the 12th, CG in peroxisome-deficient CHO mutants reported to date and represents a novel mammalian CG.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

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.

Animals↗
PubMed1999

Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly.

At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein.

Amino Acid Sequence↗
PubMed1999

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 cell's 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.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

A novel nonsense mutation of the PEX7 gene in a patient with rhizomelic chondrodysplasia punctata.

Mutations 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 Sequence↗
PubMed1999

Prenatal diagnosis of peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency.

The prenatal diagnosis of peroxisomal D-3-hydroxyacyl-coenzyme A (CoA) dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-BP) deficiency was performed by peroxisomal beta-oxidation assay, indirect immunofluorescence staining, immunoblot analysis, and gene analysis of cultured amniocytes obtained from a fetus at 16 weeks' gestational age. beta-Oxidation activity, measured by [1-14C] lignoceric acid oxidation, was markedly decreased compared with the controls. Large peroxisomes were readily identified by immunofluorescence staining with anti-human catalase, as was found in the reported patients. Immunoreactive D-BP material was absent on immunoblot analysis and immunofluorescence staining with anti-human D-BP. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed the presence of the same 237-bp deletion in the cDNA as that detected in a sibling (the proband). The autopsied fetus showed the characteristic facial appearance and D-BP was deficient on immunoblot and immunohistopathological studies of the fetal tissues. No neuronal migration disorder was identified. This seems to be the first prenatal diagnosis of D-BP deficiency.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Nonsense and temperature-sensitive mutations in PEX13 are the cause of complementation group H of peroxisome biogenesis disorders.

Peroxisome biogenesis disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease, are lethal hereditary diseases caused by abnormalities in peroxisomal assembly. To date, 12 genotypes have been identified. We now have evidence that the complete human cDNA encoding Pex13p, an SH3 protein of a docking factor for the peroxisome targeting signal 1 receptor (Pex5p), rescues peroxisomal matrix protein import and its assembly in fibroblasts from PBD patients of complementation group H. In addition, we detected mutations on the human PEX13 cDNA in two patients of group H. A severe phenotype of a ZS patient (H-02) was homozygous for a nonsense mutation, W234ter, which results in the loss of not only the SH3 domain but also the putative transmembrane domain of Pex13p. A more mildly affected NALD patient (H-01), whose fibroblasts showed the temperature-sensitive (TS) phenotype, was homozygous for a missense mutation in the SH3 domain of Pex13p, I326T. This mutant PEX13 cDNA expression in a PEX13-defective CHO mutant showed I326T to be a TS mutation and thus suggested that Pex13p with the I326T mutation in the SH3 domain is stable at 30 degrees C but is somewhat unstable at 37 degrees C.

Amino Acid Sequence↗
PubMed1999

Isolation, characterization and mutation analysis of PEX13-defective Chinese hamster ovary cell mutants.

We isolated peroxisome biogenesis mutants ZP128 and ZP150 from rat PEX2 -transformed Chinese hamster ovary (CHO) cells, by the 9-(1'-pyrene)nonanol/ultraviolet method. The mutants lacked morphologically recognizable peroxisomes and showed a typical peroxisome assembly-defective phenotype such as a high sensitivity to 12-(1'-pyrene)dodecanoic acid/UV treatment. By means of PEX cDNA transfection and cell fusion, ZP128 and ZP150 were found to belong to a recently identified complementation group H. Expression of human PEX13 cDNA restored peroxisome assembly in ZP128 and ZP150. CHO cell PEX13 was isolated; its deduced sequence comprises 405 amino acids with 93% identity to human Pex13p. Mutation in PEX13 of mutant ZP150 was determined by RT-PCR: G to A transition resulted in one amino acid substitution, Ser319Asn, in one allele and truncation of a 42 amino acid sequence from Asp265 to Lys306 in another allele. Therefore, ZP128 and ZP150 are CHO cell lines with a phenotype of impaired PEX13.

Amino Acid Sequence↗
PubMed1999

Defective PEX gene products correlate with the protein import, biochemical abnormalities, and phenotypic heterogeneity in peroxisome biogenesis disorders.

Peroxisome biogenesis disorders (PBD) comprise three phenotypes including Zellweger syndrome (ZS) (the most severe), neonatal adrenoleucodystrophy, and infantile Refsum disease (IRD) (the most mild), and can be classified into at least 12 genetic complementation groups, which are not predictive of the phenotypes. Several pathogenic genes for PBD groups have been identified, but the relationship between the defective gene products and phenotypic heterogeneity has remained unclear. We identified a mutation in the PEX2 gene in an IRD patient with compound heterozygosity for a missense mutation and the known nonsense mutation detected in ZS patients. In transfection experiments using the peroxisome deficient CHO mutant, Z65 with a nonsense mutation in the PEX2 gene, we noted the E55K mutation had mosaic activities of peroxisomal protein import machinery and residual activities of peroxisomal functions, including dihydroxyacetone phosphate acyltransferase and beta oxidation of very long chain fatty acids. The nonsense mutation severely affects these peroxisomal functions as well as the protein import. These data suggest that allelic heterogeneity of the PEX gene affects the peroxisomal protein import and functions and regulates the clinical severity in PBD.

Catalase↗
PubMed1999

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 beta-oxidation. In ALD, very long-chain acyl-coenzyme A synthetase (VLACS), which is necessary for peroxisomal beta-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 beta-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 beta-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.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed1998

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.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

A cytoplasmic AAA family peroxin, Pex1p, interacts with Pex6p.

Human PEX1 (HsPEX1) is the causative gene for peroxisome-deficiency disorders such as Zellweger syndrome of complementation group I, encoding the peroxin, Pex1p, a member of AAA family. Pex1p tagged with an epitope flag was expressed in wild-type Chinese hamster ovary (CHO) cell, CHO-K1. Pex1p was localized in the cytoplasm, as assessed by immunofluorescent microscopy. Cell-lysate of HsPEX1-transfected CHO-K1 was incubated with in vitro synthesized 35S-labelled Pex6p, an AAA family peroxin. Immunoprecipitation of Pex1p using anti-Pex1p antibody resulted in concomitant recovery of 35S-Pex6p. Conversely, 35S-Pex1p was obtained in immunoprecipitate from CHO-K1 expressing human Pex6p, using anti-Pex6p antibody. These results strongly suggest that Pex1p and Pex6p interact with each other.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I.

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged "enhanced" green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 --> Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

ATPases Associated with Diverse Cellular Activitie↗