ABCMdb

ABCD1 p.Ser606Leu

ClinVar:	c.1818G>A , p.Ser606= ? , Uncertain significance c.1817C>T , p.Ser606Leu D , Pathogenic
Predicted by SNAP2:	A: D (91%), C: D (95%), D: D (95%), E: D (95%), 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%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D,

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[hide] ABCD1 mutations and the X-linked adrenoleukodystro... Hum Mutat. 2001 Dec;18(6):499-515. Kemp S, Pujol A, Waterham HR, van Geel BM, Boehm CD, Raymond GV, Cutting GR, Wanders RJ, Moser HW
ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical correlations.
Hum Mutat. 2001 Dec;18(6):499-515., [PMID:11748843]

Abstract [show]
X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC half-transporter (ALDP) involved in the import of very long-chain fatty acids (VLCFA) into the peroxisome. The disease is characterized by a striking and unpredictable variation in phenotypic expression. Phenotypes include the rapidly progressive childhood cerebral form (CCALD), the milder adult form, adrenomyeloneuropathy (AMN), and variants without neurologic involvement. There is no apparent correlation between genotype and phenotype. In males, unambiguous diagnosis can be achieved by demonstration of elevated levels of VLCFA in plasma. In 15 to 20% of obligate heterozygotes, however, test results are false-negative. Therefore, mutation analysis is the only reliable method for the identification of heterozygotes. Since most X-ALD kindreds have a unique mutation, a great number of mutations have been identified in the ABCD1 gene in the last seven years. In order to catalog and facilitate the analysis of these mutations, we have established a mutation database for X-ALD ( http://www.x-ald.nl). In this review we report a detailed analysis of all 406 X-ALD mutations currently included in the database. Also, we present 47 novel mutations. In addition, we review the various X-ALD phenotypes, the different diagnostic tools, and the need for extended family screening for the identification of new patients.

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174 P560S 7 1678C>T n.d. # P560L 7 1679C>T Reduced P560L 7 1679C>T Reduced fs I588 7 1765delC n.d. # R591P 7 1772G>C Absent S606L 8 1817C>T Present E609K 8 1825G>A Absent E609K 8 1825G>A Absent R617C 8 1849C>T Absent R617H 8 1850G>A Absent R617H 8 1850G>A Absent A626T 9 1876G>A Absent A626T 9 1876G>A Absent A626D 9 1877C>A n.d. # E630G 9 1889A>G n.d. # C631Y 9 1892G>A n.d. # T632I 9 1895C>T n.d. # V635M 9 1903G>A n.d. # L654P 9 1961T>C Absent # R660W 9 1978C>T Absent fs L663 9 1988insT n.d. # fs L663 IVS 9 IVS9+1g>a n.d. # fs L663 IVS 9 IVS9-1g>a n.d. # H667D 10 1999C>G Absent # T668I 10 2003C>T Absent # T693M 10 2078C>T Present # exon1-5del 1-5 n.d. # The 47 mutations marked with a # are novel unique mutations reported for the first time in this paper. Login to comment
280 For two missense mutations located in functional regions of the ATP-binding domain it was demonstrated that the mutations resulted in either decreased ATP-binding capacity (S606L) or reduced ATPase activity (G512S) [Roerig et al., 2001]. Login to comment

[hide] Mutational analyses of Taiwanese kindred with X-li... Pediatr Neurol. 2006 Oct;35(4):250-6. Chiu HC, Liang JS, Wang JS, Lu JF
Mutational analyses of Taiwanese kindred with X-linked adrenoleukodystrophy.
Pediatr Neurol. 2006 Oct;35(4):250-6., [PMID:16996397]

Abstract [show]
X-linked adrenoleukodystrophy is a neurodegenerative disorder with highly variable clinical presentation, including the childhood cerebral form, adult form adrenomyeloneuropathy, and Addison disease. The biochemical hallmark of the disorder is the accumulation of saturated very long chain fatty acids in all tissues and body fluids. This accumulation results from mutations in the ABCD1 gene localized to Xq28. Using polymerase chain reaction and direct sequencing of deoxyribonucleic acid, we identified five novel mutations, including a microdeletion (1624 del ATC), a splicing site mutation (intervening sequence 1 [IVS1] -2a>c), and three missense mutations (1172 T>C, 1520 G>A, and 1754 T>C), from Taiwanese kindred with X-linked adrenoleukodystrophy. A polymorphism involving a single nucleotide deletion in the intervening sequence 5 (IVS5 -6 del c) of the ABCD1 gene, previously misattributed as a mutation in the Chinese population, was also identified. The dinucleotide deletion (1415 del AG) mutation common in Japan and Western countries was not found as frequently in the Chinese and Taiwanese populations. Instead, a higher mutation frequency was observed in exon 6 of the ABCD1 gene among Japanese, Chinese, and Taiwanese kindred with X-linked adrenoleukodystrophy, representing a potential mutational hotspot for future mutational screening among these Asian populations.

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126 However, common missense mutations, including G226R, Y296C, R518Q, and S606L [13,33,35], have been observed between Chinese and Japanese (though as yet not identified in Taiwanese) X-linked adrenoleukodystrophy patients, indicating the possibility of inheritance from common ancestors. Login to comment
124 However, common missense mutations, including G226R, Y296C, R518Q, and S606L [13,33,35], have been observed between Chinese and Japanese (though as yet not identified in Taiwanese) X-linked adrenoleukodystrophy patients, indicating the possibility of inheritance from common ancestors. Login to comment

[hide] Mammalian peroxisomal ABC transporters: from endog... Br J Pharmacol. 2011 Dec;164(7):1753-66. doi: 10.1111/j.1476-5381.2011.01435.x. Kemp S, Theodoulou FL, Wanders RJ
Mammalian peroxisomal ABC transporters: from endogenous substrates to pathology and clinical significance.
Br J Pharmacol. 2011 Dec;164(7):1753-66. doi: 10.1111/j.1476-5381.2011.01435.x., [PMID:21488864]

Abstract [show]
Peroxisomes are indispensable organelles in higher eukaryotes. They are essential for a number of important metabolic pathways, including fatty acid alpha- and beta-oxidation, and biosynthesis of etherphospholipids and bile acids. However, the peroxisomal membrane forms an impermeable barrier to these metabolites. Therefore, peroxisomes need specific transporter proteins to transfer these metabolites across their membranes. The mammalian peroxisomal membrane harbours three ATP-binding cassette (ABC) transporters. In recent years, significant progress has been made in unravelling the functions of these ABC transporters. There is ample evidence that they are involved in the transport of very long-chain fatty acids, pristanic acid, di- and trihydroxycholestanoic acid, dicarboxylic acids and tetracosahexaenoic acid (C24:6omega3). Surprisingly, only one disease is associated with a deficiency of a peroxisomal ABC transporter. Mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein are the cause for X-linked adrenoleukodystrophy, an inherited metabolic storage disorder. This review describes the current state of knowledge on the mammalian peroxisomal ABC transporters with a particular focus on their function in metabolite transport.

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259 Two X-ALD causing missense mutations located in the ATP-binding domain of ALDP resulted in either decreased ATP-binding capacity (p.Ser606Leu) or reduced ATPase activity (p.Gly512Ser), when analysed in the context of recombinant nucleotide binding domains (Roerig et al., 2001). Login to comment

[hide] Mutational analysis and genotype-phenotype correla... Arch Neurol. 1999 Mar;56(3):295-300. Takano H, Koike R, Onodera O, Sasaki R, Tsuji S
Mutational analysis and genotype-phenotype correlation of 29 unrelated Japanese patients with X-linked adrenoleukodystrophy.
Arch Neurol. 1999 Mar;56(3):295-300., [PMID:10190819]

Abstract [show]
BACKGROUND: X-linked adrenoleukodystrophy (ALD) is an inherited disease characterized by progressive neurologic dysfunction, occasionally associated with adrenal insufficiency. The classic form of ALD usually has onset in childhood (childhood cerebral ALD), with rapid neurologic deterioration leading to a vegetative state. Adult-onset cerebral ALD also presents with rapidly progressive neurologic dysfunction. Milder phenotypes such as adrenomyeloneuropathy and Addison disease only also have been recognized. Despite discovery of the causative gene, a molecular basis for the diverse clinical presentations remains to be elucidated. OBJECTIVES: To conduct mutational analyses in 29 Japanese patients with ALD from 29 unrelated families, to obtain knowledge of the spectrum of mutations in this gene, and to study genotype-phenotype correlations in Japanese patients. METHODS: The 29 patients comprised 13 patients with childhood cerebral ALD, 11 patients with adult-onset cerebral ALD, and 5 patients with adrenomyeloneuropathy. We conducted detailed mutational analyses of 29 unrelated Japanese patients with ALD by genomic Southern blot analysis and direct nucleotide sequence analysis of reverse transcriptase-polymerase chain reaction products derived from total RNA that was extracted from cultured skin fibroblasts, lymphoblastoid cells, or peripheral blood leukocytes. RESULTS: Three patients with adult-onset cerebral ALD were identified as having large genomic rearrangements. The remaining 26 patients were identified as having 21 independent mutations, including 12 novel mutations resulting in small nucleotide alterations in the ALD gene. Eighteen (69%) of 26 mutations were missense mutations. Most missense mutations involved amino acids conserved in homologous gene products, including PMP70, mALDRP, and Pxa1p. The AG dinucleotide deletion at position 1081-1082, which has been reported previously to be the most common mutation in white patients (12%-17%), was also identified as the most common mutation in Japanese patients (12%). All phenotypes were associated with mutations resulting in protein truncation or subtle amino acid changes. There were no differences in phenotypic expressions between missense mutations involving conserved amino acids and those involving nonconserved amino acids. CONCLUSIONS: There are no obvious correlations between the phenotypes of patients with ALD and their genotypes, suggesting that other genetic or environmental factors modify the phenotypic expressions of ALD.

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42 Mutations in the ALD Gene That Result in Amino Acid Substitutions or In-frame Deletions* Patient No. Phenotype Mutation† Exon Effect of Mutation‡ Position of Mutation§ Amino Acid Identityሻ Family DataPMP70 mALDRP Pxa1p Amino Acid Deletion G4010 ACALD del.1256-1258 1 del.E291 EAA-like motif E E E CCALD G4011(s) ACALD del.2146-2157¶ 7 del.HILQ587-590 Between Walker A and B# HILE HIVQ YLLK No family history Missense Mutation G4012 CCALD A829G 1 N148S TM3 N N N AMN G1986 CCALD G984A¶ 1 D200N TM4 D D D ACALD G4013 CCALD A1026G¶ 1 N214D TM4 N N N Not available G4014 AMN G1182A 1 G266R Between TM5 and EAA motif G G Non AMN G4015(s) CCALD G1182A 1 G266R Between TM5 and EAA motif G G Non No family history G4016(s) AMN G1197A 1 E271K Between TM5 and EAA motif T E R No family history G4017(s) ACALD A1273G¶ 1 Y296C EAA motif Y Y Y No family history G4018 CCALD A1273G¶ 1 Y296C EAA motif Y Y Y Not available G4019 AMN C1587T¶ 3 R401W Between TM6 and Walker A R R R Asymptomatic carrier G4020 CCALD G1906T¶ 6 G507V Walker A# G G G Not available G4021 CCALD G1939A 6 R518Q Walker A# R R R CCALD G4022 CCALD G1939A 6 R518Q Walker A# R R R Not available G4023 ACALD T2005C¶ 6 F540S Between Walker A and B# F F F Adult asymptomatic carrier G4024(s) CCALD A2017G 6 Q544R Between Walker A and B# Q Q Q No family history G4025 CCALD C2065T 7 S560L Between Walker A and B# P P P Adult asymptomatic carrier G2469(s) ACALD C2157T¶ 7 R591W Between Walker A and B# R R R No family history G2022(s) AMN C2203T 8 S606L Between Walker A and B# S S S No family history G4026 ACALD C2364T 8 R660W C-terminal to Walker B R R R ACALD *ALD indicates adrenoleukodystrophy; ACALD, adult-onset cerebral ALD; CCALD, childhood cerebral ALD; AMN, adrenomyeloneuropathy; (s), apparently sporadic patients; and del., delete. Login to comment
65 Although each of the remaining 3 mutations (E271K, R401W, and S606L) was identified in only 1 apparently sporadic case of a patient with AMN, a review of the literature indicated that the S606L mutation has been identified in 2 patients with Addison disease only but in no patients with CCALD.33,46 COMMENT MUTATIONS IN THE ALD GENE Because of the low frequency (4%-7%) of large genomic rearrangements in the ALD gene,26,33,34 a detailed nucleotide sequence analysis to detect small nucleotide alterations is required for most cases of ALD. Login to comment
85 In the present study, 1 patient with AMN with no family history was identified to have the C2203T (S606L) mutation, although it had previously been reported33,46 to beassociatedexclusivelywiththeAddisondiseaseonlyphe- notype in other studies. Login to comment

[hide] X-linked adrenoleukodystrophy: very long-chain fat... Mol Genet Metab. 2007 Mar;90(3):268-76. Epub 2006 Nov 7. Kemp S, Wanders RJ
X-linked adrenoleukodystrophy: very long-chain fatty acid metabolism, ABC half-transporters and the complicated route to treatment.
Mol Genet Metab. 2007 Mar;90(3):268-76. Epub 2006 Nov 7., [PMID:17092750]

Abstract [show]
X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in elevated levels of very long-chain fatty acids (VLCFA) and reduced VLCFA beta-oxidation in peroxisomes. The peroxisomal membrane harbors three additional closely related ABC half-transporters, ALDRP, PMP70 and PMP69 (PMP70R). ABC half-transporters must dimerize to form a functional full-transporter. Whether ALDP forms a homodimer or a heterodimer has not yet been resolved, but most indirect evidence favors homodimerization. The peroxisomal ABC half-transporters are functionally related. Over-expression of ALDRP can correct the biochemical defect both in X-ALD patients cells and the Abcd1 knockout mouse, providing an exciting new possibility for treatment of X-ALD patients. This paper provides an overview of current knowledge and the problems that have been encountered.

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57 ATP-binding and ATPase activity has been demonstrated for ALDP [17], and Roerig and coworkers demonstrated that two disease causing missense mutations located in the ATP-binding domain resulted in either decreased ATP-binding capacity (Ser606Leu), or reduced ATPase activity (Gly512Ser) [18]. Login to comment
56 ATP-binding and ATPase activity has been demonstrated for ALDP [17], and Roerig and coworkers demonstrated that two disease causing missense mutations located in the ATP-binding domain resulted in either decreased ATP-binding capacity (Ser606Leu), or reduced ATPase activity (Gly512Ser) [18]. Login to comment

[hide] Role of ATP-binding cassette transporters in brain... J Neurochem. 2008 Mar;104(5):1145-66. Epub 2007 Oct 31. Kim WS, Weickert CS, Garner B
Role of ATP-binding cassette transporters in brain lipid transport and neurological disease.
J Neurochem. 2008 Mar;104(5):1145-66. Epub 2007 Oct 31., [PMID:17973979]

Abstract [show]
The brain is lipid-rich compared to other organs and although previous studies have highlighted the importance of ATP-binding cassette (ABC) transporters in the regulation of lipid transport across membranes in peripheral tissues, very little is known regarding ABC transporter function in the CNS. In this study, we bring together recent literature focusing on potential roles for ABC transporters in brain lipid transport and, where appropriate, identify possible links between ABC transporters, lipid transport and neurological disease. Of the 48 transcriptionally active ABC transporters in the human genome, we have focused on 13 transporters (ABCA1, ABCA2, ABCA3, ABCA4, ABCA7 and ABCA8; ABCB1 and ABCB4; ABCD1 and ABCD2; ABCG1, ABCG2, and ABCG4) for which there is evidence suggesting they may contribute in some way to brain lipid transport or homeostasis. The transporters are discussed in terms of their location within brain regions and brain cell types and, where possible, in terms of their known functions and established or proposed association with human neurological diseases. Specific examples of novel treatment strategies for diseases, such as Alzheimer's disease and X-linked adrenoleukodystrophy that are based on modulation of ABC transporter function are discussed and we also examine possible functions for specific ABC transporters in human brain development.

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208 Two ABCD1 mutations associated with X-ALD (Ser606Leu and Gly512Ser) result in decreased ATP binding and reduced ATPase activity, respectively, thereby indicating that an energy dependent transport activity is crucial to the function of ABCD1 (Roerig et al. 2001). Login to comment
212 Two ABCD1 mutations associated with X-ALD (Ser606Leu and Gly512Ser) result in decreased ATP binding and reduced ATPase activity, respectively, thereby indicating that an energy dependent transport activity is crucial to the function of ABCD1 (Roerig et al. 2001). Login to comment

[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
181 These included relatively common mutants which are unstable in fibroblasts, but which can be rescued by the application of proteasome inhibitors (p.Ser606Leu, p.Arg617His and p.His667Asp), the p.Arg104Cys mutant in which degradation of ALDP cannot be prevented by proteasome inhibitors and the p.Tyr174Cys mutant which is Table 2 X-ALD mutants used for analysis of targeting in tobacco cells The occurrence is the number of documented patients bearing the mutation; source: X-ALD database (http://www.x-ald.nl). 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
182 These included relatively common mutants which are unstable in fibroblasts, but which can be rescued by the application of proteasome inhibitors (p.Ser606Leu, p.Arg617His and p.His667Asp), the p.Arg104Cys mutant in which degradation of ALDP cannot be prevented by proteasome inhibitors and the p.Tyr174Cys mutant which is c The Authors Journal compilation c Table 2 X-ALD mutants used for analysis of targeting in tobacco cells The occurrence is the number of documented patients bearing the mutation; source: X-ALD database (http://www.x-ald.nl). Login to comment

[hide] Adrenoleukodystrophy: subcellular localization and... J Neurochem. 2007 Jun;101(6):1632-43. Takahashi N, Morita M, Maeda T, Harayama Y, Shimozawa N, Suzuki Y, Furuya H, Sato R, Kashiwayama Y, Imanaka T
Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations.
J Neurochem. 2007 Jun;101(6):1632-43., [PMID:17542813]

Abstract [show]
Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.

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2 In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. Login to comment
3 When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Login to comment
4 Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Login to comment
9 These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Login to comment
35 We found that mutant ALDPs with the missense mutations in the G116R S342P Q544R R617H H667D Y174C NH2 COOH C sequence Cytosol Membrane Matrix Walker A Walker B S606P, S606L R104C Fig. 1 A putative secondary structure of adrenoleukodystrophy protein. Login to comment
71 CHO-K1 cells (5 · 105 cells) were cultured in Ham`s F-12 medium with 10% FBS, 70 lg/mL of penicillin, and 140 lg/mL of streptomycin and transfected with 5 lg of pMAM2/ Table 1 Oligonucleotide primer sequences used for the generation of mutant ALDP constructs Construct name Forward primer (5' to 3') (top) R104C GCCTTGGTGAGCTGCACCTTCCTGTCG G116R GCCCGCCTGGACAGAAGGCTGGCC Y174C GCCTACCGCCTCTGCTCCTCCCAG S342P TGGAGCGCCCCGGGCCTGCTCATG Q544R GCATGTTCTACATCCCGCGGAGGCCCTACATGTC S606P AAGGACGTCCTGCCGGGTGGCGAGAAG S606L AAGGACGTCCTGTTGGGTGGCGAGAAG R617H GCAGAGAATCGGCATGGCCCACATGTTCTACCACAGGC H667D TCCCTGTGGAAATACGACACACACTTGCTA The underlined letters indicate the single base mutation leading to an amino acid replacement. Login to comment
120 However, the ratios of certain His-ALDPs (S606L, R617H, and H667D) were significantly lower than those of wild type His-ALDP and other mutant His-ALDPs. Login to comment
125 Taken together, it appears His-ALDP (S606L, R617H, and H667D) might be degraded after translation. Login to comment
127 As shown in Fig. 3d, His-ALDPs (R104C, G116R, S342P, Q544R, S606P, and S606L) exhibited a punctate staining pattern in the cells, which was superimposable on the distribution of catalase in the same cells, suggesting that these mutant His-ALDPs were correctly localized to peroxisomes. Login to comment
135 In this experiment, we chose R104C, G116R, and S606P (with normal localization in peroxisomes), Y174C (mislocalization), and S606L and H667D (degradation). Login to comment
141 In contrast, mutant ALDP-GFP (S606L and H667D) was not detected in any subcellular fractions although PMP70 and catalase were recovered in fractions 2 and 3. Login to comment
150 The fragments were not extractable with 0.1 mol/L sodium carbonate, indicating (a) (c) (b) (d) 400 140 120 100 Expressionratio(%) 80 60 40 His-ALDP R104C G116R Y174C S342P Q544R S606P S606L R617H H667D Catalase 20 0 100 Expressionratio(%) 80 60 40 20 0 350 300 250 200 150 pmol/h/mgprotein 100 50 0 Normal (139T) X-ALD (163T) M ock M ock W ild W ild N one S606L His-ALDP GFP Catalase R 617H H 667D R 104CG 116RY174C S342PQ 544RS606PS606LR 617HH 667D M ock W ildR 104CG 116RY174CS342PQ 544RS606PS606LR 617HH 667D Fig. 3 Expression of wild type and mutant His-adrenoleukodystrophy proteins (ALDPs) in X-linked adrenoleukodystrophy (X-ALD) fibroblasts (163T). Login to comment
159 (c) Co-expression of wild or mutant His-ALDP (S606L, R617H, or H667D) with green fluorescence proteins (GFP). Login to comment
169 Effect of proteasome inhibitors on mutant ALDP The transient and stable expression experiments of mutant ALDPs suggest that mutant ALDPs such as S606L, R617H, H667D, and R104C are degraded by proteases. Login to comment
180 Degradation of mutant ALDP (S606L) was similarly inhibited by lactacystin, but not by leupeptin, wild R104C G116R ALDP-GFP ALDP PMP70 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 Non specific ALDP-GFP ALDP PMP70 Non specific ALDP-GFP ALDP PMP70 Non specific Y174C H667D S606P S606L Fig. 4 Subcellular localization of wild type and mutant adrenoleukodystrophy protein (ALDP) -green fluorescence proteins (GFP) in CHO cells. Login to comment
181 The mitochondrial and light mitochondrial fraction from CHO cells expressing wild type ALDP and ALDP-GFP or each mutant ALDP-GFP (R104C, G116R, Y174C, S606P, S606L, or H667D) was fractionated by equilibrium density centrifugation on sucrose. Login to comment
217 S606P and S606L are in the ABC signature motif. Login to comment
223 Mutant ALDP-GFP (S606L) showed similar results (date not shown). Login to comment
229 Recently Lie et al. investigated the dimerization of the COOH-terminal half of ALDP by a yeast two-hybrid assay and found that it could dimerize Table 2 Expression and localization of missense ALDPs Mutant ALDP Transient Stable Expressiona Localizationb b-Oxidationc Expressiona Localizationb Wild +++ Px + ++ Px R104Cd , G116R, S342P, Q544R, S606P +++ Px ) ++ Px Y174C +++ mis ) + mis S606L ++ Px ) ) ) R617H, H667D + ) ) ) ) Wild and mutant His-ALDPs or ALDP-GFPs were transiently expressed in X-ALD fibroblasts or stably expressed in CHO cells, respectively. Login to comment
244 In Fig. 4, the wild type ALDP would dimerize each other or with endogenous ALDP and could be stabilized, but such complex could not be detected on sucrose gradient of CHO cells expressing mutant ALDP (S606L and H667D). Login to comment
264 Recently, Roerig et al. suggested that mutation of S606L decreases affinity against ATP, although ATP hydrolyzing activity is normal using a NBD fragment expressed in Escherichia coli (Roerig et al. 2001). Login to comment
265 Our results suggest that the stability of mutant ALDP (S606L) is also affected by (the) mutation. Login to comment
271 In this study, we found that mutant ALDP (S606L, R617H, and H667D) was degraded by proteasomes together with wild type ALDP. Login to comment

[hide] Variability of endocrinological dysfunction in 55 ... Eur J Endocrinol. 1997 Jul;137(1):40-7. Korenke GC, Roth C, Krasemann E, Hufner M, Hunneman DH, Hanefeld F
Variability of endocrinological dysfunction in 55 patients with X-linked adrenoleucodystrophy: clinical, laboratory and genetic findings.
Eur J Endocrinol. 1997 Jul;137(1):40-7., [PMID:9242200]

Abstract [show]
X-linked adrenoleucodystrophy (ALD) has been shown to be one of the most frequent causes of Addison's disease in men. It is characterized by an impaired peroxisomal beta-oxidation of very long chain fatty acids and is associated with mutations of the ALD gene resulting in a defective peroxisomal membrane transport protein. There is a striking variability of endocrinological and neurological symptoms in patients with ALD, with no clearly evident correlation between mutations of the ALD gene and the different neurological phenotypes. No data on endocrinological symptoms and the ALD genotype have been published so far. We report endocrinological, clinical, laboratory and molecular genetic data from 55 patients with ALD from 34 families. Endocrinological symptoms of adrenal insufficiency were observed in 33 patients, 20 of whom showed additional neurological symptoms of cerebral ALD or adrenomyeloneuropathy. Isolated neurological symptoms were seen in 12 patients; in nine patients there were neither endocrinological nor neurological symptoms. Mutations of the ALD gene (n = 28) were detected in 50 patients (including nine sets of brothers) from 32 families. No correlation was found between the ALD gene mutation and endocrinological dysfunction. However, we found that all sets of brothers were concordant for the endocrinological phenotype (cortisol synthesis was reduced in two sets and normal in seven sets), whereas four sets showed a discordant neurological phenotype. As yet unknown hereditary factors other than mutations within the ALD gene may interfere with the endocrinological phenotype more strongly than with the neurological phenotype of ALD.

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118 Start of Cortisol Start of Age at endocrine Basal increase neurological examination symptoms ACTH cortisol after i.v. ACTH symptoms ALD gene Patient (year-month) (year-month) (pmol/l) (nmol/l) (nmol/l) (year-month) mutation 1a 9-05 6-08 223 179 0 7-02 cALD 2252-10 G-A 1b 13-03 9-05 41 331 41 9-05 cALD 2252-10 G-A 2a 26-01 - 6 386 221 - R418W 2b 27-10 - 6 461 229 11-00 cALD R418W 3a 11-06 6-00 536 138 8 - S606L 3b 11-06 6-00 423 143 11 10-10 cALD S606L 4a 4-04 2-09 >330 127 141 - R617C 4b 5-06 3-11 >330 97 50 - R617C 4c 7-10 5-05 >330 50 6 6-03 cALD R617C 5a 10-06 - 8 433 290 8-02 cALD del 1257-9 5b 17-07 - 9 637 629 - del 1257-9 6a 27-00 24-00 >320 30 11 18-03 AMN L107P 6b 32-10 - 47 279 185 19-01 AMN L107P 7a 34-08 - 48 469 350 24-00 AMN R418W 7b 36-05 - 43 486 201 28-00 AMN R418W 8a 14-05 - 33 287 94 - 1801 del AG 8b 16-10 - 14 348 149 - 1801 del AG 9a 13-07 5-10 1094 36 6 - 740 del G 9b 157-04 1241 63 11 - 740 del G cALD, cerebral adrenoleucodystrophy; AMN, adrenomyeloneuropathy; -, no symptoms. Login to comment

[hide] Functional characterization of the adrenoleukodyst... Endocr Res. 2002 Nov;28(4):741-8. Gartner J, Dehmel T, Klusmann A, Roerig P
Functional characterization of the adrenoleukodystrophy protein (ALDP) and disease pathogenesis.
Endocr Res. 2002 Nov;28(4):741-8., [PMID:12530690]

Abstract [show]
X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder characterized by abnormal accumulation of saturated very long chain fatty acids in tissues and body fluids with predominance in brain white matter and adrenal cortex. The clinical phenotype is highly variable ranging from the severe childhood cerebral form to asymptomatic persons. The responsible ALD gene encodes the adrenoleukodystrophy protein (ALDP), a peroxisomal integral membrane protein that is a member of the ATP-binding cassette (ABC) transporter protein family. The patient gene mutations are heterogeneously distributed over the functional domains of ALDP. The extreme variability in clinical phenotype, even within one affected family, indicates that besides the ALD gene mutations other factors strongly influence the clinical phenotype. To understand the cell biology and function of mammalian peroxisomal ABC transporters and to determine their role in the pathogenesis of X-ALD we developed a system for expressing functional ABC protein domains in fusion with the maltose binding protein. Wild type and mutant fusion proteins of the nucleotide-binding fold were overexpressed, purified, and characterized by photoaffinity labeling with 8-azido ATP or 8-azido GTP and a coupled ATP regenerating enzyme assay for ATPase activity. Our studies provide evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter and that ALD gene mutations alter peroxisomal transport function. The established disease model will be used further to study the influence of possible disease modifier proteins on ALDP function.

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41 The mutant constructs included missense mutations of patients with X-ALD in the nucleotide binding fold regions Walker A and 19mer (ALDP-NBF-G512S, ALDP-NBF-Q544R, ALDP-NBF-P560L, ALDP-NBF-R591Q, ALDP-NBF-S606L, and ALDP-NBF-D629H) and corresponding mutations in another ABC transporter in the peroxisome membrane, the 70 kDa peroxisomal membrane protein (PMP70; PMP70-NBF-G478R, PMP70- NBF-S572I). Login to comment
60 In contrast, the patient mutation S606L alters ATP binding affinity but has no effect on the overall ATPase activity. Login to comment
81 In contrast, missense mutations included in the second group like S606L in the ALD gene reduce ATP-binding but do not have any effect on the overall ATPase activity. Login to comment

[hide] Altered expression of ALDP in X-linked adrenoleuko... Am J Hum Genet. 1995 Aug;57(2):292-301. Watkins PA, Gould SJ, Smith MA, Braiterman LT, Wei HM, Kok F, Moser AB, Moser HW, Smith KD
Altered expression of ALDP in X-linked adrenoleukodystrophy.
Am J Hum Genet. 1995 Aug;57(2):292-301., [PMID:7668254]

Abstract [show]
X-linked adrenoleukodystrophy (ALD) is a neurodegenerative disorder with variable phenotypic expression that is characterized by elevated plasma and tissue levels of very long-chain fatty acids. However, the product of the gene defective in ALD (ALDP) is a membrane transporter of the ATP-binding cassette family of proteins and is not related to enzymes known to activate or oxidize fatty acids. We generated an antibody that specifically recognizes the C-terminal 18 amino acids of ALDP and can detect ALDP by indirect immunofluorescence. To better understand the mechanism by which mutations in ALDP lead to disease, we used this antibody to examine the subcellular distribution and relative abundance of ALDP in skin fibroblasts from normal individuals and ALD patients. Punctate immunoreactive material typical of fibroblast peroxisomes was observed in cells from seven normal controls and eight non-ALD patients. Of 35 ALD patients tested, 17 had the childhood-onset cerebral form of the disease, 13 had the milder adult phenotype adrenomyeloneuropathy, 3 had adrenal insufficiency only, and 2 were affected fetuses. More than two-thirds (69%) of all patients studied showed no punctate immunoreactive material. There was no correlation between the immunofluorescence pattern and clinical phenotype. We determined the mutation in the ALD gene in 15 of these patients. Patients with either a deletion or frameshift mutation lacked ALDP immunoreactivity, as expected. Four of 11 patients with missense mutations were also immunonegative, indicating that these mutations affected the stability or localization of ALDP. In the seven immunopositive patients with missense mutations, correlation of the location and nature of the amino acid substitution may provide new insights into the function of this peroxisomal membrane protein. Furthermore, the study of female relatives of immunonegative ALD probands may aid in the assessment of heterozygote status.

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176 In 11 patients, missense mutations that occurred throughout the protein were found: within the transmembrane domains (patients 1, 3, and 4), within the ATP-binding domain (patients 8-12), and on either side of the ATP-binding Table 3 Mutational Analysis of the ALD Gene in IS Unrelated Patients ALDP Patient Phenotype Mutation Consequence Immunoreactivity 1 .................. CALD 825 A-GG K276E + 2.................. AMN 870-2AGAGE291,& 3 .................. CALD 872 G-C E291D 4 .................. AMN 1023 T-IC S342P+ 5 .................. AMN 1166 G-C R389H + 6 .................. CALD 1201 G-AA R401Q + 7 ........ CALD 1415-6 AAG FS@472 8 ........ AMN 1771 G-AA R591Q + 9 ........ Addison 1817 C-T S606L + 10 ................ AMN 1850 G-AA R617H 11 ................ CALD 1876 G-AA A626T 12 ................ Fetus 1884 G-C D629H + 13 ................ CALD 1932 C-UT Q645X 14 ................ AMN 1978 C-OT R660W 15 ........ AMN AExon7-10 Null Mutations in the ALD gene were determined, as described in Methods, in 15 of the ALD patients reported in table 2. Login to comment
178 In 11 patients, missense mutations that occurred throughout the protein were found: within the transmembrane domains (patients 1, 3, and 4), within the ATP-binding domain (patients 8-12), and on either side of the ATP-binding Table 3 Mutational Analysis of the ALD Gene in IS Unrelated Patients ALDP Patient Phenotype Mutation Consequence Immunoreactivity 1 .................. CALD 825 A-GG K276E + 2 .................. AMN 870-2 AGAG E291,& 3 .................. CALD 872 G-C E291D 4 .................. AMN 1023 T-IC S342P + 5 .................. AMN 1166 G-C R389H + 6 .................. CALD 1201 G-AA R401Q + 7 ........ CALD 1415-6 AAG FS@472 8 ........ AMN 1771 G-AA R591Q + 9 ........ Addison 1817 C-T S606L + 10 ................ AMN 1850 G-AA R617H 11 ................ CALD 1876 G-AA A626T 12 ................ Fetus 1884 G-C D629H + 13 ................ CALD 1932 C-UT Q645X 14 ................ AMN 1978 C-OT R660W 15 ........ AMN AExon7-10 Null Mutations in the ALD gene were determined, as described in Methods, in 15 of the ALD patients reported in table 2. Login to comment

[hide] Cerebral adrenoleukodystrophy (ALD) in only one of... Ann Neurol. 1996 Aug;40(2):254-7. Korenke GC, Fuchs S, Krasemann E, Doerr HG, Wilichowski E, Hunneman DH, Hanefeld F
Cerebral adrenoleukodystrophy (ALD) in only one of monozygotic twins with an identical ALD genotype.
Ann Neurol. 1996 Aug;40(2):254-7., [PMID:8773611]

Abstract [show]
We report on monozygotic twins with different clinical phenotypes of X-linked adrenoleukodystrophy. At the age of 10 years both boys were neurologically asymptomatic. The first cranial magnetic resonance examination showed normal findings in the first twin and parietooccipital demyelination in the second. The latter developed behavioral problems 9 months later, followed by visual impairment and gait ataxia. His cranial magnetic resonance image at the age of 11 years showed progressive demyelination. In contrast, neurological status and magnetic resonance images remained normal in the first twin. The same point mutation in exon 8 of the adrenoleukodystrophy gene (C2203T) was detected in both boys. All genotype examinations were consistent with the diagnosis of monozygotic twins, suggesting that some nongenetic factors may be important for different adrenoleukodystrophy phenotypes.

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85 The S606L mutation reported here has already been described in a patient with ALD and Addison's disease [l11. Login to comment

[hide] Biochemical aspects of X-linked adrenoleukodystrop... Brain Pathol. 2010 Jul;20(4):831-7. Kemp S, Wanders R
Biochemical aspects of X-linked adrenoleukodystrophy.
Brain Pathol. 2010 Jul;20(4):831-7., [PMID:20626744]

Abstract [show]
X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder. The disease is characterized by the accumulation of very long-chain fatty acids (VLCFA; >C22) in plasma and tissues. X-ALD is caused by mutations in the ABCD1 gene encoding ALDP, an adenosine triphosphate (ATP)-binding-cassette (ABC) transporter located in the peroxisomal membrane. In this paper, we describe the current knowledge on the function of ALDP, its role in peroxisomal VLCFA beta-oxidation and the consequences of a defect in ALDP on VLCFA metabolism. Furthermore, we pay special attention to the role of the VLCFA elongation system in VLCFA homeostasis, with elongation of very long-chain fatty acids like-1 (ELOVL1) as key player, and its relevance to X-ALD.

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47 ALDP is an integral peroxisomal membrane protein with the nucleotide-binding domain located toward the cytoplasmic surface of the peroxisomal membrane (14).ATP-binding andATPase activity has been demonstrated for ALDP (71), and Roerig et al demonstrated that two disease causing missense mutations located in the ATP-binding domain resulted in either decreased ATP-binding capacity (p.Ser606Leu) or reduced ATPase activity (p.Gly512Ser) (64). Login to comment

[hide] Mutational analysis of patients with X-linked adre... Hum Mutat. 1995;6(2):104-15. Kok F, Neumann S, Sarde CO, Zheng S, Wu KH, Wei HM, Bergin J, Watkins PA, Gould S, Sack G, et al.
Mutational analysis of patients with X-linked adrenoleukodystrophy.
Hum Mutat. 1995;6(2):104-15., [PMID:7581394]

Abstract [show]
Adrenoleukodystrophy (ALD) is an X-linked neurodegenerative disorder characterized by elevated very long chain fatty acid (VLCFA) levels, reduced activity of peroxisomal VLCFA-CoA ligase, and variable phenotypic expression. A putative gene for ALD was recently identified and surprisingly encodes a protein (ALDP) that belongs to a family of transmembrane transporters regulated or activated by ATP (the ABC proteins). We have examined genomic DNA from ALD probands for mutations in the putative ALD gene. We detected large deletions of the carboxyl-terminal portion of the gene in 4 of 112 probands. Twenty-five of the ALD probands whose ALD genes appeared normal by Southern blot analysis were surveyed for mutations by Single Strand Conformation Polymorphism (SSCP) procedures and DNA sequence analysis. SSCP variants were detected in 22 probands and none in 60 X-chromosomes from normal individuals. Mutations were detected in all of the ALD probands. The mutations were distributed throughout the gene and did not correlate with phenotype. Approximately half were non-recurrent missense mutations of which 64% occurred in CpG dinucleotides. There was a cluster of frameshift mutations in a small region of exon 5, including an identical AG deletion in 7 unrelated probands. These data strongly support the supposition that mutations in the putative ALD gene result in ALD.

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131 3' deletion 3' deletion 3' deletion 3' deletion R104C A141T R152C R182P Frameshift at AA 231 G277W R389H Spl mutation at AA 408 Q466 stop Frameshift at AA 470 Frameshift at AA 470 Frameshift at AA 472 Frameshift at AA 472 Frameshift at AA 472 Frameshift at AA 472 Frameshift at AA 472 Frameshift at AA 472 Frameshift at AA 472 G512S M566K S606L L516L R617H R660W - - Exons 3-10 Exons 7-10 Exons 8-10 Exons 7-10 33 Anglos 5 Scott 8 Anglos 7 Anglos 11 Jewish 36 Irish 51 Italian 37 Filipino 28 Anglos 23 Anglos 11 Anglos 8 Anglos 40 Italian 22 German 4 Anglos 5 black 8 Anglos 31 Anglos 10 Anglos 28 Anglos 22 Italian 8 German 35 German 7 Hispanic 28 German 24 Anglos 18 Jewish 9 Hispanic AMNa C E R ~ Cer Add' Cer AMN AMN AMN AMN Cer Cer Cer Add AMN AMN Cer Cer Cer AMN Add AMN AMN Cer AMN Cer AMN AMN AMN 5 Cer,AMN,Add 4 Cer,AMN 1 Cer 5 Cer,AMN,Add 1 4 2 1 2 2 5 Adopted 5 2 15 1 13 2 2 1 Cer AMN AMN,Add AMN Cer,AMN Cer,AMN Cer,AMN,Add ? Login to comment
234 Four mutations were found within the ATP bind- ing domain, one in Walker A (G512S), one in Walker B (R617H), one in the highly conserved sequence preceding Walker B (S606L),and one in the middle of the domain. Login to comment

[hide] Characterization and functional analysis of the nu... FEBS Lett. 2001 Mar 9;492(1-2):66-72. Roerig P, Mayerhofer P, Holzinger A, Gartner J
Characterization and functional analysis of the nucleotide binding fold in human peroxisomal ATP binding cassette transporters.
FEBS Lett. 2001 Mar 9;492(1-2):66-72., [PMID:11248239]

Abstract [show]
The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.

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5 Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Login to comment
24 For the mutant constructs we selected X-ALD patient mutations in highly conserved residues in the Walker A and 19-mer region of the NBF of ALDP (G512S and S606L) and the corresponding PMP70 mutations (G478R and S572I). Login to comment
85 Additionally, we changed the conserved serine in the 19-mer motif of PMP70 and ALDP to isoleucine (S572I) and leucine (S606L), respectively. Login to comment
88 The K-subunit of L-galactosidase (L-Protein Synthetic oligonucleotide Mutation ALDP 5P-CCCCAATGGCTGCAGCAAGAGCTCCC-3P G512S 5P-GGATCCGGACAGGGAGCTCTTGCTGCAGC-3P ALDP 5P-ACTGGAAGGACGTCCTGTTGGG-3P S606L 5P-CGCCACCCAACAGGACGTCCTTCC-3P PMP70 5P-GGCTGCAGAAAGAGTTCACTTTTCCG-3P G478R 5P-GGCCATAATTCACCAAGAACACGGAAA AGTGAACTCTTTCTG-3P PMP70 5P-GACGTACTCATTGGTGGAG-3P S572I 5P-CCACCAATGAGTACGTCCATCCAATCC-3P gal) in fusion with the MBP was used as a control. Login to comment
117 G512S and S606L cause X-ALD. Login to comment
136 The S606L and G478R mutants have a decreased ATP binding a¤nity while the G512S and S572I mutants decrease the maximum velocity of ATPase activity. Login to comment
171 In contrast, the X-ALD patient mutation S606L alters ATP binding a¤nity but has no e¡ect on the overall ATPase activity. Login to comment
172 Because the carboxyl-terminal halves of ALDP, PMP70 and ALDR are involved in protein dimerization and interactions with other protein motifs like the transmembrane domain [28], it is possible that the S606L missense mutation in ALDP exerts its disease causing e¡ect in this manner. Login to comment
182 Previous studies on MDR suggest that the ATPase activity of the native protein Table 1 Kinetic parameters of ATPase activity in wild type and mutant ALDP and PMP70 NBF fusion proteins Fusion protein KM (WM) Vmax (nmol/Wmol NBF/min) Speci'c activity (1033 U/mg) ALDP (wild type) 11.5 þ 0.97 641.9 þ 10.7 10.0 þ 0.17 ALDP (G512S) 17.9 þ 1.23 279.3 þ 4.2 4.4 þ 0.06 ALDP (S606L) 45.6 þ 2.40 666.0 þ 8.7 10.4 þ 0.14 PMP70 (wild type) 8.2 þ 0.52 580.8 þ 6.7 9.0 þ 0.10 PMP70 (G478R) 161.8 þ 34.40 641.2 þ 28.2 10.0 þ 0.44 PMP70 (S572I) 9.9 þ 0.82 298.1 þ 4.7 4.6 þ 0.07 The kinetic data of all fusion proteins are mean values and standard deviations of 15^20 measurements at various protein concentrations using at least two distinct protein preparations. Login to comment
86 Additionally, we changed the conserved serine in the 19-mer motif of PMP70 and ALDP to isoleucine (S572I) and leucine (S606L), respectively. Login to comment
89 The K-subunit of L-galactosidase (LProtein Synthetic oligonucleotide Mutation ALDP 5P-CCCCAATGGCTGCAGCAAGAGCTCCC-3P G512S 5P-GGATCCGGACAGGGAGCTCTTGCTGCAGC-3P ALDP 5P-ACTGGAAGGACGTCCTGTTGGG-3P S606L 5P-CGCCACCCAACAGGACGTCCTTCC-3P PMP70 5P-GGCTGCAGAAAGAGTTCACTTTTCCG-3P G478R 5P-GGCCATAATTCACCAAGAACACGGAAA AGTGAACTCTTTCTG-3P PMP70 5P-GACGTACTCATTGGTGGAG-3P S572I 5P-CCACCAATGAGTACGTCCATCCAATCC-3P gal) in fusion with the MBP was used as a control. Login to comment
118 G512S and S606L cause X-ALD. Login to comment
137 The S606L and G478R mutants have a decreased ATP binding a&#a4;nity while the G512S and S572I mutants decrease the maximum velocity of ATPase activity. Login to comment
173 Because the carboxyl-terminal halves of ALDP, PMP70 and ALDR are involved in protein dimerization and interactions with other protein motifs like the transmembrane domain [28], it is possible that the S606L missense mutation in ALDP exerts its disease causing e&#a1;ect in this manner. Login to comment
183 Previous studies on MDR suggest that the ATPase activity of the native protein Table 1 Kinetic parameters of ATPase activity in wild type and mutant ALDP and PMP70 NBF fusion proteins Fusion protein KM (WM) Vmax (nmol/Wmol NBF/min) Speci'c activity (1033 U/mg) ALDP (wild type) 11.5 &#fe; 0.97 641.9 &#fe; 10.7 10.0 &#fe; 0.17 ALDP (G512S) 17.9 &#fe; 1.23 279.3 &#fe; 4.2 4.4 &#fe; 0.06 ALDP (S606L) 45.6 &#fe; 2.40 666.0 &#fe; 8.7 10.4 &#fe; 0.14 PMP70 (wild type) 8.2 &#fe; 0.52 580.8 &#fe; 6.7 9.0 &#fe; 0.10 PMP70 (G478R) 161.8 &#fe; 34.40 641.2 &#fe; 28.2 10.0 &#fe; 0.44 PMP70 (S572I) 9.9 &#fe; 0.82 298.1 &#fe; 4.7 4.6 &#fe; 0.07 The kinetic data of all fusion proteins are mean values and standard deviations of 15^20 measurements at various protein concentrations using at least two distinct protein preparations. Login to comment

[hide] Identification of mutations in the putative ATP-bi... J Clin Invest. 1994 Aug;94(2):516-20. Fanen P, Guidoux S, Sarde CO, Mandel JL, Goossens M, Aubourg P
Identification of mutations in the putative ATP-binding domain of the adrenoleukodystrophy gene.
J Clin Invest. 1994 Aug;94(2):516-20., [PMID:8040304]

Abstract [show]
The recently identified adrenoleukodystrophy (ALD) gene is predicted to encode a peroxisomal protein of 745 amino acids that includes one domain for ATP-binding, termed nucleotide-binding fold (NBF). To determine whether mutations occur in the putative NBF of ALD protein, we analyzed by denaturing gradient gel electrophoresis (DGGE) exon 6 and 8 that encode most part of this domain in 50 ALD patients. Four amino acid substitutions, three frameshift mutations leading to premature termination signal, and a splicing mutation were identified. These amino acid substitutions occurred at residues highly conserved in other ATP-binding cassette (ABC) proteins. In addition, a nonsense mutation was detected in exon 4.

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48 Asterisks indicate the posi- *l I U13 | tion of the four missense mutations in ALD protein (ALDP): R518W substitution occurs at the same amino acid position as in the CFTR mutant S1255P, and S606L at the same position as in the S5491 or S549R CFTR mutants; R617C and R617H have no equivalents in CFTR mutants. Login to comment
88 The C2203 -+ T (Ser6O6 -+ Leu or S606L) mutation was identified in a patient who had Addison's disease without neurologic involvement at 20 years. Login to comment
127 Three of them (S606L, R617C, and R617H) involve invariant residues in all ABC proteins studied so far (Fig. 1). Login to comment
143 The CFTR mutants S5491 and S549R, that involve the same amino acid position as in the ALD mutant S606L, fail to produce mature CFTR and therefore prevent trafficking to the correct cellular localization ( 12). Login to comment
145 Mutations Detected in the ALD Gene Name Nucleotide change Effect on coding sequence Exon Clinical phenotype R464X C - T at 1776 Arg - Stop at 464 4 AMN* 1937delC Deletion of C at 1937 Frameshift 6 cerebral ALD R518W CT at 1938 Arg-Trpat 518 6 AMN 2020 + I G - A G - A at 2020 + 1 5' splice signal Intron 6 ACMN2 2177delTA Deletion of TA at 2177 Frameshift 8 cerebral ALD S606L C - T at 2203 Ser - Leu at 606 8 Addison 2204delG Deletion of G at 2204 Frameshift 8 Addison R617C C - T at 2235 Arg - Cys at 617 8 cerebral ALD R617H G - A at 2236 Arg - His at 617 8 ACMN * Adrenomyeloneuropathy; tadrenomyeloneuropathy with cerebral involvement. Login to comment

[hide] ABCD1 gene mutations in Chinese patients with X-li... Pediatr Neurol. 2005 Aug;33(2):114-20. Pan H, Xiong H, Wu Y, Zhang YH, Bao XH, Jiang YW, Wu XR
ABCD1 gene mutations in Chinese patients with X-linked adrenoleukodystrophy.
Pediatr Neurol. 2005 Aug;33(2):114-20., [PMID:16087056]

Abstract [show]
X-linked adrenoleukodystrophy is a neurodegenerative disorder caused by mutations in the adrenoleukodystrophy (ALD) protein gene ABCD1. This study used direct sequencing of genomic polymerase chain reaction products to perform mutational analysis of ABCD1 in 34 unrelated Chinese X-linked adrenoleukodystrophy patients and 27 of their maternal relatives. Thirty-two different mutations were identified in 34 patients. Most of the mutations (62.5%, 20/32) were missense mutations, six of which are novel. One novel single nucleotide polymorphism, c.1047 C>A, was also found in three patients and their mothers, which can also be observed in 1 of 120 normal control alleles. Two synonymous mutations (p.L516L and p.V349V) appeared in two unrelated patients, and no other mutations were evident after screening the gene's 10 exons. Seventeen of the probands' mothers were found to be heterozygous for the same mutations present in their sons' ABCD1 gene. Eight of the 10 screened sisters and cousins were identified as carriers. There were no hot spot mutations in the ABCD1 gene of Chinese patients with X-linked adrenoleukodystrophy. However, over half of the mutations (19/34) were located in exon 1 and exon 6, suggesting possible hot exons. No obvious relationship between genotype and phenotype was observed.

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94 RFLP Associated Phenotype Missense mutations A30 1 421 GϾA A141T CCALD A23 1 545 GϾC R182P CCALD A14 1 796 GϾA G266R‡ CCALD A18 1 847 CϾG H283D†§ - Nsp I CCALD A32 1 871 GϾA E291K‡ CCALD A28 1 887 AϾG Y296C ACALD A21 2 1028 GϾT G343V*‡§ - Ava I ACALD A20 2, 3 1047 CϾA V349V*§ ϩ Rsa I CCALD 1210 TϾC S404P†‡ A6 6 1526 AϾT N509I†‡ CCALD A26 6 1529 GϾA G510D*§ - Bg1 I ACALD A1 6 1552 CϾG R518G†‡§ - Msp I CCALD A24 6 1548 GϾA L516L‡ CCALD 1553 GϾA R518Q‡ A10 6 1553 GϾA R518Q‡ CCALD A7 6 1559 TϾA L520Q CCALD A12 7 1661 GϾA R554H‡ CCALD A19 7 1667 AϾG Q556R‡ AMN A16 8 1814 TϾA L605Q*§ ϩ BstX I CCALD A17 8 1817 CϾT S606L CCALD A2 8 1849 CϾT R617C‡ AO A15 8 1849 CϾG R617G CCALD Nonsense mutations A11 1 396 GϾA W132X‡ CCALD A3 1 726 GϾA W242X CCALD A34 4 1390 CϾT R464X‡ CCALD A8 8 1785 GϾA W595X‡ CCALD Frameshift mutations A29 1 385 ins G fs R128* ACALD A27 2 937 del C fs D312 CCALD A13 5 1415 del AG fs E471 ACALD A22 6 1603 del CC fs P534* CCALD Amino acid insertion A33 1 240-241ins9 R80-L81insPAA* CCALD Splicing defect A5 IVS1 IVS1 ϩ1 gϾt CCALD A31 IVS3 IVS3 ϩ2 cϾt CCALD A25 IVS5 IVS5 -6 delc†‡ ACALD Synonymous mutation A4 2, 6 1047 CϾA V349V‡ CCALD 1548 GϾA L516L‡ A9 2, 6 1047 CϾA V349V‡ CCALD 1548 GϾA L516L‡ * The mutation was novel. Login to comment
95 RFLP Associated Phenotype Missense mutations A30 1 421 Gb0e;A A141T CCALD A23 1 545 Gb0e;C R182P CCALD A14 1 796 Gb0e;A G266Rߥ CCALD A18 1 847 Cb0e;G H283Dߤ&#a7; afa; Nsp I CCALD A32 1 871 Gb0e;A E291Kߥ CCALD A28 1 887 Ab0e;G Y296C ACALD A21 2 1028 Gb0e;T G343V*ߥ&#a7; afa; Ava I ACALD A20 2, 3 1047 Cb0e;A V349V*&#a7; af9; Rsa I CCALD 1210 Tb0e;C S404Pߤߥ A6 6 1526 Ab0e;T N509Iߤߥ CCALD A26 6 1529 Gb0e;A G510D*&#a7; afa; Bg1 I ACALD A1 6 1552 Cb0e;G R518Gߤߥ&#a7; afa; Msp I CCALD A24 6 1548 Gb0e;A L516Lߥ CCALD 1553 Gb0e;A R518Qߥ A10 6 1553 Gb0e;A R518Qߥ CCALD A7 6 1559 Tb0e;A L520Q CCALD A12 7 1661 Gb0e;A R554Hߥ CCALD A19 7 1667 Ab0e;G Q556Rߥ AMN A16 8 1814 Tb0e;A L605Q*&#a7; af9; BstX I CCALD A17 8 1817 Cb0e;T S606L CCALD A2 8 1849 Cb0e;T R617Cߥ AO A15 8 1849 Cb0e;G R617G CCALD Nonsense mutations A11 1 396 Gb0e;A W132Xߥ CCALD A3 1 726 Gb0e;A W242X CCALD A34 4 1390 Cb0e;T R464Xߥ CCALD A8 8 1785 Gb0e;A W595Xߥ CCALD Frameshift mutations A29 1 385 ins G fs R128* ACALD A27 2 937 del C fs D312 CCALD A13 5 1415 del AG fs E471 ACALD A22 6 1603 del CC fs P534* CCALD Amino acid insertion A33 1 240-241ins9 R80-L81insPAA* CCALD Splicing defect A5 IVS1 IVS1 af9;1 gb0e;t CCALD A31 IVS3 IVS3 af9;2 cb0e;t CCALD A25 IVS5 IVS5 afa;6 delcߤߥ ACALD Synonymous mutation A4 2, 6 1047 Cb0e;A V349Vߥ CCALD 1548 Gb0e;A L516Lߥ A9 2, 6 1047 Cb0e;A V349Vߥ CCALD 1548 Gb0e;A L516Lߥ * The mutation was novel. Login to comment

[hide] [Adrenoleukodystrophy: structure and function of A... Yakugaku Zasshi. 2007 Jan;127(1):163-72. Takahashi N, Morita M, Imanaka T
[Adrenoleukodystrophy: structure and function of ALDP, and intracellular behavior of mutant ALDP with naturally occurring missense mutations].
Yakugaku Zasshi. 2007 Jan;127(1):163-72., [PMID:17202797]

Abstract [show]
Adrenoleukodystrophy (ALD) is an inherited disorder characterized by progressive demyelination of the central nervous system and adrenal dysfunction. The biochemical characterization is based on the accumulation of pathgnomonic amounts of saturated very long-chain fatty acid (VLCFA; C>22) in all tissues, including the brain white matter, adrenal glands, and skin fibroblasts, of the patients. The accumulation of VLCFA in ALD is linked to a mutation in the ALD (ABCD1) gene, an ABC subfamily D member. The ALD gene product, so-called ALDP (ABCD1), is thought to be involved in the transport of VLCFA or VLCFA-CoA into the peroxisomes. ALDP is a half-sized peroxisomal ABC protein and it has 745 amino acids in humans. ALDP is thought to be synthesized on free polysomes, posttranslationally transported to peroxisomes, and inserted into the membranes. During this process, ALDP interacts with Pex19p, a chaperone-like protein for intracellular trafficking of peroxisomal membrane protein (PMP), the complex targets Pex3p on the peroxisomal membranes, and ALDP is inserted into the membranes. After integration into the membranes, ALDP is thought to form mainly homodimers. Here, we chose nine arbitrary mutations of human ALDP with naturally occurring missense mutations and examined the intracellular behavior of their ALDPs. We found that mutant ALDP (S606L, R617H, and H667D) was degraded together with wild-type ALDP by proteasomes. These results suggest that the complex of mutant and wild-type ALDP is recognized as misfolded proteins and degraded by the protein quality control system associated with proteasomes. Further, we found fragmentation of mutant ALDP (R104C) on peroxisomes and it was not inhibited by proteasomes inhibitors, suggesting that an additional protease(s) is also involved in the quality control of mutant ALDP. In addition, mutation of ALDP (Y174C) suggests that a loop between transmembrane domains 2 and 3 is important for the targeting of ALDP to peroxisomes.

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8 We found that mutant ALDP (S606L, R617H, and H667D) was degraded together with wild-type ALDP by proteasomes. Login to comment
19 S606P and S606L are in ABC signature motif. Login to comment
28 ミスセンス変異を持つ ALDP の細胞内動態 ―一過性発現による解析 ALD 患者の持つ変異 ALDP の機能,細胞内局在性,細胞内における安定性を解析することは, ALDP の各ドメインの機能を知る上で有用な情報を提供すると思われる．特にミスセンス変異は,たった 1 つのアミノ酸変異による異常であるので特に興味深い．われわれは ALD 患者で報告されているミスセンス変異の中から,TMD から 4 つ(R104C, G116R, Y174C, S342P),NBD から 4 つ(Q544R, S606P, S606L, R617H),C 末端部位から 1 つ (H667D)を任意に選び(Fig. 1),その機能と細胞内動態を解析した．これらの実験は,大学院シンポジウムで報告したので,詳しく述べたいと思う． ALDP はペルオキシソームにおける極長鎖脂肪酸の b 酸化に関与していることが知られている．実際に ALD 患者由来の繊維芽細胞では極長鎖脂肪酸の b 酸化活性が正常な線維芽細胞と比べて約 50 ―70%程度減少している．そこで野生型及び変異型 ALDP の機能を確認するため,ALDP を発現していない ALD 患者由来線維芽細胞に,N 末端に His タグを付加した野生型と変異型 ALDP を一過性に発現し,［1-14 C]lignoceric acid を基質として極長鎖脂肪酸 b 酸化活性の測定を行った．その結果, ALDP 欠損線維芽細胞の極長鎖脂肪酸 b 酸化活性は,正常細胞の約 50%まで減少していたが,野生型 His-ALDP を発現させると正常と同程度にまで活性が回復した．このことから発現させた野生型 His-ALDP は ALDP と同等の機能を持つことが確認された．一方,9 種類のミスセンス変異 ALDP を発現した線維芽細胞では極長鎖脂肪酸 b 酸化活性の増加は認められなかった．よって,これらのミスセンス変異 ALDP は機能を欠くことが確認された．ついで,野生型及び変異型 His-ALDP を発現した ALD 患者線維芽細胞を回収し,変異型 ALDP の発現量を immunoblotting により定量化し解析した(Table 1)．なお ALDP の発現量は,ペルオキシソームの指標酵素であるカタラーゼの発現量で補正した．その結果,変異型 ALDP(R104C, G116R, Y174C, S342P, Q544R, S606P)は,野生型とほぼ同程度の発現量を示した．一方,変異型 ALDP (S606L, R617H, H667D)では発現量が野生型の発 167 Table 1. Login to comment
29 Expression and Localization of Missense ALDPs Mutant Transient Stable Expression Localization b-Oxidation Expression Localization Wild Z Px ＋ Z Px R104C, G116R S342P, Q544R S606P Z Px － Z Px Y174C Z mis － Z mis S606L ＋ Px －－－ R617H ± －－ na na H667D ＋－－－－ Wild and Mutant His-ALDPs or ALDP-GFPs were transiently expressed in X-ALD ˆbroblasts and stably expressed in CHO cells, respectively. Login to comment
36 1 現量と比べて約 50%程度減少していた．なお,各 ALDP ポジティブの細胞は約 30%程度であり,各細胞間での発現効率に有意な差は認められなかった．このことから,ALDP の発現量が減少していた 3 つの変異 ALDP は細胞内での安定性が低下していると推察された．また興味深いことに S606P と S606L は同じ部位の変異にも係わらず,置換したアミノ酸によって発現量には差が認められた．ついで,変異型 His-ALDP の細胞内局在を蛍光抗体法で確認した．変異型 ALDP(R104C, G116R, S342P, Q544R, S606P, S606L)では ALDP がカタラーゼの局在と一致したことから,正常にペルオキシソームへ局在していることが確認された．一方, 変異型 ALDP(Y174C, H667D)では局在が一致せず,ALDP が他の細胞内小器官へ間違って輸送されていると考えられた．変異型 ALDP(R617H) では ALDP の発現が認められなかった．変異型 ALDP(R104C, G116R, S342P, Q544R, S606P)では野生型とほぼ同程度のタンパク量が発現し,ペルオキシソームへの局在も確認されたので,これらの変異型 ALDP は合成されたのちに正常にペルオキシソームに運ばれるが,ペルオキシソーム膜においてその機能(ATP 結合・加水分解若しくは基質輸送)に異常を持つことが推察された．特に R104C, G116R, S342P は TMD に存在することから ALDP の基質輸送能が変化していると考えられる．一方, NBD に存在する Q544R, S606P は ATP 結合・加水分解に影響を与えている可能性が考えられる．また S606P, S606L は変異が同じ部位でも構造的に安定性が異なっていた．Roerig らは S606L の変異型 ALDP は,ATP との親和性が低下している一方で ATP 加水分解は正常に行われていると報告している．29) このことは ALDP と ATP の親和性が ALDP の安定性にも影響を及ぼしている可能性を示している．S606L と S606P の安定性の違いと機能の関係は ALDP の機能を知る上でも興味深い点であり, 今後さらに検討を行う必要がある．一方,Y174C の変異型 ALDP は正常に発現するにも係わらず, ペルオキシソームへ局在せず他の細胞内小器官へミスターゲッティングした．これまでにペルオキシソームへの局在化シグナルを欠くペルオキシソーム膜タンパク質は,非特異的にミトコンドリアや小胞体に移行することが知られている．30,31) よって, ALDP の TMD2―3 の間のループは,ペルオキシソームへの局在化に重要な役割を果たしている可能性が推察される．Pex19p 存在化での in vitro タンパク質翻訳系において,ALDP(Y174C)は Pex19p に結合できるので,ALDP の N 末端 67―164 に存在するペルオキシソーム移行に係わる領域が ALDP の何らかの構造変化によってマスクされるのかもしれない． 5. Login to comment
43 168 Vol. 127 (2007) ALDP については ALDP の C 末端に GFP(green ‰uorescent protein)を融合させた変異 ALDP-GFP を発現させるとともに,野生型ヒト ALDP を CHO 細胞に共発現させた(CHO 細胞にも内在性の ALDP が発現しているが,本実験に用いた抗体が交差しないため,ヒト ALDP を発現させた)．GFP 融合タンパク質は内在性のタンパク質との区別が容易であること,安定発現細胞の取得が容易にできることなどの利点がある．この実験では,ペルオキシソームに正常に輸送される変異型 ALDP(R104C, G116R, S606P),ペルオキシソームに局在しない変異型 ALDP(Y174C),発現量が低下している変異型 ALDP(H667D)を選んだ．これら安定過剰発現細胞における ALDP-GFP の細胞内局在性をみると,その分布は一過性発現させた His-ALDP と同様であった．得られた安定発現細胞よりオルガネラ粗分画を調製し,ショ糖密度勾配遠心分離法により各フラクションに分けたのち,SDS-PAGE 及び immunoblotting により変異 ALDP の局在について解析を行った(Fig. 3)．ペルオキシソームマーカーとしてペルオキシソーム膜タンパク質である PMP70 とペルオキシソームの主要なマトリックスタンパク質であるカタラーゼを用いた．野生型 ALDP を安定過剰発現している細胞において,カタラーゼ活性並びに PMP70 が主としてフラクション 3 及び 4 に存在することより,この分画にペルオキシソームが回収されたことが示唆された．また約 110 kDa の分子サイズを持つ ALDP-GFP 並びに 83 kDa の野生型 ALDP は,ペルオキシソームマーカーとほぼ同じ分布を示していたことからペルオキシソームに局在していることが示唆された．また変異型 ALDP-GFP(G116R, S606P)も同様の分布を示した．一方,変異型 ALDP-GFP(H667D)を安定過剰発現している細胞の場合は,ALDP-GFP のバンドは検出されなかった(Fig. 3)．興味深いことに, PMP70 は検出されたが , 共発現させた野生型 ALDP のバンドも検出されなかった．また変異型 ALDP(S606L)についても同様であった．これらの結果は , 変異型 ALDP ( H667D, S606L ) は PMP70 とではなく,野生型 ALDP と複合体を形成し,両者が分解される可能性を示唆している． ABC タンパク質の機能発現に重要である TMD や NBD 以外の C 末端部位での変異がタンパク質の安定性に影響を及ぼすことは興味深い．ALDP の C 末端部位である 600―700 アミノ酸での変異が X-ALD を引き起こす頻度が高いことから,ALDP の C 末端部位はタンパク質の安定性に重要な役割を担っている可能性がある．Liu らは ALDP のダイマー化には C 末端部位(AA.631―745)が重要であると報告している．13) H667D や S606L のような変異は,それ自身あるいは野生型 ALDP とミスフォールドしたダイマーを形成し,異常タンパク質として認識され分解されると考えられる．一方,変異型 ALDP-GFP(R104C)はペルオキシソーム分画に回収されるものの,フラグメント化していること 169 Fig. 4. Login to comment
49 変異型 ALDP の分解過程の解析新生タンパク質が正し;044;フォールディングを受けることは,そのタンパク質の正常な機能発現のために必須である．遺伝子変異などが存在すると,タンパク質がミスフォールディングされる．このミスフォールドタンパクが細胞外へ分泌されたり,細胞内に蓄積したりすると生体にとって極めて有害になるため,このようなタンパクはプロテアソーム,リソソーム等によって迅速に分解される．ちなみに,嚢胞性線維症の原因タンパク質 CFTR は細胞膜イオンチャネルとして機能する ABC タンパク質であるが,変異 CFTR は小胞体膜からプロテアソームにリクルートされ分解されることが報告されている．32,33) しかしながら,変異型 ALDP を始めとして,ペルオキシソーム膜タンパク質についての解析はほとんど行われていない．変異型 ALDP の一過性発現と安定過剰発現実験より,ALDP(S606L, R617H, H667D, R104C)は, プロテアーゼにより分解されていると推定された．そこで,ALDP-GFP(H667D)を発現している CHO 細胞に各種プロテアーゼ阻害剤を処理し,解析を行った．その結果,プロテアソーム阻害剤である lactacystin を処理した細胞では ALDP-GFP 及び ALDP のバンドが出現した ( Fig. 4 )．一方 , leupeptin, AEBSF, E64d には効果がなかった．また他のプロテアソーム阻害剤である MG132 も有効であった．さらにプロテアソーム阻害剤により分解を逃れた変異型 ALDP-GFP(H667D)の細胞内局在を蛍光抗体法で観察すると,ペルオキシソームに局在していることが確認された．一方,変異型 ALDP(R104C)のフラグメント化は上記プロテアーゼ処理では阻害されなかった．さらに ALD 患者由来細胞の内因性変異 ALDP の分解とプロテアソーム分解系の関与について確認するため,変異型 ALDP(R617H)を持つ患者由来線維芽細胞を用いてタンパク分解の阻害実験を行った．その結果,lactacystin と MG132 処理により, ALDP のバンドが出現した．以上の結果より,ペルオキシソーム膜上にはミスフォールドしたタンパク質を認識する仕組みが存在し,プロテアソーム及び他のプロテアーゼを介して排除していることが示唆された．一方,山田らは ALD 患者線維芽細胞を［35 S］メチオニンでパルスチェイスすることにより,変異型 ALDP(G512S, R660W)の分解が E-64 と leupepu- tin により抑制されることを報告している．34) 彼らの実験ではプロテアソーム阻害剤については実験していないので,プロテアソームの関与は不明であるが,変異型 ALDP の分解には,複数のプロテアーゼが関与している可能性がある． 7. Login to comment
52 Some mutant ALDPs (R104C, G116R, S342P, Q544R and S606P) are normally inserted into the peroxisomal membrane, and others were mislocalized (Y174C) or degraded by proteasome (S606L, R617H and H667D). Login to comment
53 170 Vol. 127 (2007) その結果より,ミスセンス変異 ALDP は以下に示すように 4 種類の細胞内動態を持つことが示された(Fig. 5)．1) 野生型と同様にペルオキシソームに局在するがその機能が阻害されている変異 (R104C, G116R, S342P, Q544R, S606P),2) ペルオキシソームへの局在化に障害がある変異(Y174C), 3) 変異によりタンパク質の安定性が低下しプロテアソームでの分解を受けるが,一部ペルオキシソームに局在する変異(S606L),4) 変異によりタンパク質の安定性が低下しプロテアソームで選択的に分解を受け,細胞内でほとんど確認できない変異 (R617H, H667D)の 4 種類のパターンである．発現量も局在化も正常な変異では,ABC タンパク質としての機能に直接関与している機能ドメインの障害が起こっていると推察される．この中で G116R, S342P は TMD に位置しており,基質の認識や輸送に障害があると推察される．また Q544R, S606P は ATP との結合・加水分解に関与する NBD に位置している．このような変異は,ALDP の ABC タンパク質としての機能を解析するために有益と考えられる．発現量は正常だが局在化に異常が認められた Y174C は,TMD2 と 3 の間のループ 2 に位置しており,この領域が ALDP のペルオキシソームへのターゲッティングに必要であることを示している． ALDP のターゲッティングに必要な領域は 67―164 番目のアミノ酸に存在することが報告されている．20) このことから,Y174C の変異による構造変化のため,ターゲッティングシグナルがマスクされているのかもしれない．このタイプの変異は ALDP のペルオキシソームへの局在化を調べる上で重要と考えられる． ALDP の変異で最も多いミスセンス変異ではその多くが細胞内で分解を受けている．R617H 及び H667D では発現量の著しい低下が認められる．特に安定発現した CHO では immunoblot で検出できなかった．ミスフォールドタンパク質の分解システムの 1 つにプロテアソームによる分解系がある．このタンパク質分解は,生物の様々な高次機能の制御や環境ストレスに応答した恒常性の維持(ストレス応答,タンパク質の品質管理など)に必須な役割を担っている．しかし,小胞体を経由して合成される分泌タンパク質や膜タンパク質に比べて,小胞体を経由しない細胞内タンパク質の品質管理機構はあまり報告されていない．ALDP は遊離のポリソームから直接ペルオキシソームに輸送されるが,この過程でどのように R617H, H667D などの変異が認識され,プロテアソーム系が働いているか興味深い． 171171No. Login to comment
27 df;b9;bb;f3;b9;᜕ᶒఔᢝ௸ ALDP Ĳe;d30;Pde;ᑁ4d5;ɦb; ߟe00;Έe;ឋ˿a;Ife;Ĳb;ఐĴb;Ye3;᪆ ALD <a3;ὅĲe;ᢝ௸᜕ᶒ ALDP Ĳe;a5f;Pfd;,d30;Pde;ᑁc40;ᙠ ឋ,d30;Pde;ᑁĲb;İa;௫Ĵb;b89;b9a;ឋఔYe3;᪆௳Ĵb;௭௼Ĳf;, ALDP Ĳe;ᔜc9;e1;a4;f3;Ĳe;a5f;Pfd;ఔMe5;Ĵb;e0a;௻ᨵᵨĲa;<c5;ᛇ ఔ?d0;f9b;௳Ĵb;௼əd;Ĵf;Ĵc;Ĵb;&#ff0e;ᱯĲb;df;b9;bb;f3;b9;᜕ᶒĲf;,ıf; ௷ıf; 1 ௸Ĳe;a2;df;ce;⏚᜕ᶒĲb;ఐĴb;ᶒe38;௻௢Ĵb;Ĳe;௻ᱯĲb; ‐ᕡdf1;௤&#ff0e;Ĵf;Ĵc;Ĵf;Ĵc;Ĳf; ALD <a3;ὅ௻ᛇȠa;௯Ĵc;௺௤Ĵb; df;b9;bb;f3;b9;᜕ᶒĲe;e2d;İb;఑,TMD İb;఑ 4 ௸(R104C, G116R, Y174C, S342P) ,NBD İb;఑ 4 ௸(Q544R, S606P, S606L, R617H) ,C ʠb;aef;Ze8;f4d;İb;఑ 1 ௸ (H667D)ఔefb;ɢf;Ĳb;⍶ఁ(Fig. 1) ,ıd;Ĳe;a5f;Pfd;௼d30;Pde; ᑁ4d5;ɦb;ఔYe3;᪆௱ıf;&#ff0e;௭Ĵc;఑Ĳe;b9f; a13;Ĳf;,ᜧb66;▾b7;f3;dd; b8;a6;e0;௻ᛇȠa;௱ıf;Ĳe;௻,a73;௱İf;ff0;ఇıf;௤௼əd;௦&#ff0e; ALDP Ĳf;da;eb;aa;ad;b7;bd;fc;e0;Ĳb;İa;௫Ĵb;ᬿ╩⒴ᾦPaa; ⏚Ĳe; b ⏚ᓄĲb;_a2;e0e;௱௺௤Ĵb;௭௼İc;Me5;఑Ĵc;௺௤Ĵb;&#ff0e; b9f;ωb;Ĳb; ALD <a3;ὅᵫᩭĲe;e4a;dad;Rbd;d30;Pde;௻Ĳf;ᬿ╩⒴ᾦPaa; ⏚Ĳe; b ⏚ᓄd3b;ឋİc;b63;e38;Ĳa;dda;dad;Rbd;d30;Pde;௼bd4;ఇ௺d04; 50 ߟ70%a0b;ea6;e1b;c11;௱௺௤Ĵb;&#ff0e;ıd;௭௻[ce;˯f;ɂb;5ca;ఁ᜕ᶒɂb; ALDP Ĳe;a5f;Pfd;ఔNba;a8d;௳Ĵb;ıf;ఉ,ALDP ఔ˿a;Ife;௱௺ ௤Ĳa;௤ ALD <a3;ὅᵫᩭdda;dad;Rbd;d30;Pde;Ĳb;,N ʠb;aef;Ĳb; His bf;b0;ఔed8;4a0;௱ıf;[ce;˯f;ɂb;௼᜕ᶒɂb; ALDP ఔe00;Έe;ឋĲb; ˿a;Ife;௱, &#ff3b;1-14 C]lignoceric acid ఔ9fa;cea;௼௱௺ᬿ╩⒴ ᾦPaa;⏚ b ⏚ᓄd3b;ឋĲe;e2c;b9a;ఔʹc;௷ıf;&#ff0e;ıd;Ĳe;d50;ʧc;, ALDP b20;ʀd;dda;dad;Rbd;d30;Pde;Ĳe;ᬿ╩⒴ᾦPaa;⏚ b ⏚ᓄd3b;ឋ Ĳf;,b63;e38;d30;Pde;Ĳe;d04; 50%ĳe;௻e1b;c11;௱௺௤ıf;İc;,[ce;˯f; ɂb; His-ALDP ఔ˿a;Ife;௯ıb;Ĵb;௼b63;e38;௼Ȝc;a0b;ea6;Ĳb;ĳe;௻ d3b;ឋİc;8de;fa9;௱ıf;&#ff0e;௭Ĳe;௭௼İb;఑˿a;Ife;௯ıb;ıf;[ce;˯f;ɂb; His-ALDP Ĳf; ALDP ௼Ȝc;b49;Ĳe;a5f;Pfd;ఔᢝ௸௭௼İc;Nba; a8d;௯Ĵc;ıf;&#ff0e;e00;Ab9;,9 a2e;ϙe;Ĳe;df;b9;bb;f3;b9;᜕ᶒ ALDP ఔ˿a;Ife;௱ıf;dda;dad;Rbd;d30;Pde;௻Ĳf;ᬿ╩⒴ᾦPaa;⏚ b ⏚ᓄd3b; ឋĲe;ᜉ4a0;Ĳf;a8d;ఉ఑Ĵc;Ĳa;İb;௷ıf;&#ff0e;ఐ௷௺,௭Ĵc;఑Ĳe;df; b9;bb;f3;b9;᜕ᶒ ALDP Ĳf;a5f;Pfd;ఔb20;İf;௭௼İc;Nba;a8d;௯Ĵc; ıf;&#ff0e; ௸௤௻,[ce;˯f;ɂb;5ca;ఁ᜕ᶒɂb; His-ALDP ఔ˿a;Ife;௱ ıf; ALD <a3;ὅdda;dad;Rbd;d30;Pde;ఔ8de;5ce;௱,᜕ᶒɂb; ALDP Ĳe;˿a;Ife;[cf;ఔ immunoblotting Ĳb;ఐĴa;b9a;[cf;ᓄ௱Ye3;᪆௱ ıf;(Table 1) &#ff0e;Ĳa;İa; ALDP Ĳe;˿a;Ife;[cf;Ĳf;,da;eb;aa;ad; b7;bd;fc;e0;Ĳe;ᢣa19;⏗d20;௻௢Ĵb;ab;bf;e9;fc;bc;Ĳe;˿a;Ife;[cf;௻Xdc; b63;௱ıf;&#ff0e;ıd;Ĳe;d50;ʧc;,᜕ᶒɂb; ALDP(R104C, G116R, Y174C, S342P, Q544R, S606P)Ĳf;,[ce;˯f;ɂb;௼ĳb;ĳc; Ȝc;a0b;ea6;Ĳe;˿a;Ife;[cf;ఔ̙a;௱ıf;&#ff0e;e00;Ab9;,᜕ᶒɂb; ALDP (S606L, R617H, H667D)௻Ĳf;˿a;Ife;[cf;İc;[ce;˯f;ɂb;Ĳe;˿a; 167 Table 1. Login to comment
34 167 No. 1 Ife;[cf;௼bd4;ఇ௺d04; 50%a0b;ea6;e1b;c11;௱௺௤ıf;&#ff0e;Ĳa;İa;,ᔜ ALDP dd;b8;c6;a3;d6;Ĳe;d30;Pde;Ĳf;d04; 30%a0b;ea6;௻௢Ĵa;,ᔜ d30;Pde;╹௻Ĳe;˿a;Ife;4b9;᳛Ĳb;ᨵɢf;Ĳa;dee;Ĳf;a8d;ఉ఑Ĵc;Ĳa;İb;௷ ıf;&#ff0e;௭Ĳe;௭௼İb;఑,ALDP Ĳe;˿a;Ife;[cf;İc;e1b;c11;௱௺௤ ıf; 3 ௸Ĳe;᜕ᶒ ALDP Ĳf;d30;Pde;ᑁ௻Ĳe;b89;b9a;ឋİc;f4e;e0b;௱ ௺௤Ĵb;௼?a8;bdf;௯Ĵc;ıf;&#ff0e;ĳe;ıf;‐ᕡdf1;௤௭௼Ĳb; S606P ௼ S606L Ĳf;Ȝc;௲Ze8;f4d;Ĳe;᜕ᶒĲb;ఊfc2;Ĵf;఑ıa;,f6e;?db;௱ ıf;a2;df;ce;⏚Ĳb;ఐ௷௺˿a;Ife;[cf;Ĳb;Ĳf;dee;İc;a8d;ఉ఑Ĵc;ıf;&#ff0e; ௸௤௻,᜕ᶒɂb; His-ALDP Ĳe;d30;Pde;ᑁc40;ᙠఔVcd;ᐝ ᢙf53;cd5;௻Nba;a8d;௱ıf;&#ff0e;᜕ᶒɂb; ALDP(R104C, G116R, S342P, Q544R, S606P, S606L)௻Ĳf; ALDP İc;ab;bf; e9;fc;bc;Ĳe;c40;ᙠ௼e00;Qf4;௱ıf;௭௼İb;఑,b63;e38;Ĳb;da;eb;aa;ad; b7;bd;fc;e0;ఆc40;ᙠ௱௺௤Ĵb;௭௼İc;Nba;a8d;௯Ĵc;ıf;&#ff0e;e00;Ab9;, ᜕ᶒɂb; ALDP(Y174C, H667D)௻Ĳf;c40;ᙠİc;e00;Qf4;ıb; ıa;,ALDP İc;ed6;Ĳe;d30;Pde;ᑁc0f;ᘤb98;ఆ╹⍟௷௺f38;〈௯ Ĵc;௺௤Ĵb;௼ὃ௨఑Ĵc;ıf;&#ff0e;᜕ᶒɂb; ALDP(R617H) ௻Ĳf; ALDP Ĳe;˿a;Ife;İc;a8d;ఉ఑Ĵc;Ĳa;İb;௷ıf;&#ff0e;᜕ᶒɂb; ALDP(R104C, G116R, S342P, Q544R, S606P)௻ Ĳf;[ce;˯f;ɂb;௼ĳb;ĳc;Ȝc;a0b;ea6;Ĳe;bf;f3;d1;af;[cf;İc;˿a;Ife;௱,da;eb; aa;ad;b7;bd;fc;e0;ఆĲe;c40;ᙠఊNba;a8d;௯Ĵc;ıf;Ĳe;௻,௭Ĵc;఑Ĳe; ᜕ᶒɂb; ALDP Ĳf;ᔠᡂ௯Ĵc;ıf;Ĳe;௵Ĳb;b63;e38;Ĳb;da;eb;aa;ad; b7;bd;fc;e0;Ĳb;Έb;௾Ĵc;Ĵb;İc;,da;eb;aa;ad;b7;bd;fc;e0;̳c;Ĳb;İa;௤ ௺ıd;Ĳe;a5f;Pfd;(ATP d50;ᔠfb;4a0;c34;ᑖYe3;Re5;௱İf;Ĳf;9fa;cea;f38; 〈)Ĳb;ᶒe38;ఔᢝ௸௭௼İc;?a8;bdf;௯Ĵc;ıf;&#ff0e;ᱯĲb; R104C, G116R, S342P Ĳf; TMD Ĳb;b58;ᙠ௳Ĵb;௭௼İb;఑ ALDP Ĳe;9fa;cea;f38;〈Pfd;İc;᜕ᓄ௱௺௤Ĵb;௼ὃ௨఑Ĵc;Ĵb;&#ff0e;e00;Ab9;, NBD Ĳb;b58;ᙠ௳Ĵb; Q544R, S606P Ĳf; ATP d50;ᔠfb;4a0;c34; ᑖYe3;Ĳb;f71;aff;ఔe0e;௨௺௤Ĵb;5ef;Pfd;ឋİc;ὃ௨఑Ĵc;Ĵb;&#ff0e;ĳe;ıf; S606P, S606L Ĳf;᜕ᶒİc;Ȝc;௲Ze8;f4d;௻ఊEcb;⌼ḄĲb;b89;b9a; ឋİc;ᶒĲa;௷௺௤ıf;&#ff0e;Roerig ఑Ĳf; S606L Ĳe;᜕ᶒɂb; ALDP Ĳf;,ATP ௼Ĳe;Yaa;Ȥc;ឋİc;f4e;e0b;௱௺௤Ĵb;e00;Ab9;௻ ATP 4a0;c34;ᑖYe3;Ĳf;b63;e38;Ĳb;ʹc;Ĵf;Ĵc;௺௤Ĵb;௼ᛇȠa;௱௺௤ Ĵb;&#ff0e; 29) ௭Ĳe;௭௼Ĳf; ALDP ௼ ATP Ĳe;Yaa;Ȥc;ឋİc; ALDP Ĳe;b89;b9a;ឋĲb;ఊf71;aff;ఔ5ca;ĳc;௱௺௤Ĵb;5ef;Pfd;ឋఔ̙a;௱௺௤ Ĵb;&#ff0e;S606L ௼ S606P Ĳe;b89;b9a;ឋĲe;⍟௤௼a5f;Pfd;Ĳe;_a2;fc2; Ĳf; ALDP Ĳe;a5f;Pfd;ఔMe5;Ĵb;e0a;௻ఊ‐ᕡdf1;௤Fb9;௻௢Ĵa;, eca;f8c;௯఑Ĳb;ʳc;a0e;ఔʹc;௦fc5;⌕İc;௢Ĵb;&#ff0e;e00;Ab9;,Y174C Ĳe;᜕ᶒɂb; ALDP Ĳf;b63;e38;Ĳb;˿a;Ife;௳Ĵb;Ĳb;ఊfc2;Ĵf;఑ıa;, da;eb;aa;ad;b7;bd;fc;e0;ఆc40;ᙠıb;ıa;ed6;Ĳe;d30;Pde;ᑁc0f;ᘤb98;ఆdf; b9;bf;fc;b2;c3;c6;a3;f3;b0;௱ıf;&#ff0e;௭Ĵc;ĳe;௻Ĳb;da;eb;aa;ad;b7; bd;fc;e0;ఆĲe;c40;ᙠᓄb7;b0;ca;eb;ఔb20;İf;da;eb;aa;ad;b7;bd;fc;e0; ̳c;bf;f3;d1;af;cea;Ĳf;,Ϗe;ᱯᶒḄĲb;df;c8;b3;f3;c9;ea;a2;ఌc0f;Pde; f53;Ĳb;Ofb;ʹc;௳Ĵb;௭௼İc;Me5;఑Ĵc;௺௤Ĵb;&#ff0e; 30,31) ఐ௷௺, ALDP Ĳe; TMD2ߟ3 Ĳe;╹Ĳe;eb;fc;d7;Ĳf;,da;eb;aa;ad;b7; bd;fc;e0;ఆĲe;c40;ᙠᓄĲb;[cd;⌕Ĳa;f79;ᒘఔʧc;ıf;௱௺௤Ĵb;5ef;Pfd; ឋİc;?a8;bdf;௯Ĵc;Ĵb;&#ff0e;Pex19p b58;ᙠᓄ௻Ĳe; in vitro bf;f3; d1;af;cea;ffb;a33;cfb;Ĳb;İa;௤௺,ALDP(Y174C)Ĳf; Pex19p Ĳb;d50;ᔠ௻İd;Ĵb;Ĳe;௻,ALDP Ĳe; N ʠb;aef; 67ߟ164 Ĳb;b58;ᙠ௳Ĵb;da;eb;aa;ad;b7;bd;fc;e0;Ofb;ʹc;Ĳb;fc2;Ĵf;Ĵb;♚9df;İc; ALDP Ĳe;f55;఑İb;Ĳe;Ecb;⌼᜕ᓄĲb;ఐ௷௺de;b9;af;௯Ĵc;Ĵb; Ĳe;İb;ఊ௱Ĵc;Ĳa;௤&#ff0e; 5. Login to comment
41 168 Vol. 127 (2007) ALDP Ĳb;௸௤௺Ĳf; ALDP Ĳe; C ʠb;aef;Ĳb; GFP(green ߮uorescent protein)ఔͮd;ᔠ௯ıb;ıf;᜕ᶒ ALDP-GFP ఔ˿a;Ife;௯ıb;Ĵb;௼௼ఊĲb;,[ce;˯f;ɂb;d2;c8; ALDP ఔ CHO d30;Pde;Ĳb;ᐳ ˿a;Ife;௯ıb;ıf;(CHO d30;Pde;Ĳb;ఊ ᑁᙠឋĲe; ALDP İc;˿a;Ife;௱௺௤Ĵb;İc;,ʠc;b9f; a13;Ĳb;ᵨ௤ıf;ᢙf53;İc; ea4;dee;௱Ĳa;௤ıf;ఉ,d2;c8; ALDP ఔ˿a;Ife;௯ıb;ıf;) &#ff0e;GFP ͮd;ᔠbf;f3;d1;af;cea;Ĳf;ᑁᙠឋĲe;bf;f3;d1;af;cea;௼Ĳe;ȕa;ᑩİc;bb9; ᧕௻௢Ĵb;௭௼,b89;b9a;˿a;Ife;d30;Pde;Ĳe;5d6;f97;İc;bb9;᧕Ĳb;௻İd;Ĵb; ௭௼Ĳa;௽Ĳe;ᑭFb9;İc;௢Ĵb;&#ff0e;௭Ĳe;b9f; a13;௻Ĳf;,da;eb;aa;ad;b7; bd;fc;e0;Ĳb;b63;e38;Ĳb;f38;〈௯Ĵc;Ĵb;᜕ᶒɂb; ALDP(R104C, G116R, S606P) ,da;eb;aa;ad;b7;bd;fc;e0;Ĳb;c40;ᙠ௱Ĳa;௤᜕ ᶒɂb; ALDP(Y174C) ,˿a;Ife;[cf;İc;f4e;e0b;௱௺௤Ĵb;᜕ᶒ ɂb; ALDP(H667D)ఔ⍶క௴&#ff0e;௭Ĵc;఑b89;b9a;Έe;ᒖ˿a; Ife;d30;Pde;Ĳb;İa;௫Ĵb; ALDP-GFP Ĳe;d30;Pde;ᑁc40;ᙠឋఔĳf;Ĵb; ௼,ıd;Ĳe;ᑖe03;Ĳf;e00;Έe;ឋ˿a;Ife;௯ıb;ıf; His-ALDP ௼Ȝc; Ed8;௻௢௷ıf;&#ff0e; f97;఑Ĵc;ıf;b89;b9a;˿a;Ife;d30;Pde;ఐĴa;aa;eb;ac;cd;e9;c97;ᑖ˱b;ఔabf; Xfd;௱,b7;e7;cd6;bc6;ea6;4fe;Βd;⍤fc3;ᑖ`e2;cd5;Ĳb;ఐĴa;ᔜd5;e9;af;b7; e7;f3;Ĳb;ᑖ௫ıf;Ĳe;௵,SDS-PAGE 5ca;ఁ immunoblotting Ĳb;ఐĴa;᜕ᶒ ALDP Ĳe;c40;ᙠĲb;௸௤௺Ye3;᪆ఔʹc;௷ ıf;(Fig. 3) &#ff0e;da;eb;aa;ad;b7;bd;fc;e0;de;fc;ab;fc;௼௱௺da; eb;aa;ad;b7;bd;fc;e0;̳c;bf;f3;d1;af;cea;௻௢Ĵb; PMP70 ௼da;eb; aa;ad;b7;bd;fc;e0;Ĳe;e3b;⌕Ĳa;de;c8;ea;c3;af;b9;bf;f3;d1;af;cea;௻௢ Ĵb;ab;bf;e9;fc;bc;ఔᵨ௤ıf;&#ff0e;[ce;˯f;ɂb; ALDP ఔb89;b9a;Έe;ᒖ ˿a;Ife;௱௺௤Ĵb;d30;Pde;Ĳb;İa;௤௺,ab;bf;e9;fc;bc;d3b;ឋe26;ఁĲb; PMP70 İc;e3b;௼௱௺d5;e9;af;b7;e7;f3; 3 5ca;ఁ 4 Ĳb;b58;ᙠ௳ Ĵb;௭௼ఐĴa;,௭Ĳe;ᑖ˱b;Ĳb;da;eb;aa;ad;b7;bd;fc;e0;İc;8de;5ce;௯ Ĵc;ıf;௭௼İc;̙a;ᖂ௯Ĵc;ıf;&#ff0e;ĳe;ıf;d04; 110 kDa Ĳe;ᑖb50;b5; a4;ba;ఔᢝ௸ ALDP-GFP e26;ఁĲb; 83 kDa Ĳe;[ce;˯f;ɂb; ALDP Ĳf;,da;eb;aa;ad;b7;bd;fc;e0;de;fc;ab;fc;௼ĳb;ĳc;Ȝc;௲ ᑖe03;ఔ̙a;௱௺௤ıf;௭௼İb;఑da;eb;aa;ad;b7;bd;fc;e0;Ĳb;c40;ᙠ ௱௺௤Ĵb;௭௼İc;̙a;ᖂ௯Ĵc;ıf;&#ff0e;ĳe;ıf;᜕ᶒɂb; ALDP-GFP(G116R, S606P)ఊȜc;Ed8;Ĳe;ᑖe03;ఔ̙a;௱ıf;&#ff0e; e00;Ab9;,᜕ᶒɂb; ALDP-GFP(H667D)ఔb89;b9a;Έe;ᒖ ˿a;Ife;௱௺௤Ĵb;d30;Pde;Ĳe;ᛊᔠĲf;,ALDP-GFP Ĳe;d0;f3;c9; Ĳf;ʳc;3fa;௯Ĵc;Ĳa;İb;௷ıf;(Fig. 3) &#ff0e;‐ᕡdf1;௤௭௼Ĳb;, PMP70 Ĳf; ʳc; 3fa; ௯ Ĵc; ıf; İc; , ᐳ ˿a; Ife; ௯ ıb; ıf; [ce; ˯f; ɂb; ALDP Ĳe;d0;f3;c9;ఊʳc;3fa;௯Ĵc;Ĳa;İb;௷ıf;&#ff0e;ĳe;ıf;᜕ᶒɂb; ALDP(S606L)Ĳb;௸௤௺ఊȜc;Ed8;௻௢௷ıf;&#ff0e;௭Ĵc;఑ Ĳe; d50; ʧc; Ĳf; , ᜕ ᶒ ɂb; ALDP ( H667D, S606L ) Ĳf; PMP70 ௼௻Ĳf;Ĳa;İf;,[ce;˯f;ɂb; ALDP ௼⋋ᔠf53;ఔf62;ᡂ ௱,e21;ὅİc;ᑖYe3;௯Ĵc;Ĵb;5ef;Pfd;ឋఔ̙a;ᖂ௱௺௤Ĵb;&#ff0e; ABC bf;f3;d1;af;cea;Ĳe;a5f;Pfd;˿a;Ife;Ĳb;[cd;⌕௻௢Ĵb; TMD ఌ NBD ee5;᜜Ĳe; C ʠb;aef;Ze8;f4d;௻Ĳe;᜕ᶒİc;bf;f3;d1;af;cea;Ĳe;b89; b9a;ឋĲb;f71;aff;ఔ5ca;ĳc;௳௭௼Ĳf;‐ᕡdf1;௤&#ff0e;ALDP Ĳe; C ʠb;aef;Ze8;f4d;௻௢Ĵb; 600ߟ700 a2;df;ce;⏚௻Ĳe;᜕ᶒİc; X-ALD ఔf15;İd;d77;௭௳ϗb;ea6;İc; ad8;௤௭௼İb;఑,ALDP Ĳe; C ʠb;aef;Ze8;f4d;Ĳf;bf;f3;d1;af;cea;Ĳe;b89;b9a;ឋĲb;[cd;⌕Ĳa;f79;ᒘఔ>c5; ௷௺௤Ĵb;5ef;Pfd;ឋİc;௢Ĵb;&#ff0e;Liu ఑Ĳf; ALDP Ĳe;c0;a4; de;fc;ᓄĲb;Ĳf; C ʠb;aef;Ze8;f4d;(AA.631ߟ745)İc;[cd;⌕௻ ௢Ĵb;௼ᛇȠa;௱௺௤Ĵb;&#ff0e; 13) H667D ఌ S606L Ĳe;ఐ௦Ĳa; ᜕ᶒĲf;,ıd;Ĵc;Qea;eab;௢Ĵb;௤Ĳf;[ce;˯f;ɂb; ALDP ௼df;b9;d5; a9;fc;eb;c9;௱ıf;c0;a4;de;fc;ఔf62;ᡂ௱,ᶒe38;bf;f3;d1;af;cea;௼ ௱௺a8d;b58;௯Ĵc;ᑖYe3;௯Ĵc;Ĵb;௼ὃ௨఑Ĵc;Ĵb;&#ff0e;e00;Ab9;,᜕ᶒ ɂb; ALDP-GFP(R104C)Ĳf;da;eb;aa;ad;b7;bd;fc;e0;ᑖ˱b; Ĳb;8de;5ce;௯Ĵc;Ĵb;ఊĲe;Ĳe;,d5;e9;b0;e1;f3;c8;ᓄ௱௺௤Ĵb;௭௼ 169 Fig. 4. Login to comment
46 ᜕ᶒɂb; ALDP Ĳe;ᑖYe3;Έe;a0b;Ĳe;Ye3;᪆ Ab0;˯f;bf;f3;d1;af;cea;İc;b63;௱௤d5;a9;fc;eb;c7;a3;f3;b0;ఔ5d7;௫ Ĵb;௭௼Ĳf;,ıd;Ĳe;bf;f3;d1;af;cea;Ĳe;b63;e38;Ĳa;a5f;Pfd;˿a;Ife;Ĳe;ıf;ఉ Ĳb;fc5;♐௻௢Ĵb;&#ff0e;΋a;f1d;b50;᜕ᶒĲa;௽İc;b58;ᙠ௳Ĵb;௼,bf;f3; d1;af;cea;İc;df;b9;d5;a9;fc;eb;c7;a3;f3;b0;௯Ĵc;Ĵb;&#ff0e;௭Ĳe;df;b9;d5; a9;fc;eb;c9;bf;f3;d1;af;İc;d30;Pde;᜜ఆᑖccc;௯Ĵc;ıf;Ĵa;,d30;Pde;ᑁ Ĳb;Tc4;a4d;௱ıf;Ĵa;௳Ĵb;௼˯f;f53;Ĳb;௼௷௺ᬿఉ௺ᨵbb3;Ĳb;Ĳa;Ĵb; ıf;ఉ,௭Ĳe;ఐ௦Ĳa;bf;f3;d1;af;Ĳf;d7;ed;c6;a2;bd;fc;e0;,ea;bd; bd;fc;e0;b49;Ĳb;ఐ௷௺fc5;΅f;Ĳb;ᑖYe3;௯Ĵc;Ĵb;&#ff0e;௵Ĳa;ĳf;Ĳb;,8a2; Pde;ឋdda;dad;Kc7;Ĳe;țf;8e0;bf;f3;d1;af;cea; CFTR Ĳf;d30;Pde;̳c;a4;aa; f3;c1;e3;cd;eb;௼௱௺a5f;Pfd;௳Ĵb; ABC bf;f3;d1;af;cea;௻௢Ĵb; İc;,᜕ᶒ CFTR Ĳf;c0f;Pde;f53;̳c;İb;఑d7;ed;c6;a2;bd;fc;e0;Ĳb; ea;af;eb;fc;c8;௯Ĵc;ᑖYe3;௯Ĵc;Ĵb;௭௼İc;ᛇȠa;௯Ĵc;௺௤ Ĵb;&#ff0e; 32,33) ௱İb;௱Ĳa;İc;఑,᜕ᶒɂb; ALDP ఔ;cb;ఉ௼௱ ௺,da;eb;aa;ad;b7;bd;fc;e0;̳c;bf;f3;d1;af;cea;Ĳb;௸௤௺Ĳe;Ye3;᪆ Ĳf;ĳb;௼క௽ʹc;Ĵf;Ĵc;௺௤Ĳa;௤&#ff0e; ᜕ᶒɂb; ALDP Ĳe;e00;Έe;ឋ˿a;Ife;௼b89;b9a;Έe;ᒖ˿a;Ife;b9f; a13; ఐĴa;,ALDP (S606L, R617H, H667D, R104C) Ĳf;, d7;ed;c6;a2;fc;bc;Ĳb;ఐĴa;ᑖYe3;௯Ĵc;௺௤Ĵb;௼?a8;b9a;௯Ĵc;ıf;&#ff0e; ıd;௭௻,ALDP-GFP(H667D)ఔ˿a;Ife;௱௺௤Ĵb; CHO d30;Pde;Ĳb;ᔜa2e;d7;ed;c6;a2;fc;bc;σb;bb3;ᒐఔ3e6;ᳮ௱,Ye3; ᪆ఔʹc;௷ıf;&#ff0e;ıd;Ĳe;d50;ʧc;,d7;ed;c6;a2;bd;fc;e0;σb;bb3;ᒐ௻௢ Ĵb; lactacystin ఔ3e6;ᳮ௱ıf;d30;Pde;௻Ĳf; ALDP-GFP 5ca;ఁ ALDP Ĳe; d0; f3; c9; İc; 3fa; Ife; ௱ ıf; ( Fig. 4 ) &#ff0e; e00; Ab9; , leupeptin, AEBSF, E64d Ĳb;Ĳf;4b9;ʧc;İc;Ĳa;İb;௷ıf;&#ff0e;ĳe; ıf;ed6;Ĳe;d7;ed;c6;a2;bd;fc;e0;σb;bb3;ᒐ௻௢Ĵb; MG132 ఊᨵ4b9; ௻௢௷ıf;&#ff0e;௯఑Ĳb;d7;ed;c6;a2;bd;fc;e0;σb;bb3;ᒐĲb;ఐĴa;ᑖYe3; ఔ⌫Ĵc;ıf;᜕ᶒɂb; ALDP-GFP(H667D)Ĳe;d30;Pde;ᑁc40; ᙠఔVcd;ᐝᢙf53;cd5;௻Yb3;bdf;௳Ĵb;௼,da;eb;aa;ad;b7;bd;fc;e0;Ĳb; c40;ᙠ௱௺௤Ĵb;௭௼İc;Nba;a8d;௯Ĵc;ıf;&#ff0e;e00;Ab9;,᜕ᶒɂb; ALDP (R104C) Ĳe;d5;e9;b0;e1;f3;c8;ᓄĲf;e0a;a18;d7;ed;c6;a2;fc; bc;3e6;ᳮ௻Ĳf;σb;bb3;௯Ĵc;Ĳa;İb;௷ıf;&#ff0e; ௯఑Ĳb; ALD <a3;ὅᵫᩭd30;Pde;Ĳe;ᑁ8e0;ឋ᜕ᶒ ALDP Ĳe;ᑖYe3;௼d7;ed;c6;a2;bd;fc;e0;ᑖYe3;cfb;Ĳe;_a2;e0e;Ĳb;௸௤௺Nba;a8d; ௳Ĵb;ıf;ఉ,᜕ᶒɂb; ALDP(R617H)ఔᢝ௸<a3;ὅᵫ ᩭdda;dad;Rbd;d30;Pde;ఔᵨ௤௺bf;f3;d1;af;ᑖYe3;Ĳe;σb;bb3;b9f; a13;ఔʹc; ௷ıf;&#ff0e;ıd;Ĳe;d50;ʧc;,lactacystin ௼ MG132 3e6;ᳮĲb;ఐĴa;, ALDP Ĳe;d0;f3;c9;İc;3fa;Ife;௱ıf;&#ff0e;ee5;e0a;Ĳe;d50;ʧc;ఐĴa;,da; eb;aa;ad;b7;bd;fc;e0;̳c;e0a;Ĳb;Ĳf;df;b9;d5;a9;fc;eb;c9;௱ıf;bf;f3;d1; af;cea;ఔa8d;b58;௳Ĵb;ed5;d44;ĳf;İc;b58;ᙠ௱,d7;ed;c6;a2;bd;fc;e0;5ca; ఁed6;Ĳe;d7;ed;c6;a2;fc;bc;ఔecb;௱௺᣸◀௱௺௤Ĵb;௭௼İc;̙a; ᖂ௯Ĵc;ıf;&#ff0e; e00;Ab9;,c71;ᵪ఑Ĳf; ALD <a3;ὅdda;dad;Rbd;d30;Pde;ఔ &#ff3b;35 S&#ff3d; e1;c1; aa;cb;f3;௻d1;eb;b9;c1;a7;a4;b9;௳Ĵb;௭௼Ĳb;ఐĴa;,᜕ᶒɂb; ALDP (G512S, R660W) Ĳe;ᑖYe3;İc; E-64 ௼ leupepu- tin Ĳb;ఐĴa;ᢓᑴ௯Ĵc;Ĵb;௭௼ఔᛇȠa;௱௺௤Ĵb;&#ff0e; 34) f7c;఑ Ĳe;b9f; a13;௻Ĳf;d7;ed;c6;a2;bd;fc;e0;σb;bb3;ᒐĲb;௸௤௺Ĳf;b9f; a13;௱ ௺௤Ĳa;௤Ĳe;௻,d7;ed;c6;a2;bd;fc;e0;Ĳe;_a2;e0e;Ĳf;e0d;ʔe;௻௢Ĵb; İc;,᜕ᶒɂb; ALDP Ĳe;ᑖYe3;Ĳb;Ĳf;,⋋ᦪĲe;d7;ed;c6;a2;fc; bc;İc;_a2;e0e;௱௺௤Ĵb;5ef;Pfd;ឋİc;௢Ĵb;&#ff0e; 7. Login to comment

[hide] Mutations, clinical findings and survival estimate... PLoS One. 2012;7(3):e34195. doi: 10.1371/journal.pone.0034195. Epub 2012 Mar 29. Pereira Fdos S, Matte U, Habekost CT, de Castilhos RM, El Husny AS, Lourenco CM, Vianna-Morgante AM, Giuliani L, Galera MF, Honjo R, Kim CA, Politei J, Vargas CR, Jardim LB
Mutations, clinical findings and survival estimates in South American patients with X-linked adrenoleukodystrophy.
PLoS One. 2012;7(3):e34195. doi: 10.1371/journal.pone.0034195. Epub 2012 Mar 29., [PMID:22479560]

Abstract [show]
In this study, we analyzed the ABCD1 gene in X-linked adrenoleukodystrophy (X-ALD) patients and relatives from 38 unrelated families from South America, as well as phenotypic proportions, survival estimates, and the potential effect of geographical origin in clinical characteristics. METHODS: X- ALD patients from Brazil, Argentina and Uruguay were invited to participate in molecular studies to determine their genetic status, characterize the mutations and improve the genetic counseling of their families. All samples were screened by SSCP analysis of PCR fragments, followed by automated DNA sequencing to establish the specific mutation in each family. Age at onset and at death, male phenotypes, genetic status of women, and the effect of family and of latitude of origin were also studied. RESULTS: We identified thirty-six different mutations (twelve novel). This population had an important allelic heterogeneity, as only p.Arg518Gln was repeatedly found (three families). Four cases carried de novo mutations. Intra-familiar phenotype variability was observed in all families. Out of 87 affected males identified, 65% had the cerebral phenotype (CALD). The mean (95% CI) ages at onset and at death of the CALD were 10.9 (9.1-12.7) and 24.7 (19.8-29.6) years. No association was found between phenotypic manifestations and latitude of origin. One index-case was a girl with CALD who carried an ABCD1 mutation, and had completely skewed X inactivation. CONCLUSIONS: This study extends the spectrum of mutations in X-ALD, confirms the high rates of de novo mutations and the absence of common mutations, and suggests a possible high frequency of cerebral forms in our population.

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No. Sentence Comment
24 Family/Index case Phenotype at diagnosis Mutation Exon/IVS Mutation type Effect on protein (cDNA) Effect on protein (mRNA) Protein localization Origin of mutations Origin of family 1/Female asymptomatic p.Gly512Ser (Feigenbaum V et al. 1996) E6 Missense c.1534G.A GGC.AGC NBF de novo Southern Brazil 2/Female asymptomatic p.Ser606Leu (Fanen P et al., 1994) E8 Missense c.1817C.T UCG.UUG NBF Inherited Southern Brazil 3/Male AMN p.Trp601X (Gartner J et al.,1998) E8 Stop codon c.1802C.A Truncated NBF Inherited Southern Brazil 4/Female asymptomatic p.Arg617His (Fanen P et al., 1994) E8 Missense c.1850G.A CGC.CAC NBF ND Southern Brazil 5/Male AMN p.Pro623Leu # E9 Missense c.1868C.T CCC.CUC NBF Inherited Southern Brazil 6/Male AO p.Trp326X (Barcelo A et al, 1996) E2 Stop codon c.978G.A Truncated TMD Inherited Southern Brazil 8/Female asymptomatic p.Glu577X # E7 Stop codon c.1729G.T Truncated NBF Inherited Southern Brazil 9/Male asymptomatic p.Arg554His (Smith KD et al., 1999) E7 Missense c.1661G.A CGU.CAU NBF Inherited Southern Brazil 10/Male CALD p.Arg518Gln (Imamura A et al., 1997) E6 Missense c.1553G.A CGG.CAG NBF Inherited Southern Brazil 11/Male AO p.Tyr33_Pro34fsX34 # E1A Frameshift+stop codon c.99_102delC Truncated - Inherited Southern Brazil 12/Female asymptomatic p.Gly266Arg (Fuchs S et al., 1994) E7 Missense c.1653insG Truncated TMD ND Southern Brazil 20/Male CALD p.Arg538fs # E6 Frameshift c.1614_1615dup27 Elonged NBF de novo Southern Brazil 21/Male CALD p.Ala232fsX64 # E2 Frameshift+stop codon c.696_697del11 Truncated TMD Inherited Southern Brazil 22/Male CALD p.Trp137fsX57 # E1B Frameshift+stop codon c.411_412insC Truncated TMD Inherited Northern Brazil 23/Male asymptomatic p.Trp679X (Waterham HR et al, 1998) E10 Stop codon c.2037G.A Truncated NBF ND Southern Brazil 24/Male AO p.Tyr296Cys (Takano H et al., 1999) E2 Missense c.887A.G UAU.UGU TMD Inherited Southern Brazil 27/Male CALD p.Leu628Glu # E9 Missense c.1883T.A CUG.GAG NBF Inherited Southern Brazil 29/Male CALD p.Pro546fsX? Login to comment