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

Abstract

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.

Figure 1

Complementation of peroxisomes in CG-I CHO mutant cells. (a–c) Intracellular location of EGFP in ZP107EG1 cells, ZP107 stably expressing EGFP-PTS1, was monitored on unfixed cells grown on a coverslip, under a fluorescence microscope. (d–f) Immunofluorescent staining of peroxisomes in 107P1 cells, stable HsPEX1-transformants of ZP107. (a) Peroxisome-deficient mutant ZP107EG1 cells. (b) Peroxisome-restored ZP107EG1, after lipofection with a subpool (F6-17) of human cDNA library. Arrows indicate the complemented cells. Cytosolic appearance of EGFP-PTS1 was apparent in the other cells. (c) ZP107EG1 transfected with pUcD2Hyg⋅HsPEX1 plasmid. (d and e) 107P1 cells were stained with goat antibody to rat catalase plus donkey antibody to goat IgG conjugated to rhodamine (Chemicon) and antibody to PMP70, respectively. Note that punctate structures (peroxisomes) stained in d and e are superimposable. (f) 107P1 cells stained with antibody to 3-ketoacyl-CoA thiolase. (×630; bar = 20 μm.)

Figure 2

Deduced amino acid sequence alignment of human (Hs) PEX1 protein and Pex1p from the yeasts Pichia pastoris (Pp) and Saccharomyces cerevisiae (Sc). A hyphen indicates a space. Amino acids identical between human and other species are shaded. Boxed are ATP/GTP-binding Walker motifs A (A-1 and A-2: A/GX₄GKS/T; X, any residue), motifs B (B-1 and B-2: R/K/HX_n>5ΦXΦ₂D/E; Φ, hydrophobic residue), and AAA-protein family signature (C: A/G/M/ΦXTXR/H/ΦXD/E/N/SXL/I/V/MDXAL/I/V/MXRXGRL/IV/M/Φ/YD/E), respectively. Open and solid arrowheads indicate the position of mutation in each allele from a CG-I patient, PBDE-04 (see Fig. 5A). The database accession number for the human PEX1 cDNA is AB008112.

Figure 3

Restoration of peroxisome biogenesis. (A) Latency of catalase activity in CHO-K1, ZP107, and 107P1 cells. Catalase: ○, CHO-K1; ▵, ZP107; □, 107P1. Lactate dehydrogenase in ZP107 is shown by ⋄. Relative free enzyme activity is expressed as a percentage of the total activity measured in the presence of 1% Triton X-100 (9). The results represent a mean of duplicate assays. (B) Biogenesis of peroxisomal proteins. Cell lysates (≈1.8 × 10⁵ cells) were subjected to SDS/PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. Cell types are indicated at the top. Immunoblot analysis used rabbit antibodies to rat acyl-CoA oxidase (AOx) and 3-ketoacyl-CoA thiolase (Thiolase). Arrows show the positions of AOx polypeptide components A, B, and C; open and solid arrowheads indicate a larger precursor (P) and mature protein (M) of 3-ketoacyl-CoA thiolase, respectively. Dots indicate nonspecific bands.

Figure 4

Complementation of fibroblasts from a CG-I ZS patient. (A) Transfection of PEX1 from a normal control and a CG-I patient (PBDE-04) with ZS. (a) PBDE-04 fibroblasts. (b) PBDE-04 fibroblasts were transfected with pUcD2Hyg⋅HsPEX1. (c) ZP107 was transfected with PEX1 cDNA, PEX1L664P, derived from PBDE-04. (d) PBDE-04 fibroblasts were back-transfected with PBDE-04-derived PEX1 cDNA, PEX1e/s. Cells were stained with antisera to human catalase (a, b, and d) or PTS1 peptide (c). Note that peroxisomes were restored in b, but not in c and d. (×630; bar, 20 μm.) (B) Northern blot analysis of PEX1 mRNA. Poly(A)⁺ RNA (1 μg) from fibroblasts of a control (lane 1) and the patient PBDE-04 (lane 2) was separated, transferred to Zeta-Probe GT membrane (Bio-Rad), and hybridized with ³²P-labeled cDNA probes for human PEX1. Washing was done twice with 0.15 M NaCl/10 mM sodium phosphate, pH 7.4/1 mM EDTA/0.5% SDS at 55°C. Arrow indicates PEX1 mRNA; the identity of the 2.5-kb band in the control is presently unknown. Exposure, 4 days.

Figure 5

Mutation analysis of PEX1 from a CG-I patient. (A) Nucleotide sequence analysis of PBDE-04 PEX1. Partial sequence and deduced amino acid sequence of PEX1 cDNA isolated from patient PBDE-04 and a normal control are shown. (Left) One-base point mutation (shaded), T to C in the codon for Leu-664, resulting in a codon for Pro-664 in one allele (see the open arrowhead in Fig. 2). (Right) A 171-bp deletion of nucleotide residues 1900–2070 (boxed), in another allele (solid arrowheads in Fig. 2). (B) RT-PCR analysis of PEX1 transcript from a control and PBDE-04. RT-PCR of poly(A)⁺ RNA was done with two sets of primers, RT1 and RT2 to amplify full-length PEX1 (lanes 1 and 2), and F5 and R6 to amplify a partial PEX1 fragment, nucleotide residues from 1276 to 2152 (lanes 3 and 4). Lanes: 1 and 3, a control; 2 and 4, PBDE-04. Note that two products with a normal size, ≈0.9 kb (solid arrowhead), and a smaller, ≈0.7 kb (open arrowhead), were obtained in PBDE-04 (lane 4).