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Temperature sensitive acyl-CoA oxidase import in group A peroxisome biogenesis disorders

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Editor—Peroxisome biogenesis disorders (PBDs) are lethal genetic diseases characterised by a number of peroxisomal metabolic abnormalities, including the oxidation of very long chain fatty acids (VLCFAs), biosynthesis of bile acids and plasmalogen, and detoxification of H2O2. Peroxisomal matrix proteins are synthesised on free polyribosomes and directed to the organelle by cis acting peroxisome targeting signals (PTSs). PTS1 is a C-terminal tripeptide Ser-Lys-Leu (SKL) sequence and later the consensus sequence was broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L, based on subsequent studies. Acyl-CoA oxidase (AOX) has SKL and D bifunctional protein has AKL.1-4 PTS2 is an N-terminal cleavable peptide (-R/KLX5Q/HL) that resides in peroxisomal 3-ketoacyl CoA thiolase (PT), alkyl-dihydroxyacetonephosphate synthase, and phytanoyl-CoA hydroxylase.5-9 PBDs are genetically classified into at least 12 complementation groups (CGs) and each CG contains various clinical phenotypes, for example, Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD), and infantile Refsum disease (IRD).10 11 ZS patients have severe neurological defects, liver dysfunction, and renal cysts and die before 1 year of age. NALD patients have symptoms similar to ZS patients, but they survive a little longer, and IRD patients show milder abnormalities in the central nervous system and survive even longer. We identified the restoration of peroxisome biogenesis in a temperature sensitive (TS) manner in fibroblasts from milder forms of PBDs, that is, all IRD patients and some NALD patients belonging to groups CG-A (CG8), CG-C (CG4), CG-E (CG1), CG-F (CG10), CG-H, and CG6.12-15 In these cells, peroxisomes were formed at 30°C and biochemical activities of peroxisomes, including the oxidation of VLCFAs and dihydroxyacetonephosphate acyltransferase (DHAP-AT), and the import of peroxisomal enzymes, were also restored.16 However, virtually no peroxisomes were formed in ZS cells at 30°C and import of peroxisomal enzymes did not improve.16 Here, we elucidate temperature dependent import and processing of AOX at 30°C which is unique to fibroblasts from ZS patients belonging to CG-A. Correlation between the import of peroxisomal enzymes and biochemical functions of peroxisomes is also discussed.

Materials and methods


Skin fibroblasts from the patients belonging to CG-A (CG8) including four with ZS (A-02, 06, 10, and 14), two with NALD (A-05 and 08), and one with IRD (A-04) were cultured at 37°C or 30°C in an atmosphere of 5% CO2 in MEM supplemented with 10% fetal calf serum. A-02, A-06, and A-14 were Japanese babies diagnosed as ZS with typical dysmorphic features, who died at a few months of age. The clinical data of A-04 and A-08 have been previously reported, whereas those of A-05 and A-10 have not.12 In addition, ZS fibroblasts belonging to CG-C (C-08), CG-E (E-14), and CG-F (F-01) were cultured under the same condition (the numbers and clinical data of these patients have been previously described12). All cell lines were classified by complementation analysis as previously described.10 17 18


For the detection of peroxisomes and the import of PTSs, cells were fixed after 72 hours' incubation at either 37°C or 30°C, permeabilised with 0.1% Triton X-100, and processed for indirect immunofluorescence staining.19 The first antibodies we used were rabbit antibodies to human catalase, AOX, D bifunctional protein, and PT, and in double immunofluorescence rabbit anti-rat PMP70 antibody was used.


Peroxisomal VLCFA oxidation in fibroblasts was assessed by the ratio of lignoceric acid (C24:0)/palmitic acid (C16:0) oxidation activity.20 The activity of DHAP-AT, the first enzyme in the pathway leading to plasmalogen biosynthesis, was measured as described previously21 using 14C labelled DHAP as substrate. Continuous cell labelling with 35S-methionine and immunoprecipitation of AOX with rabbit anti-human AOX antibody was performed as described previously.19 22



The fibroblasts from PBD patients belonging to the CG-A were examined by immunofluorescence microscopy to determine the import of PTS1 and PTS2 containing peroxisomal matrix proteins. The immunoreactivity of these proteins in control cells showed the same punctate pattern as previous reports (data not shown).23The most striking result was that in the fibroblasts from ZS patients, the import of AOX was rescued apparently after incubation at 30°C, whereas it was severely reduced or absent at 37°C (fig 1C, D, table1). The AOX and 70 kDa peroxisomal membrane protein (PMP70) were co-localised in these cells (data not shown). In other CGs, none of the ZS cell lines showed such TS characteristics. There was a little cross reactivity of AOX antibody with mitochondria (fig 1C) co-localised with anti-mitochondria antibody (data not shown). The import of catalase (fig 1A, B), D bifunctional protein (fig 1E, F), and PT (fig 1G, H) was defective after incubation at 30°C (table 1). In cell lines from NALD and IRD patients, the import of catalase, AOX, D bifunctional protein, and PT greatly improved after incubation at 30°C, whereas import of these enzymes was deficient at 37°C (table1).

Figure 1

Immunofluorescent staining for antibodies of peroxisome matrix proteins in fibroblasts from a ZS patient with CG-A (A-06). Cells were cultured for 72 hours at both 37°C (panels A, C, E, and G) and 30°C (panels B, D, F, and H), and stained with anti-human catalase (panels A and B), AOX (panels C and D), D bifunctional protein (panels E and F), and PT (panels G and H) rabbit antibody. Bar=10 μm.

Table 1

The import of peroxisome matrix proteins of fibroblasts from complementation group A (CG-A) patients


The relative VLCFA oxidation capacity was expressed as a ratio of C24:0/C16:0 fatty acid oxidation activity of the cell line. Using this assay, a lower ratio of C24:0/C16:0 fatty acid oxidation indicates lower peroxisomal VLCFA oxidation activity.20 In the same way, the lower activity of DHAP-AT indicates lower plasmalogen biosynthesis capacity of peroxisomes.21 The VLCFA oxidation ratio and the activity of DHAP-AT in ZS patients belonging to CG-A showed a very low level after incubation at 30°C, although a slight increase in activities was observed. Cell lines from NALD patients, showing TS import of PTSs, showed marked improvement in activities after incubation at 30°C (table 2). AOX, the first enzyme of the peroxisomal beta oxidation system, is processed into two smaller polypeptides in peroxisomes, and this processing was defective in the fibroblasts from patients with PBDs.22 On continuous labelling of the cells from a ZS patient (A-06) at 30°C, the processed form of AOX (subunit B, 53 kDa) was identified in addition to the unprocessed form of AOX (subunit A, 75 kDa), as was the case with those from the control and NALD patient (A-05). These results indicate that at least AOX is processed in a temperature dependent manner even in ZS cells. In ZS cells belonging to CG-E and CG-F, subunit B was not detected at 30°C, indicating that these ZS cells were not temperature dependent (fig 2).

Table 2

Biochemical assay of fibroblasts from complementation group A (CG-A) patients

Figure 2

Biosynthesis and processing of AOX. Fibroblasts were cultured for 72 hours and then incubated for 24 hours in the presence of 35S-methionine at either 37°C (lanes 1, 3, 5, 7, and 9) or 30°C (lanes 2, 4, 6, 8, and 10). The cell lysate, the same amount of protein, was immunoprecipitated with anti-human AOX antibody and subjected to SDS-PAGE and fluorography. Lanes 1 and 2, control fibroblasts; 3 and 4, ZS with CG-A (A-06) fibroblasts; 5 and 6, NALD with CG-A (A-05) fibroblasts; 7 and 8, ZS with CG-E fibroblasts; 9 and 10, ZS with CG-F fibroblasts.


The diagnosis in PBD patients is based on clinical features combined with a series of tests to assess peroxisomal function and structure, and the absence or reduction of both PTS1 and PTS2 protein imports are observed in the vast majority of cells from patients with PBDs.24 In fibroblasts from milder forms of PBDs, we elucidated the TS restoration of peroxisome biogenesis including the import of PTSs after incubation at 30°C.12-16 The temperature dependent manner of peroxisome biogenesis has been identified in cells from mildly affected patients belonging to various different CGs including CG-A in PBDs, whereas it has never been seen in those from ZS patients with these CGs.12-15 In this study, we clarified the restoration of AOX import and processing after incubation at 30°C in fibroblasts from patients with ZS, NALD, and IRD belonging to CG-A. AOX is one of the PTS1 proteins, and is transported to the peroxisome by binding with Pex5p, a PTS1 receptor.25 26 Pex5p is severely reduced in the fibroblasts of patients with CG-A, C, and E. However, cells with those CGs share the ability to import small amounts of both PTS1 and PTS2 proteins.27 The genes responsible for CG-C and E,PEX6 and PEX1, encode Pex6p and Pex1p, respectively. These proteins belong to the AAA (ATPases associated with diverse cellular activities) family of proteins and are required for stability of the PTS1 receptor in yeast as well as in human cells.28-32 The product of the CG-A gene, which is still unknown, may also physically function with Pex5p. The biochemical functions of peroxisome as measured by VLCFA oxidation and DHAP-AT activity are restored apparently in the fibroblasts from milder NALD patients, whereas they showed lower levels in the ZS cells even at 30°C as was the case with other CGs. This might be caused by the defective import of peroxisomal beta oxidation enzymes other than AOX. The impairment in fibroblast VLCFA beta oxidation and the clinical manifestations were more severe in bifunctional protein deficient than AOX deficient patients.33 Therefore, ZS patients with CG-A showed as severe a clinical course and prognosis as those with other CGs, notwithstanding the AOX import was rescued at 30°C in the cells from patients with CG-A.12 34 TS import of AOX in ZS fibroblasts is unique to CG-A. A peroxin encoded by the candidate gene for CG-A may interact with Pex5p independently or with the Pex5p-AOX complex, and may lead to the characteristic import of AOX at 30°C. Isolation of the PEX gene for CG-A will help to clarify the function of peroxin for CG-A and mechanism of the selective TS import of AOX in CG-A patients.


We thank T Hashimoto for anti-human catalase, acyl-CoA oxidase, and D bifunctional protein antibodies. This work was supported in part by a Grant in Aid for Scientific Research (10670718, 10670721) from the Ministry of Education, Science, Sports and Culture of Japan, by Health Science Research Grants from the Ministry of Health and Welfare of Japan, by the Mother and Child Health Foundation, and by the Ichiro Kanehara Foundation.