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Human Molecular Genetics Pages 239-247  

Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins
Introduction
Results
   Complementation of VLCFA [beta]-oxidation defect in X-ALD fibroblasts following expression of ALDP
   PMP70 overexpression complements VLCFA [beta]-oxidation in X-ALD fibroblasts
   ALDP or PMP70 expression restores peroxisome biogenesis in cells deficient in the 35 kDa peroxisomal protein Pex2p
Discussion
Materials And Methods
   Bacterial strains and patient cell lines
   Library screening and cDNA cloning
   Immunoflourescence studies and [beta]-oxidation determinations
Acknowledgements
References
Footnote

Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins

Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins

Lelita T. Braiterman1,2,*, Siqun Zheng1, Paul A. Watkins1,3, Michael T. Geraghty2, Gerald Johnson4, Martina C. McGuinness1,3, Ann B. Moser1,3, Kirby D. Smith1,2

1Kennedy Krieger Research Institute, 707 North Broadway, Baltimore, MD 21205, USA, 2Department of Pediatrics and 3Department of Neurology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA and 4Department of Biology and Molecular Biology, San Diego State University, 550 Campanile, San Diego, CA 92182, USA

Received September 4, 1997; Revised and Accepted November 5, 1997

X-Linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by reduced peroxisomal very long chain fatty acid (VLCFA) [beta]-oxidation. The X-ALD gene product (ALDP) is a peroxisomal transmembrane protein with an ATP binding cassette (ABC). ALDP and three other ABC proteins (PMP70, ALDR, P70R) localize to the peroxisomal membrane. The function of this family of peroxisomal membrane proteins is unknown. We used complementation studies to begin analysis of their role in VLCFA [beta]-oxidation and on the peroxisomal membrane. Expression of either ALDP or PMP70 restores VLCFA [beta]-oxidation in X-ALD fibroblasts, indicating overlapping functions. Their expression also restores peroxisome biogenesis in cells that are deficient in the peroxisomal membrane protein Pex2p. Thus it is likely that complex protein interactions are involved in the function and biogenesis of peroxisomal membranes that may contribute to disease heterogeneity.

INTRODUCTION

X-Linked adrenoleukodystrophy (X-ALD) is a phenotypically variable neurodegenerative disorder characterized by elevated plasma and tissue levels of very long chain fatty acids (VLCFA) (1). Failure of peroxisomal very long chain acyl-CoA synthetase (VLCS) to activate these fatty acids prevents their degradation by peroxisomal [beta]-oxidation. A candidate gene for X-ALD was isolated by positional cloning. Its deduced amino acid sequence had no homology to VLCS (2) but did have homology to a family of transmembrane transporter proteins, the ATP binding cassette (ABC) proteins (3). Studies in our laboratory (4,5) and by others (6-10) have demonstrated mutations in the X-ALD gene of all X-ALD patients reported to date. Localization studies revealed that ALDP is a peroxisomal membrane protein (5,11) and that 70% of X-ALD patients lacked immunoreactive ALDP (5). Furthermore, we (12) and others (13,14) have demonstrated correction of the VLCFA [beta]-oxidation defect in SV40-transformed X-ALD fibroblasts. However, neither the function of ALDP nor its relationship to VLCS is known.

ABC transporter protein family members have the ability to transport a wide variety of substrates (15). The typical eukaryotic ABC transporter protein consists of two hydrophobic transmembrane domains and two hydrophilic nucleotide binding folds (NBF). In contrast, ALDP has only one hydrophobic and one hydrophilic domain harboring an NBF and is thus designated a half-transporter. At least three other half-transporter ABC protein family members are present in the peroxisomal membrane, ALDR (16), PMP70 (17) and P70R (18). There is a high degree of homology among these genes, suggesting that functional similarity could exist between them. Since most other half-transporters identified to date dimerize to form a functional transporter, it has been suggested that these peroxisomal half-transporters may dimerize in the peroxisome membrane to form a whole transporter (18,19). Heterodimerization for two peroxisomal half-transporter ABC proteins has been demonstrated in yeast (20). Because X-ALD results from mutations in the X-ALD gene and not in any of the other peroxisomal half-transporters, ALDP may function as a homodimer, possibly binding or translocating VLCS itself (11), CoA, VLCFA-CoA or the VLCFA substrate for activation by VLCS inside the peroxisome.

Since knowledge of ALDP function is essential to understanding the molecular mechanisms and pathophysiology of X-ALD and to developing approaches for clinical therapy, we have begun studies to systematically examine the function of ALDP, its role in X-ALD pathology and the functional relationship of ALDP to VLCS and the other peroxisomal membrane half-transporters. In this report we examine the ability of ALDP and PMP70 cDNA expression to complement the [beta]-oxidation defect in cultured skin fibroblasts from X-ALD patients. In addition, we investigate the ability of ALDP and PMP70 to restore peroxisome biogenesis in cells deficient in the 35 kDa peroxisomal membrane protein Pex2p (21), a member of the zinc ring family of zinc binding proteins and formerly known as PMP35 (22). Pex2p is deficient in a subgroup of patients with Zellweger syndrome, a peroxisome biogenesis disorder (PBD) (23). PBDs are a phenotypically heterogenous group of neurological diseases that result from defects in peroxisome biogenesis, a subgroup of peroxisomal diseases that are classified into 16 different complementation groups (CGs) (24).

RESULTS

Complementation of VLCFA [beta]-oxidation defect in X-ALD fibroblasts following expression of ALDP

For these complementation studies we used skin fibroblasts from two previously characterized unrelated X-ALD patients (5). X-ALD-1 fibroblasts with the missense mutation A626T lack the punctate immunoreactive staining pattern characteristic of ALDP while X-ALD-2 fibroblasts with the missense mutation R591Q retain expression of ALDP. Primary fibroblasts rather than SV40-transformed fibroblasts were used for these studies because results from our laboratory demonstrated that the VLCFA [beta]-oxidation pathway was down-regulated in SV-40 transformed fibroblasts (manuscript in preparation). Because of the finite lifetime of primary cells, drug selection strategies to obtain stably expressing cell lines cannot be used. We describe here the use of transient transfection studies to examine the effects of gene expression in various human skin fibroblasts.

Restoration of ALDP in peroxisomes of the X-ALD-1 cell line following transient expression of the ALDP cDNA is demonstrated (Fig. 1). The punctate peroxisomal staining pattern typically observed with the anti-ALDP peptide antibody (Fig. 1a) and the co-localization of catalase (Fig. 1b), an enzyme known to be targeted to peroxisomes, in control human skin fibroblasts are shown (5). The absence of punctate staining and the non-specific nuclear staining seen with anti-ALDP in the X-ALD-1 cell line (Fig. 1c) and the presence of peroxisomes following immunostaining with anti-catalase (Fig. 1d) are shown. Following transfection with ALDP cDNA X-ALD-1 fibroblasts immunostained with anti-ALDP exhibited a punctate staining pattern typical of peroxisomes (Fig. 1g) that co-localized with structures immunostained with anti-catalase (Fig. 1h). In contrast, no immunoreactive ALDP material was observed in the cells transfected with the pCDNA3 vector (Fig. 1e) but we observed the same punctate staining pattern as observed in Figure 1d after immunostaining with anti-catalase (Fig. 1f).


Figure 1 ALDP expression following transfection of primary skin fibroblasts from an X-ALD patient. Fibroblasts were transfected with the pCDNA3 vector alone or with pCDNA3 expressing wt ALDP. Three days later the transfected cell population was processed for indirect immunofluorescence microscopy using anti-ALDP (5) and anti-catalase to identify peroxisomes. (a) Control primary human skin fibroblasts stained with anti-ALDP. The peroxisomal pattern and non-specific nuclear staining typically obtained with this antibody are shown. (b) The same cells stained with anti-catalase. (c) X-ALD-1 cell line stained with anti-ALDP. (d) The same cells stained with anti-catalase. (e) X-ALD-1 cell line transfected with pCDNA3 vector and stained with anti-ALDP. (f) The same cells stained with anti-catalase. (g) X-ALD-1 cell line transfected with pCDNA3 expressing wt ALDP and stained with anti-ALDP. (h) The same cells stained with anti-catalase.

Functional complementation was examined following transient expression of ALDP cDNA in the X-ALD-2 cell line. Lignoceric acid (C24:0) [beta]-oxidation activities in control and X-ALD-2 fibroblasts were determined to be 0.85 ± 0.19 and 0.16 ± 0.05 nmol/h/mg respectively (Table 1). Consistent with previous results (25,26), the X-ALD-2 cell line exhibited residual VLCFA [beta]-oxidation activity that is 19% of the control activity. Following transfection by electroporation we routinely observed a small but consistent increase in VLCFA [beta]-oxidation activity in the vector-transfected cells. However, when wild-type (wt) X-ALD cDNA was transfected into the X-ALD-2 cell line we observed a 2-fold increase in C24:0 [beta]-oxidation activity over vector-transfected cells following subtraction of the residual C24:0 [beta]-oxidation activity typical for this cell line (Table 1). Furthermore, the rate of VLCFA [beta]-oxidation, 0.9 nmol/h/mg for transfected cells when normalized for the number of cells overexpressing ALDP (10%), is comparable with that seen in control cells (0.7 nmol/h/mg). To confirm that complementation of the VLCFA [beta]-oxidation defect was due to wt ALDP expression, two different X-ALD cDNA constructs harboring naturally occuring X-ALD patient mutations were tested. With 4-15% of cells overexpressing the mutant ALDP construct R591Q no complementation of VLCFA [beta]-oxidation in transfected X-ALD-2 cells was seen (Table 1). A second mutant ALDP (R617H) construct tested in X-ALD-2 fibroblasts did not result in overexpression of ALDP, as judged by immunofluorescence (data not shown), or complementation of VLCFA [beta]-oxidation (Table 1). The lack of ALDP expression by this mutant construct was not surprising because ALDP protein expression in the patient fibroblast cell line with the R617H mutation was undetectable (5).

Table 1. [beta]-Oxidation of VLCFA in X-ALD primary fibroblasts following expression of wt ALDP and mutant ALDP
Fibroblast cell line n C24:0 specific activity (mean ± SD) nmol/h/mg Normalized C24:0 specific activitya nmol/h/mg C24:0 specific activity adjusted for the no. of expressing cellsb nmol/h/mg
Control 13 0.85 ± 0.19 0.69 0.7
X-ALD-2 4 0.16 ± 0.05c 0  
X-ALD-2 vector transfected 5 0.22 ± 0.06 0.08  
X-ALD-2 expressing wt ALDP 5 0.32 ± 0.07d 0.17 0.9
X-ALD-2 expressing mALDP (R591Q) 5 0.21 ± 0.05 0.07  
X-ALD-2 expressing mALDP (R617H) 5 0.22 ± 0.06 0.08  
aThe rates of VLCFA [beta]-oxidation observed have been normalized by subtracting the observed level of C24:0 oxidation typical for the X-ALD-2 cell line.
bThe rates of VLCFA [beta]-oxidation observed after subtracting C24:0 activity routinely observed in vector transfected cells, then adjusted for the fraction (10%, range 4-15%) of cells overexpressing ALDP as detected by immunofluorescence.
cSkin fibroblasts from X-ALD patients demonstrate a dramatic reduction in the capacity to [beta]-oxidize VLCFA. Rates of VLCFA oxidation are routinely 10-30% of those observed for normal human skin fibroblasts (control). The X-ALD mutation in the X-ALD-2 cells line is R591Q, a missense mutation that does not dramatically alter the expression of ALDP and has 19% of the activity determined for control fibroblasts.
dThe C24:0 specific activity after expression of wt ALDP differs from C24:0 specific activity of the vector transfected cells (paired t-test = 6.32; d/f = 4; P = 0.0016).

PMP70 overexpression complements VLCFA [beta]-oxidation in X-ALD fibroblasts

PMP70 was the first ABC protein identified in the peroxisomal membrane (17) and has 38.5% overall amino acid identity to ALDP and 79% sequence similarity when conservative amino acid substitutions are considered (3). Therefore, to test the ability of PMP70 to complement VLCFA [beta]-oxidation we overexpressed PMP70 in fibroblasts from X-ALD patients. Because fibroblasts from X-ALD patients normally have PMP70, human PMP70 cDNA encoding a human c-myc epitope at its C-terminus (PMP70-c-myc) was used to allow detection of PMP70-c-myc with anti-c-myc antibody. Following transfection of the X-ALD-1 cell line with the PMP70-c-myc cDNA we observed a punctate staining pattern typical of peroxisomes in fibroblasts after immunostaining with anti-c-myc (Fig. 2a). The anti-c-myc staining co-localized with structures immunostained with anti-catalase (Fig. 2b).

Table 2. [beta]-Oxidation of VLCFA in X-ALD primary fibroblasts following expression of PMP70
Fibroblast cell line n C24:0 specific activitya nmol/h/mg C24:0 specific activity normalized and adjusted for no. of expressing cellsb nmol/h/mg
Control 13 0.85 ± 0.19 0.74
X-ALD-1 9 0.11 ± 0.05c 0
X-ALD-1 vector transfected 6 0.13 ± 0.02  
X-ALD-1 expressing PMP70 6 0.16 ± 0.05d 0.26
X-ALD-1 expressing ALDP 6 0.18 ± 0.05d 0.41
X-ALD-1 expressing Pex2p 3 0.12 ± 0.02 0
aMean ± SD.
bThe rates of VLCFA [beta]-oxidition observed have been normalized by subtracting either the level of C24:0 oxidation typical for the X-ALD-1 cell line or the value observed after mock transfection, then adjusted for the fraction (13%, range 9-22%) of cells expressing ALDP as detected by immunofluorescence.
cSkin fibroblasts from X-ALD paitents demonstrate a dramatic reduction in the capacity to [beta]-oxidize VLCFA. Rates of VLCFA oxidation are routinely 10-30% of those observed for normal human skin fibroblasts (control). The X-ALD mutation in the X-ALD-1 cell line is A626T, a missense mutation which destabilizes protein expression.
dThe C24:0 specific activity after expression of either PMP70 c-myc or wt ALDP differs from C24:0 specific activity of the vector transfected cells (paired t-test = 2.07; d/f = 5; P < 0.05 and paired t-test = 3.9; d/f = 5; P < 0.06, respectively).

Functional complementation of the VLCFA [beta]-oxidation defect in X-ALD-1 cells was examined following transient transfection of PMP70-c-myc cDNA as well as ALDP cDNA (Table 2). Residual C24:0 [beta]-oxidation activity for the X-ALD-1 cell line was less than that observed for the X-ALD-2 cell line, 13% of the amount determined for control cells (Table 2). Lipid-mediated transient transfection of the X-ALD-1 cell line by ALDP cDNA results in increased ALDP expression as judged by immunofluorescence (13% of cells, range 9-22%), with an increase in C24:0 [beta]-oxidation activity over vector-transfected cells (Table 2). Following expression of PMP70-c-myc cDNA we observed a significant increase (P < 0.05) in C24:0 [beta]-oxidation activity over that of vector-transfected cells (0.13 and 0.16 nmol/h/mg for vector and PMP70 expressing cells respectively). The statistical significance of the small but consistent difference between the vector-transfected and PMP70-c-myc-transfected cell populations was determined before normalization. The levels of C24:0 [beta]-oxidation following ALDP expression in this cell line are comparable with those observed for PMP70 (0.18 and 0.16 nmol/h/mg respectively). When C24:0 [beta]-oxidation specific activities determined for PMP70 and ALDP are normalized for the number of ALDP-expressing cells the corrected levels of C24:0 [beta]-oxidation are 35 and 55% respectively of the specific activity of the normalized control cells. As a control we examined the effect of overexpressing an unrelated peroxisomal membrane protein, Pex2p, a C3CH4 zinc binding integral peroxisomal membrane protein of 35 kDa (see below). As shown in Table 2, VLCFA metabolism was not altered following overexpression of Pex2p cDNA.


Figure 2 PMP70 expression following transfection of primary skin fibroblasts from an X-ALD patient. Fibroblasts were transfected with the pCDNA3 vector alone or pCDNA1/Amp expressing PMP70-c-myc. Three days later the transfected cell population was processed for indirect immunofluorescence microscopy using anti-c-myc and anti-catalase to identify peroxisomes. (a) X-ALD-1 cell line transfected with pCDNA1/Amp expressing PMP70-c-myc stained with anti-c-myc. (b) The same cells stained with anti-catalase.



Because the majority of cells in these primary fibroblast cultures are not expressing the various cDNAs in these transient transfection studies we designed a mixing experiment to confirm that the C24:0 [beta]-oxidation specific activity observed in these experiments can be detected when a small subpopulation of cells expresses ALDP. [beta]-Oxidation activity was determined when X-ALD-1 cell suspensions were supplemented with 5, 10, 15, 20, 25 and 50% control fibroblasts (Fig. 3). As the contribution of control cells decreased the C24:0 [beta]-oxidation specific activity was reduced, showing good agreement with the expected value (r2 = 0.997). Thus the large number of non-transfected cells in these experiments did not hinder our ability to detect [beta]-oxidation activity of the transfected cell population.

ALDP or PMP70 expression restores peroxisome biogenesis in cells deficient in the 35 kDa peroxisomal protein Pex2p

It has been demonstrated that overexpression of PMP70 restores peroxisome biogenesis in the peroxisome-deficient Chinese hamster ovary (CHO) 78/C cell line (27). These cells have a defect in the PEX2 gene and typically contain a small number of structures that resemble peroxisomes but contain no catalase or other peroxisomal matrix proteins (28). Therefore, we tested the ability of wt ALDP cDNA to restore peroxisome biogenesis in CHO 78/C cells. Following transient transfection of ALDP cDNA the transfected CHO cells contained more typical peroxisomal structures, as indicated by a punctate immunostaining pattern observed with anti-ALDP (Fig. 4a) and by immunostaining with antibody to the tripeptide SKL (Fig. 4b), the targeting signal 1 (PTS1) of most peroxisomal matrix proteins (29). The fields shown display both transfected and non-transfected cells. The cells expressing human ALDP have peroxisomes containing matrix proteins, consistent with restoration of peroxisome biogenesis following expression of ALDP.


Figure 3 C24:0 [beta]-oxidation activity in a mixed cell population. Control human skin fibroblasts and X-ALD-1 cells were harvested by trypsinization, counted then mixed in the ratios indicated and the C24:0 [beta]-oxidation activity of the mixture determined. The C24:0 activity per 106 cells is plotted versus the percent control cells in the mixture.


Figure 4 CHO cells (ZR78/C) following expression of either wt ALDP or PMP70-c-myc. ZR78/C cells were transfected with either pCDNA3 expressing wt ALDP or pCDNA1/Amp expressing PMP70-c-myc. Three days later the transfected cell populations were processed for indirect immunofluorescence microscopy using anti-ALDP, anti-c-myc and anti-SKL to identify peroxisomes. The staining patterns of non-transfected cells are indicated by an asterisk (*). (a) ZR78/C cells transfected with pCDNA3 expressing wt ALDP stained with anti-ALDP. (b) The same cells stained with anti-SKL. (c) ZR78/C cells transfected with pCDNA1/Amp expressing PMP70-c-myc stained with anti-c-myc. (d) The same cells stained with anti-SKL.

Confirming the first observation that an ABC protein can restore peroxisome biogenesis in this cell line (27), we show that expression of PMP70-c-myc also has the ability to restore peroxisome biogenesis in the CHO 78/C cell line. Following expression of the PMP70-c-myc cDNA a punctate immunostaining pattern typical of peroxisomes was observed with anti-c-myc (Fig. 4c) and peroxisomal matrix proteins were imported, as evidenced by immunostaining with antibody to the tripeptide SKL only in PMP70-expressing cells (Fig. 4d).

The PEX2 gene is also defective in CG 10 of human peroxisome biogenesis disorders (24,30). Therefore, the effects of ALDP and PMP70 overexpression on peroxisome biogenesis were examined in a primary skin fibroblast cell line from a CG 10 Zellweger syndrome patient. Typically fibroblasts from this CG 10 Zellweger patient, PBD0094 (31), lacked visible punctate structures after immunostaining with antibody to the tripeptide SKL, indicating the absence of matrix proteins in peroxisomes (Fig. 5a). Following transient expression studies of Pex2p cDNA in this CG 10 cell line we observed a punctate staining pattern following immunostaining with anti-SKL antibody (Fig. 5b), indicating restoration of peroxisome biogenesis. A punctate staining pattern with anti-SKL antibody was always observed following Pex2p expression; however, the transient transfection efficiencies were quite variable (1-50%) for this cell line. To investigate the effects of ALDP and PMP70 on human peroxisome biogenesis disorders we examined the targeting of per-oxisome matrix proteins following transient transfection of ALDP and PMP70 cDNAs. Following transfection of the ALDP cDNA we routinely observed cells exhibiting a punctate staining pattern after immunostaining with anti-SKL antibody (Fig. 5c) or anti-catalase (Fig. 5d), indicating the restoration of peroxisome biogenesis following ALDP overexpression. A punctate staining pattern was observed in 1/200-1/300 fewer cells following ALDP expression than following Pex2p expression.


Figure 5 Zellweger (CG 10) patient primary skin fibroblasts following expression of Pex2p or wt ALDP. Fibroblasts were transfected with either Pex2p cDNA or pCDNA3 expressing wt ALDP. Three days later the transfected cell populations were processed for indirect immunofluorescence microscopy using anti-SKL or anti-catalase to identify peroxisomes. (a) Primary skin fibroblasts from a Zellweger (CG 10) patient stained with anti-SKL. (b) Primary skin fibroblasts from a Zellweger (CG 10) patient following transfection with Pex2p cDNA stained with anti-SKL. (c) Primary skin fibroblasts from a Zellweger (CG 10) patient following transfection with pCDNA3 expressing wt ALDP stained with anti-SKL. (d) Primary skin fibroblasts from a Zellweger (CG 10) patient following transfection with pCDNA3 expressing wt ALDP stained with anti-catalase. The number of peroxisomes observed in transfected cells was variable.


Figure 6 Zellweger (CG 10) patient primary skin fibroblasts following expression of PMP70-c-myc. Fibroblasts were transfected with pCDNA1/Amp expressing PMP70-c-myc. Three days later the transfected cell populations were processed for indirect immunofluorescence microscopy using anti-c-myc and anti-SKL to identify peroxisomes. (a) Endogenous PMP70 in primary skin fibroblasts from a Zellweger (CG 10) patient stained with anti-PMP70. (b) Primary skin fibroblasts from a Zellweger (CG 10) patient following transfection with PMP70-c-myc cDNA stained with anti-c-myc. (c) The same cells stained with anti-SKL.

Endogenous PMP70 expression was detectable in fibroblasts from this CG 10 Zellweger patient using anti-PMP70 antibody (Fig. 6a), but the number of punctate structures was reduced compared with control fibroblasts (see Fig. 1b). Following overexpression of PMP70-c-myc cDNA we also observed a small population of cells with increased numbers of peroxisomes following immunostaining with anti-c-myc antibody (Fig. 6b). All cells expressing PMP70-c-myc now contain peroxisomal matrix proteins, as revealed by immunostaining using anti-SKL (Fig. 6c), a pattern quite different from the anti-SKL pattern typical for this cell line (Fig. 5a). Thus PMP70 overexpression in this cell line from a CG 10 patient also results in restoration of peroxisome biogenesis.

DISCUSSION

The role of ABC half-transporter peroxisomal membrane proteins in mammals is unknown. Prior to cloning of the X-ALD gene the requirement for a transporter protein essential for VLCFA [beta]-oxidation had not been hypothesized. A role for PMP70 in peroxisome biogenesis in CG 1 has been suggested (32), but recent results (33) raise questions about the role of PMP70. Thus functional roles for PMP70 and ALDR and P70R have yet to be established. The high degree of sequence similarity among these proteins suggests that they may have related functions in the peroxisomal membrane. It has been suggested that ALDP and PMP70 may interact to form heterodimers (19). Because all ALD patients reported to date (>150 found worldwide) have mutations in the X-ALD gene, there is no evidence for a non-X-linked disease resembling X-ALD and as ALDP and PMP70 have different patterns of expression (34,35), it seems likely that ALDP and PMP70 function independently as homodimers. In this report we demonstrate that overexpression of either ALDP or PMP70 corrects the VLCFA [beta]-oxidation defect in primary skin fibroblasts from X-ALD patients. Thus it is possible that they have separate but overlapping functions in the peroxisomal membrane, perhaps in the transport of VLCFAs in a chain length-specific manner like the different acyl-CoA ligases that activate fatty acids of different chain length (36,37). Based on these functional complementation studies suggesting the involvement of PMP70 in peroxisomal VLCFA [beta]-oxidation, it can be hypothesized that PMP70 might contribute to the residual 10-30% VLCFA [beta]-oxidation activity observed in fibroblasts from X-ALD patients (25). Variable expression of PMP70 among X-ALD patients might also contribute to the marked phenotypic heterogeneity observed in this disorder (1).

We further demonstrate that overexpression of either ALDP or PMP70 can restore peroxisomal biogenesis in cells deficient for the 35 kDa peroxisomal membrane protein Pex2p. Defects in PEX2 result in Zellweger disease (CG 10) (30). Although the function of Pex2p is unknown, fibroblasts from these Zellweger patients and CHO cells defective in PEX2 have small numbers of large peroxisomes that do not import peroxisomal matrix proteins (38,39). These observations demonstrate the requirement of PEX2 for matrix protein import and support its possible role in peroxisome biogenesis. It was reported previously that expression of PMP70 in CHO cells (78/C) defective in PEX2 restored peroxisome biogenesis (27). We show here that overexpression of ALDP also restores peroxisome biogenesis in CHO cells where PEX2 is inactivated by a missense mutation in the putative zinc binding region (28). In addition, we demonstrate that both ALDP and PMP70 are capable of restoring peroxisome biogenesis and matrix protein import in skin fibroblasts from a CG 10 Zellweger patient with a homozygous nonsense mutation (R119ter) (23). These observations suggest that ABC proteins as well as Pex2p may play a role in peroxisomal membrane biogenesis, a still poorly understood process. Though Pex2p is likely involved in peroxisome matrix protein import in normal circumstances, the presence of these overexpressed proteins demonstrates that import of matrix proteins can occur in the absence of Pex2p. The restoration of peroxisomal biogenesis by overexpression of the ABC proteins tested in this report may result from high copy suppression, a phenomenon frequently observed for yeast mutants (40). The mechanism of membrane biogenesis in general is not understood; however, it has been observed that overexpression of hydrophobic membrane proteins in yeast (41,42) and mammalian cells (43-45) can result in membrane proliferation. Furthermore, peroxisomal membrane proliferation in rodent liver is well documented following treatment with certain xenobiotic substances known as peroxisome proliferators (46,47). Taken together the results of these suppression studies suggest the likelihood of complex protein interactions involved in the biogenesis and function of peroxisomal membranes. Similar cross-correction studies may enhance understanding of the disease mechanisms and peroxisome function and biogenesis.

MATERIALS AND METHODS

Bacterial strains and patient cell lines

Escherichia coli DH10B [F- mcrA [Delta](mrr-hsdRMS-mcrBC)l [phis]80dlacZ[Delta]M15 [Delta]lacX74 endA1 recA1 deoR [Delta](ara-leu)7697 ara139 galU galK nupG rpsL [lambda]-], INV[alpha]F[prime] (Invitrogen) [F[prime] endA1 recA1 hsdR17(rk- mk+) supE44 thi-1 gyrA96 recA1 [phis]80lacZ[Delta](lacZYA-argF)U169] and TOP10F[prime] (Invitrogen) [F[prime] {lacIq Tn10(TetR)} mcrA [Delta](mrr-hsdRMS-mcrBC) [phis]80lacZ[Delta]M15 [Delta]lacX74 deoPrecA1 araD139 [Delta](ara-leu)7697 galU galK rpsL (StrR) endA1 nupG] were used for transformation of plasmid constructs and plasmid DNA purification. Zellweger syndrome and X-ALD patients whose skin fibroblasts were used in this study were diagnosed on the basis of clinical history and elevated plasma levels of VLCFA and obtained through the Mental Retardation and Development Disabilities Research Center of the Kennedy Krieger Institute. Patient skin fibroblasts, orginially obtained for diagnostic purposes, were grown in culture as previously described (48). The CHO mutant cell line ZR-78, harboring the C246T missense mutation, was a kind gift of A.Zoeller (28).

Library screening and cDNA cloning

A human X chromosome-specific cosmid reference library (no. 104-L4/FSC X), constructed by Dean Nizetic from flow sorted X chromosomes digested and ligated into the Lawrist 4 vector (49), was obtained from the Imperial Cancer Research Fund (ICRF) Genome Analysis Laboratory (50). A filter containing the cosmid library was probed with a partial ALD cDNA probe using standard techniques (51); two clones of the estimated 20 736 clones screened gave a positive signal. Clone 1, the ICRF104cM105 cosmid, contains [sim]45 kb of human genomic DNA and was initially shown to contain ALD gene sequences based on PCR analysis of exons 1-9 using primer pairs as described (4). Subsequently three BamHI fragments extending from [sim]1 kb upstream of exon 1 to 4 kb downstream of exon 10 (3,52) were identified, indicating that this clone contains the entire ALD gene. Clone 2, the ICRF104cM1013 cosmid, contains [sim]35 kb of human genomic DNA and was initially shown to contain ALD gene sequences based on positive PCR reactions for primer pairs for exons 1-7. Clone 2 was used as the template for amplification of exon 1; the cloned PCR product was used for the X-ALD cDNA construct described.

Using an X-ALD probe, EX13 (3), we screened a recombinant phage retinal cDNA library (kindly provided by Jeremy Nathans) and identified one clone encoding bp 1137-2275 of the ALD cDNA (note that base pair number 1 of the ALD cDNA designates the A of the ATG throughout this manuscript). The EcoRI insert fragment was subcloned into pBluescript (Stratagene) and designated MGcDNA. In order to generate a full-length cDNA a series of overlapping clones was generated and assembled in pUC19 to give pCJ3. For expession studies the full-length X-ALD cDNA was cloned into pCDNA3 (Invitrogen) to give pLB741. The integrity of this cDNA was confirmed by sequence analysis using gene-specific primers used by our laboratory for patient mutation detection (4).

Mutant ALDP cDNAs were generated by TA cloning (Invitrogen) of RT-PCR products generated from RNA isolated from patient fibroblast cell lines harboring either the R617H mutation that both destablizes and inactivates ALDP or the missense mutation R591Q that inactivates ALDP without altering stability of the protein (5). The missense mutations were confirmed by sequence analysis, then subcloned into pLB741 to give pLB743 and pLB748 respectively.

The PMP70 cDNA open reading frame was extended by 10 amino acids to encode the c-myc epitope EQKLISEEDL and cloned into pCDNA1/Amp (Invitrogen), designated pSG683, a generous gift of Stephen J.Gould (Johns Hopkins University).

The PEX2 cDNA was a generous gift of D.Valle (Johns Hopkins University).

Sequencing. DNA samples were sequenced using the fluorescent dideoxy terminator method of cycle sequencing on a Perkin Elmer Applied Biosystems Division (PE/ABd) 373a automated DNA sequencer, following the ABd protocols at the DNA Analysis Facility of Johns Hopkins University.

Transfection. Human primary skin fibroblast cell lines were transfected with plasmid DNA using either electroporation or Lipofectamine. For electroporation, cells (1-2 × 106) were resuspended in either 0.5 ml HEPES-buffered saline (HBS) or Opti-MEM I (Life Technologies, Gaithersburg, MD); 700 V/cm was delivered to the cells at a capacitance of 330 µF using a Cell-porator (Life Technologies). However, we observed both high levels of cell death resulting in a small number of cells available for biochemical analysis and variability in the number of cells expressing the various cDNA constructs. Subsequently we optimized a transfection technique yielding higher transfection efficiencies and more reproducible results. Briefly, human primary skin fibroblast cells (1-2 × 106) were harvested by trypsinization, rinsed and resuspended in 0.5 ml Opti-MEM I (Life Technologies). The cells were incubated at room temperature for 30 min with plasmid DNA that was pre-incubated with Lipofectamine (Life Technologies) for 15-45 min in 500 µl Opti-MEM I according to the manufacturer's instructions. Following treatment with Lipofectamine, cells were plated on glass coverslips and in 100 mm tissue culture dishes containing 10% fetal bovine serum in MEM. Medium was replaced after 40-48 h.

Antibodies. Polyclonal rabbit antibody to the 20 amino acid synthetic peptide KKHVPAPSPQGPGGLQGAST-COOH containing the C-terminal 18 amino acids of the predicted amino acid sequence of ALDP (residues 728-745) was used as described (5). A 1:100 dilution of rabbit polyclonal antibody to the tripeptide SKL, a peroxisomal matrix protein import signal, was a generous gift from Stephen J.Gould (Johns Hopkins University School of Medicine) (53) and the antisera to endogenous human PMP70 was a generous gift from Suresh Subramani (University of California, San Diego).

Sheep anti-human catalase IgG (The Binding Site), affinity purified rabbit anti-c-myc raised against a peptide corresponding to amino acids 408-421 of human c-myc (Santa Cruz Biotechnology), Lissamine rhodamine (LRSC)-conjugated affinity purified donkey anti-rabbit IgG, donkey anti-sheep IgG and fluorescein (FITC)-conjugated affinity purified goat anti-rabbit F(ab[prime])2 fragment (Jackson Immunological Laboratories) were obtained commercially.

Immunoflourescence studies and [beta]-oxidation determinations

Immunostaining was used to evaluate ALDP and PMP70 expression. Human skin fibroblasts were plated on glass coverslips and 3 days after transfection were processed for immunoflourescence studies as described (5). Fibroblasts were harvested by trypsinization 3 or 4 days following transfection and processed for [beta]-oxidation as described (54). The average number of d.p.m. detected in the blank was 179. Routinely we observed 216-500, 313-672 and 345-735 d.p.m. above the blank for the vector, ALDP cDNA and PMP70-c-myc cDNA respectively.

ACKNOWLEDGEMENTS

This work was supported by grants P30HD10981 and PO1HD10981 from the National Institutes of Health and by the John Hirshbeck Memorial Fund. We thank Janet Hanseth for help with the immunostaining, Cindy James for help with construction of the ALDP cDNA, He-Ming Wei for RT-PCR sample preparation, Hugo Moser for support and helpful discussions, Dave Valle for the PEX2 cDNA and reading the manuscript and Stephen Gould for pSG683, anti-SKL and reading the manuscript.

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*To whom correspondence should be addressed. Tel. +1 410 502 8149; Fax: +1 410 502 9839; Email: lita@welchlink.welch.jhu.edu

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