ABCMdb

ABCD1 p.Asp194His

Predicted by SNAP2:	A: D (91%), C: D (91%), E: D (91%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Conservation of targeting but divergence in functi... Biochem J. 2011 Jun 15;436(3):547-57. Zhang X, De Marcos Lousa C, Schutte-Lensink N, Ofman R, Wanders RJ, Baldwin SA, Baker A, Kemp S, Theodoulou FL
Conservation of targeting but divergence in function and quality control of peroxisomal ABC transporters: an analysis using cross-kingdom expression.
Biochem J. 2011 Jun 15;436(3):547-57., [PMID:21476988]

Abstract [show]
ABC (ATP-binding cassette) subfamily D transporters are found in all eukaryotic kingdoms and are known to play essential roles in mammals and plants; however, their number, organization and physiological contexts differ. Via cross-kingdom expression experiments, we have explored the conservation of targeting, protein stability and function between mammalian and plant ABCD transporters. When expressed in tobacco epidermal cells, the mammalian ABCD proteins ALDP (adrenoleukodystrophy protein), ALDR (adrenoleukodystrophy-related protein) and PMP70 (70 kDa peroxisomal membrane protein) targeted faithfully to peroxisomes and P70R (PMP70-related protein) targeted to the ER (endoplasmic reticulum), as in the native host. The Arabidopsis thaliana peroxin AtPex19_1 interacted with human peroxisomal ABC transporters both in vivo and in vitro, providing an explanation for the fidelity of targeting. The fate of X-linked adrenoleukodystrophy disease-related mutants differed between fibroblasts and plant cells. In fibroblasts, levels of ALDP in some 'protein-absent' mutants were increased by low-temperature culture, in some cases restoring function. In contrast, all mutant ALDP proteins examined were stable and correctly targeted in plant cells, regardless of their fate in fibroblasts. ALDR complemented the seed germination defect of the Arabidopsis cts-1 mutant which lacks the peroxisomal ABCD transporter CTS (Comatose), but neither ALDR nor ALDP was able to rescue the defect in fatty acid beta-oxidation in establishing seedlings. Taken together, our results indicate that the mechanism for trafficking of peroxisomal membrane proteins is shared between plants and mammals, but suggest differences in the sensing and turnover of mutant ABC transporter proteins and differences in substrate specificity and/or function.

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153 Approximately 60% of X-ALD ABCD1 mutations are missense mutations, 65% of which result in no detectable ALDP, based on IF (immunofluorescence), indicating that they affect protein Table 1 Quantification of ALDP levels in X-ALD fibroblasts ALDP Mutation IF Immunoblot (% of control) p.Arg74Trp Absent 7.5 + - 0.6 p.Arg104Cys Reduced 35 + - 3.0 p.Ser149Asn Present 77 + - 3.0 p.Asp194His Present 60 + - 13.6 p.Leu220Pro Reduced 21.8 + - 5.4 p.Arg389His Present 40.6 + - 3.6 p.Arg554His Absent 1.0 + - 0.5 p.Ser606Leu Present 25 + - 1.5 p.Glu609Gly Absent 2.1 + - 1.3 p.Glu609Lys Absent 1.8 + - 0.9 p.Ala616Thr Absent 4.3 + - 1.7 p.Leu654Pro Absent 1.5 + - 1.3 p.Arg660Trp Absent 1.6 + - 0.8 p.His667Asp Absent 2.9 + - 1.0 p.Arg113fs Absent - Figure 3 Interaction of mammalian ABCD proteins with Arabidopsis Pex19 in vivo Tobacco plants stably expressing CFP-SKL were co-transfected with 35S::ABCD-YFP fusions andNLS-Pex19constructs.Leafepidermalcellswereimagedusingconfocalmicroscopy:(A-D) ALDP-YFP plus NLS-HsPex19; (E-H) ALDP-YFP plus NLS-AtPex19_1; (I-L) ALDR-YFP plus NLS-AtPex19_1. Login to comment
172 ALDP was increased from 2-4% to ~20% of wild-type levels in cell lines bearing the mutations p.Glu609Gly, p.Ala616Thr and p.Arg660Trp, from 1 to 10% in p.Glu609Lys and p.Arg554His cells and from 45 to 75% in the p.Asp194His cell line (Figure 4A). Login to comment
173 VLCFA β-oxidation was measured in cells that were cultured at 30◦ C for 72 h, but in only one case (p.Ala616Thr) was the capacity to degrade VLCFA restored to near-control levels (Figure 4C). Login to comment
176 Lines bearing the p.Glu609Gly and p.Asp194His mutations did not show any correction of the C26:0/C22:0 ratio (Figure 4D). Login to comment
154 Approximately 60% of X-ALD ABCD1 mutations are missense mutations, 65% of which result in no detectable ALDP, based on IF (immunofluorescence), indicating that they affect protein Table 1 Quantification of ALDP levels in X-ALD fibroblasts ALDP Mutation IF Immunoblot (% of control) p.Arg74Trp Absent 7.5 + - 0.6 p.Arg104Cys Reduced 35 + - 3.0 p.Ser149Asn Present 77 + - 3.0 p.Asp194His Present 60 + - 13.6 p.Leu220Pro Reduced 21.8 + - 5.4 p.Arg389His Present 40.6 + - 3.6 p.Arg554His Absent 1.0 + - 0.5 p.Ser606Leu Present 25 + - 1.5 p.Glu609Gly Absent 2.1 + - 1.3 p.Glu609Lys Absent 1.8 + - 0.9 p.Ala616Thr Absent 4.3 + - 1.7 p.Leu654Pro Absent 1.5 + - 1.3 p.Arg660Trp Absent 1.6 + - 0.8 p.His667Asp Absent 2.9 + - 1.0 p.Arg113fs Absent - Figure 3 Interaction of mammalian ABCD proteins with Arabidopsis Pex19 in vivo Tobacco plants stably expressing CFP-SKL were co-transfected with 35S::ABCD-YFP fusions andNLS-Pex19constructs.Leafepidermalcellswereimagedusingconfocalmicroscopy:(A-D) ALDP-YFP plus NLS-HsPex19; (E-H) ALDP-YFP plus NLS-AtPex19_1; (I-L) ALDR-YFP plus NLS-AtPex19_1. Login to comment
177 Lines bearing the p.Glu609Gly and p.Asp194His mutations did not show any correction of the C26:0/C22:0 ratio (Figure 4D). Login to comment

[hide] Co-expression of mutated and normal adrenoleukodys... Hum Mol Genet. 2000 Nov 1;9(18):2609-16. Unterrainer G, Molzer B, Forss-Petter S, Berger J
Co-expression of mutated and normal adrenoleukodystrophy protein reduces protein function: implications for gene therapy of X-linked adrenoleukodystrophy.
Hum Mol Genet. 2000 Nov 1;9(18):2609-16., [PMID:11063720]

Abstract [show]
Inherited defects in the X-chromosomal adrenoleukodystrophy (ALD; ABCD1) gene are the genetic cause of the severe neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). Biochemically the accumulation of very long-chain fatty acids, caused by impaired peroxisomal beta-oxidation, is the pathognomonic characteristic of the disease. Due to the X-chromosomal inheritance of X-ALD no data are available to clarify the question whether mutated adrenoleukodystrophy proteins (ALDPs) can negatively influence normal ALDP function. Here we show that restoration of beta-oxidation in X-ALD fibroblasts following transient transfection with normal ALD cDNA is more effective in ALDP-deficient fibroblasts compared with fibroblasts expressing normal amounts of mutated ALDP. Furthermore, we utilized the HeLa Tet-on system to construct a stable HeLa cell line expressing a constant level of endogenous ALDP and doxycycline-inducible levels of mutated ALDP. The induction was doxycycline dosage-dependent and the ALDP correctly localized. Interestingly, although mutated ALDP increased >6-fold in a dosage-dependent manner the total amount of ALDP (mutated and normal) remained approximately even as demonstrated by western blot and flow cytometric analyses. Thus, apparently mutated and normal ALDP compete for integration into a limited number of sites in the peroxisomal membrane. Consequently, increased amounts of mutated ALDP resulted in decreased peroxisomal beta-oxidation and accumulation of very long-chain fatty acids. These findings have direct implications on future gene therapy approaches for treatment of X-ALD, since in some patients a non-functional endogenous protein could act in a dominant negative way or displace the introduced, normal protein.

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45 We also transfected one of these cell lines, D194H, with an haemagglutinin (HA)-tagged normal construct to investigate the influence of mutated ALDP on correct peroxisomal targeting of normal ALDP when both are present in the same cell, a situation expected after gene transfer into autologous bone marrow cells of some X-ALD patients. Login to comment
49 Average transfection efficiency was determined in three independent experiments and was 14% for the ALDP-deficient cell line (range 10-17%), 20% for the D194H cell line (range 15-23%), 26% for the S213C cells and 24% for the N148S cell line (range 22-30%). Login to comment
51 Transient transfection of normal ALD cDNA into X-ALD fibroblast cell lines: A626T lack detectable ALDP, whereas N148S, D194H and S312C cell lines produce stable, mutated ALDP. Login to comment
56 To be able to further distinguish between endogenously expressed normal ALDP and mutated ALDP, a 9 amino acid motif from the influenza haemagglutinin protein was added to the C-terminus of D194H ALDP. Login to comment
59 HeLa Tet-on cells were transfected with HA-tagged D194H-ALD cDNA and stable transformants were selected. Login to comment
60 PCR analysis confirmed the presence of both endogenous ALD gene and integrated D194H-ALD cDNA. Login to comment
61 Based on indirect immunofluorescence studies one clone, HeLa-D194H, which showed low background expression and high inducibility, was finally selected. Login to comment
63 No staining outside the peroxisomes above typical cellular background staining could be observed, indicating that overexpression of D194H-ALDP did not alter its localization. Login to comment
66 Induction of D194H-ALD expression was verified by analysing mRNA and protein levels of both normal and mutated ALDP. Login to comment
67 Northern blot analysis of the HeLa-D194H clone at various concentrations of doxycycline showed robust induction even at the lowest concentration (Fig. 3a). Login to comment
70 Concerning the ratio of normal to mutated ALD mRNA, in HeLa-D194H without doxycycline the level of mutated ALD mRNA was ~30% of the level of endogenous ALD mRNA. Login to comment
75 Western blot analysis was performed with HeLa-D194H cells at varying concentrations of doxycycline (Fig. 4). Login to comment
77 Indirect immunofluorescence analysis of the HA-tagged D194H-ALDP in doxycycline-induced HeLa-D194H cells. Login to comment
80 Northern blot analysis of HeLa cells stably expressing mutated D194H-ALDP. Login to comment
99 Mutated D194H-ALDP was induced by different doxycycline concentrations (Fig. 5a). Login to comment
100 A slight decrease in β-oxidation was observed for uninduced HeLa-D194H cells and after induction with the lowest doxycycline concentration. Login to comment
101 Compared with normal HeLa Tet-on cells, the decrease for both uninduced and slightly induced HeLa-D194H cells was not statistically significant. Login to comment
102 When expression of D194H-ALDP was further upregulated by higher doxycycline concentrations the rate of β-oxidation was markedly reduced and this reduction was statistically significant compared with normal HeLa Tet-on cells. Login to comment
103 Thus, overexpression of mutated D194H-ALDP is sufficient to reduce β-oxidation in HeLa Tet-on cells. Login to comment
104 In the induced HeLa-D194H cells the overall reduction in the rate of β-oxidation was ~45%, which is a less severe effect than the reduction of ~80% for X-ALD fibroblasts compared with healthy fibroblasts (Fig. 5a). Login to comment
106 Western blot analysis of HeLa-D194H cells induced with increasing concentrations of doxycycline. Login to comment
118 (b) Gas chromatographic analysis of VLCFA levels displayed as ratio of C26:0/C22:0 accumulation in HeLa-D194H and HeLa Tet-on control cells cultured in the presence of the indicated doxycycline concentrations for 1 week. Login to comment
120 To establish whether the decreased rate of β-oxidation also leads to intracellular accumulation of very long chain fatty acids, expression of D194H-ALDP was induced in HeLa-D194H cells for 1 week and the content of VLCFA was determined by gas chromatography. Login to comment
123 In contrast, more pronounced expression of mutated D194H-ALDP at 2 or 5 µg/ml doxycycline resulted in a 7-fold increase in the accumulation of VLCFA compared with untreated cells. Login to comment
137 To further elucidate the mechanism of interaction between normal and mutated ALDP, we constructed a HeLa Tet-on cell line with D194H mutated ALDP under a strong inducible promoter. Login to comment
138 Northern blot analysis of HeLa-D194H cells at different levels of induction revealed that the amount of endogenous ALD mRNA remains constant, independent of the level of overexpression of mutated ALD mRNA. Login to comment
148 However, direct interaction between D194H mutated ALDP and normal ALDP is still hypothetical and there is no direct evidence that such heterodimers are completely non-functional. Login to comment
149 Of the three mutations analysed in transient transfection assays, N148S and S213C are located in transmembrane domains, whereas D194H is in the first cytosolic loop of the ALDP (Fig. 6). Login to comment
150 In yeast two-hybrid experiments performed with ALDP lacking the N-terminal 361 amino acids, homodimers still formed (13), arguing against the possibility that the three mutations N148S, D194H and S213C interfere with dimerization of ALDP. Login to comment
155 Missense mutations N148S and S213C are both located within putative transmembrane domains and D194H is located in the first cytosolic loop. Login to comment
160 Three different fibroblast cell lines described as having normal levels of non-functional ALDP were obtained: fibroblasts N148S (26-29, patient 55 in ref. 26) were kindly provided by Dr Aubourg (Paris), D194H (kindred no. Login to comment
169 To introduce the point mutation D194H into the pTRE-ALD expression plasmid, in vitro mutagenesis was performed using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Login to comment
176 For stable transfection of 1 × 106 HeLa Tet-on cells, 40 µg of D194H-ALD expression plasmid plus 2 µg of hygromycin resistance-conferring plasmid (pTK-Hyg; Clontech) were co-transfected at 220 V with a capacitance of 950 µF (Gene pulser and capacitance extender plus; BioRad, Hercules, CA) in 0.4 cm cuvettes. Login to comment
180 Identification of transfected clones by PCR DNA from 24 HeLa Tet-on clones was subjected to PCR analysis, resulting in two fragments of different sizes: the endogenously expressed genomic ALD DNA was amplified as a 334 bp fragment (including 145 bp from intron 6) whereas clones with the D194H-ALD cDNA gave an additional fragment of 189 bp. Login to comment
182 Of 24 clones analysed, 10 carried the mutated D194H-ALD cDNA. Login to comment
185 Finally, one clone, HeLa-D194H, was selected that showed high inducibility and no background expression. Login to comment
186 Northern blot D194H-ALDP expression was induced by different doxycycline concentrations. Login to comment

[hide] A novel cell model to study the function of the ad... Biochem Biophys Res Commun. 2006 Mar 3;341(1):150-7. Epub 2006 Jan 6. Gueugnon F, Volodina N, Taouil JE, Lopez TE, Gondcaille C, Grand AS, Mooijer PA, Kemp S, Wanders RJ, Savary S
A novel cell model to study the function of the adrenoleukodystrophy-related protein.
Biochem Biophys Res Commun. 2006 Mar 3;341(1):150-7. Epub 2006 Jan 6., [PMID:16412981]

Abstract [show]
X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder due to mutations in the ABCD1 (ALD) gene. ALDRP, the closest homolog of ALDP, has been shown to have partial functional redundancy with ALDP and, when overexpressed, can compensate for the loss-of-function of ALDP. In order to characterize the function of ALDRP and to understand the phenomenon of gene redundancy, we have developed a novel system that allows the controlled expression of the ALDRP-EGFP fusion protein (normal or non-functional mutated ALDRP) using the Tet-On system in H4IIEC3 rat hepatoma cells. The generated stable cell lines express negligible levels of endogenous ALDRP and doxycycline dosage-dependent levels of normal or mutated ALDRP. Importantly, the ALDRP-EGFP protein is targeted correctly to peroxisome and is functional. The obtained cell lines will be an indispensable tool in our further studies aimed at the resolution of the function of ALDRP to characterize its potential substrates in a natural context.

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43 This mutation in ALDRP, located in a region highly conserved between ALDRP and ALDP, corresponds to the D194H mutation in ALDP, a mutation found in X-ALD patients (see www.x-ald.nl), which disrupts the function without alteration of the peroxisomal localization and probably of the dimerization ability [30]. Login to comment
214 The D207H mutation in ALDRP is similar to the D194H mutation in ALDP that has been previously studied by stable transfection and has been shown to disrupt function without suppressing the capacity of dimerization and of import in the peroxisomal membrane [30]. Login to comment

[hide] Spectrum of mutations in the gene encoding the adr... Am J Hum Genet. 1995 Jan;56(1):44-50. Ligtenberg MJ, Kemp S, Sarde CO, van Geel BM, Kleijer WJ, Barth PG, Mandel JL, van Oost BA, Bolhuis PA
Spectrum of mutations in the gene encoding the adrenoleukodystrophy protein.
Am J Hum Genet. 1995 Jan;56(1):44-50., [PMID:7825602]

Abstract [show]
X-linked adrenoleukodystrophy (ALD) has been associated with mutations in a gene encoding an ATP-binding transporter, which is located in the peroxisomal membrane. Deficiency of the gene leads to impaired peroxisomal beta-oxidation. Systematic analysis of the open reading frame of the ALD gene, using reverse transcriptase-PCR, followed by direct sequencing, revealed mutations in all 28 unrelated kindreds analyzed. No entire gene deletions or drastic promoter mutations were detected. In only one kindred did the mutation involve multiple exons. The other mutations were small alterations leading to missense (13 of 28) or nonsense mutations, a single amino acid deletion, frameshifts, or splice acceptor-site defects. Mutations affecting a single amino acid were concentrated in the region between the third and fourth putative transmembrane domains and in the ATP-binding domain. Mutations were detected in all investigated ALD kindreds, suggesting that this gene is the only gene responsible for X-linked ALD. This overview of mutations is useful in the determination of structurally and functionally important regions and provides an efficient screening strategy for identification of mutations in the ALD gene.

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85 The mutation T1045C created a novel HpaII site, which was confirmed Table 2 Mutations in the Putative ALD Gene in Patients Studied Genomic- Kindred Type of Mutation and Amino Acid Genomic-PCR Mutation Reference cDNA Alterationa Alterationb Exonc Primers Detectiond Phenotype' Number Missense: C696Tf ................ R104C (R) 1 303F + 821R 303F, 821R AMN 17 G832A ................ S149N (N) 1 702F + 1145R 702F, 931R AMN 8 G841C ................ R152P (K) 1 702F + 1145R 702F, 931R ChALD 27 G874Af ................ R163H (R) 1 702F + 931R 702F, 931R SympCar 14 G966C ................ D194H (D) 1 685F + 1145R 914F, 1145R ChALD 12 T1045C ................ L220P (L) 1 914F + 1145R HpaII AMN 7 G1182A ................. G266R (G) 1 702F + 1231R 914F,1231R AMN 24 G1552A ................. R389H (R) 3 1479F + 1861R 1479F,1752R AMN 20 (2X): G2211A................. E609K(E) 8 544F*+ 1078R*h 544F*, 876R* AMN 13,18 A2212G ................ E609G (E) 8 544F*+ 1078R*h 544F*, 876R* ChALD 5 C2235Tf................ R617C (R) 8 544F* + 2742R 544F*, 876R* ChALD 23 C2364Tf................ R660W (R) 9 544F* + 2742R 2312F, 1078R* AMN 21 Amino acid deletion: del 2355-2357 ........... del 1657(V) 9 849F* + 2478Rh 2312F,1078R* ChALD 6 Nonsense: C783Tf ................ Q133h 1 702F + 931R 702F, 931R ChALD 26 G797A ................ W137h 1 685F +1145R 702F,931R ChALD 10 C855T ................ Q157h 1 702F + 1145R 702F,931R AMN 9 C929A ................ Y181h 1 702F + 1145R HpaIl ChALD 15 Frameshift: delC442 ................ A19> 1 303F + 821R 303F,593R ChALD 2 del C663 ................ G92> 1 303F + 840R 576F, 821R ChALD 22 dell71-1178 ........... F261> 1 702F + 1231R 914F,1231R ChALD 28 (4X): del 1801-1802 ........... E471> 5 1781F + 1861R Polyacrylamide gel ChALD, AMN 3,4,16,25 alt 1989-2377 ........... P534> 6-9 1890F +2669R 1890F,1078R* AMN 11 Splice defect: de12021-2054 ........... R545> SA 7 1880F +2132R 1880F,2114R ChALD 1 ins 8 bp 2251f ............ R622> SA 9 849F* + 1078R*h 849F*, 1078R* AMN 19 a Nucleotide numbers refer to Mosser et al. (1993), EMBL database Z21876. Login to comment