ABCMdb

ABCA1 p.Arg587Trp

Predicted by SNAP2:	A: D (66%), C: D (71%), D: D (85%), E: D (80%), F: D (75%), G: D (75%), H: D (66%), I: D (71%), K: D (59%), L: D (71%), M: D (63%), N: D (75%), P: D (85%), Q: D (71%), S: D (75%), T: D (71%), V: D (71%), W: D (80%), Y: D (71%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: N, S: D, T: D, V: D, W: D, Y: D,

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[hide] Ubiquitin-mediated proteasomal degradation of ABC ... J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12. Nakagawa H, Toyoda Y, Wakabayashi-Nakao K, Tamaki H, Osumi M, Ishikawa T
Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts.
J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12., [PMID:21567408]

Abstract [show]
The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.

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No. Sentence Comment
155 Effect of Mutations and Nonsynonymous SNPs on Protein Trafficking, Maturation, or ERAD of ABC Transporters Protein AA Mutation/SNP Effect on Protein Reference ABCA1 W590S Mutation Functional defect 115 R587W Mutation Impaired glycol processing 115 Q597R Mutation Impaired glycol processing, ERAD 115,116 Y1532C Mutation Altered protein trafficking 117 R1925Q Mutation Altered protein trafficking 118 ABCA3 R43L Mutation Altered protein trafficking 119 L101P Mutation Altered protein trafficking 119 R280C Mutation Altered protein trafficking 119 ABCA4 L541P Mutation Mislocalization 120 R602W Mutation Mislocalization 120 A1038V Mutation Mislocalization 120 C1490Y Mutation Mislocalization 120 ABCB1a G268V Mutation ERAD 121 G341C Mutation ERAD 121 I1196S Mutation Reduced glycosylation 122 ABCB4 I541F Mutation Accumulation in ER 123 ABCB11a E135K Mutation Reduced level of mature protein 124 L198P Mutation Reduced level of mature protein 124 E297G Mutation Reduced level of mature protein 124 L413W Mutation Reduced level of mature protein 124 R432T Mutation Reduced level of mature protein 124 D482G Mutation Immature protein in ER 124,125 N490D Mutation Reduced level of mature protein 124 A570T Mutation Reduced level of mature protein 124 T655I Mutation Reduced level of mature protein 124 Y818F SNP Moderate reduction of protein 124 G982R Mutation Retention in ER 125 R1153C Mutation ERAD 125 R1286Q Mutation Retention in ER 125 ABCC2a R768W Mutation Impaired protein trafficking 126 I1173F Mutation Impaired protein maturation 127 R1392 Mutation Impaired protein maturation 128 M1393 Mutation Impaired protein maturation 129 ABCC4a E757K SNP Altered protein trafficking 23 ABCC7 F508 Mutation Misfolding, ERAD 36-39,130 G85E Mutation Impaired protein maturation 130-132 G91R Mutation Impaired protein maturation 130-132 N1303K Mutation Impaired protein maturation 130-132 ABCC8 WT Wild type Ubiquitin-proteasome degradation 133 A116P Mutation Ubiquitin-proteasome degradation 133 V187D Mutation Ubiquitin-proteasome degradation 133 F1388 Mutation Impaired protein trafficking 134 L1544P Mutation Impaired protein trafficking 135,136 ABCC11a G180R SNP Ubiquitin-proteasome degradation 50 27 Mutation Ubiquitin-proteasome degradation 50 ABCG2a V12M SNP Altered protein localization 96 Q141K SNP Ubiquitin-proteasome degradation 102 F208S SNP Ubiquitin-proteasome degradation 78,99 S441N SNP Ubiquitin-proteasome degradation 78,99 Mutations of ABCA1, ABCA3, ABCA4, ABCB4, ABCB11, ABCC2, ABCC7 (CFTR), and ABCC8 are associated with Tangier disease, fatal surfactant deficiency, Stargardt disease, progressive familial intrahepatic cholestasis type 3 (PFIC-3), progressive familial intrahepatic cholestasis type 2 (PFIC-2), Dubin-Johnson syndrome, cystic fibrosis, and familial hyperinsulinism, respectively. Login to comment

[hide] Increased risk of coronary artery disease in Cauca... Biochim Biophys Acta. 2012 Mar;1821(3):416-24. doi: 10.1016/j.bbalip.2011.08.006. Epub 2011 Aug 19. Tietjen I, Hovingh GK, Singaraja R, Radomski C, McEwen J, Chan E, Mattice M, Legendre A, Kastelein JJ, Hayden MR
Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT.
Biochim Biophys Acta. 2012 Mar;1821(3):416-24. doi: 10.1016/j.bbalip.2011.08.006. Epub 2011 Aug 19., [PMID:21875686]

Abstract [show]
Mutations in ABCA1, APOA1, and LCAT reduce HDL cholesterol (HDLc) in humans. However, the prevalence of these mutations and their relative effects on HDLc reduction and risk of coronary artery disease (CAD) are less clear. Here we searched for ABCA1, APOA1, and LCAT mutations in 178 unrelated probands with HDLc <10th percentile but no other major lipid abnormalities, including 89 with >/=1 first-degree relative with low HDLc (familial probands) and 89 where familial status of low HDLc is uncertain (unknown probands). Mutations were most frequent in LCAT (15.7%), followed by ABCA1 (9.0%) and APOA1 (4.5%), and were found in 42.7% of familial but only 14.6% of unknown probands (p=2.44 *10(-5)). Interestingly, only 16 of 24 (66.7%) mutations assessed in families conferred an average HDLc <10th percentile. Furthermore, only mutation carriers with HDLc <5th percentile had elevated risk of CAD (odds ratio (OR)=2.26 for 34 ABCA1 mutation carriers vs. 149 total first-degree relative controls, p=0.05; OR=2.50 for 26 APOA1 mutation carriers, p=0.04; OR=3.44 for 38 LCAT mutation carriers, p=1.1 *10(-3)). These observations show that mutations in ABCA1, APOA1, and LCAT are sufficient to explain >40% of familial hypoalphalipoproteinemia in this cohort. Moreover, individuals with mutations and large reductions in HDLc have increased risk of CAD. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

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117 COOHA B T929I H2N R587W B A M1091T C1477R K776N N935S S1181F IVS24+1 G>C V2244I R282X D571G M640L S930F M968T R1615WIVS16-5 CA>del ABCA1 Transmembrane domain ATP-binding domain Q597R A) AA 1 AA 267 K130del L202P 74 90 98 112 122 145 167 189 211 215 233 253 APOA1 Negative charge domain Alpha-helix E222K E134DT37M 140 178 206 41127 104 121 165 200 229 360 391 Y135N V246F 127 206 369 401 Catalytic triad R322C L338H V371MV52M Y107X A117T T147I V333M Phe Leu Asp His AA 1 AA 440 R159Q I202T LCAT Alpha helixBeta sheet B) 419I.Tietjenetal./BiochimicaetBiophysicaActa1821(2012)416-424 3.4. Login to comment
169 Effects of some ABCA1 mutations described here are also consistent with previous in vitro cellular efflux studies; for example, the highly expressive ABCA1 mutations R587W, Q597R, N935S, and M1091T cause ~10-25% of wild-type efflux in vitro, while the less expressive mutations K776N and T929I cause 62 and 80% efflux, respectively [35-37]. Login to comment

[hide] Carriers of loss-of-function mutations in ABCA1 di... Diabetes Care. 2010 Apr;33(4):869-74. Epub 2010 Jan 12. Vergeer M, Brunham LR, Koetsveld J, Kruit JK, Verchere CB, Kastelein JJ, Hayden MR, Stroes ES
Carriers of loss-of-function mutations in ABCA1 display pancreatic beta-cell dysfunction.
Diabetes Care. 2010 Apr;33(4):869-74. Epub 2010 Jan 12., [PMID:20067955]

Abstract [show]
OBJECTIVE: Abnormal cellular cholesterol handling in islets may contribute to beta-cell dysfunction in type 2 diabetes. beta-Cell deficiency for the ATP binding cassette transporter A1 (ABCA1), which mediates the efflux of cellular cholesterol, leads to altered intracellular cholesterol homeostasis and impaired insulin secretion in mice. We aimed to assess the impact of ABCA1 dysfunction on glucose homeostasis in humans. RESEARCH DESIGN AND METHODS: In heterozygous carriers of disruptive mutations in ABCA1 and family-based noncarriers of similar age, sex, and BMI, we performed oral glucose tolerance tests (OGTTs) (n = 15 vs. 14) and hyperglycemic clamps (n = 8 vs. 8). RESULTS: HDL cholesterol levels in carriers were less than half those in noncarriers, but LDL cholesterol levels did not differ. Although fasting plasma glucose was similar between groups, glucose curves after an OGTT were mildly higher in carriers than in noncarriers. During hyperglycemic clamps, carriers demonstrated lower first-phase insulin secretion than noncarriers but no difference in insulin sensitivity. The disposition index (a measure of beta-cell function adjusted for insulin sensitivity) of the carriers was significantly reduced in ABCA1 heterozygotes. CONCLUSIONS: Carriers of loss-of-function mutations in ABCA1 show impaired insulin secretion without insulin resistance. Our data provide evidence that ABCA1 is important for normal beta-cell function in humans.

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46 Subjects included in the present study were heterozygous carriers of the following extremely rare ABCA1 mutations: C1477R, M1091T, and R587W (4,7-10) and L996P and Q978X (C. Candini et al., manuscript in preparation). Login to comment
48 C1477R has been reported in nine heterozygous individuals, M1091T in four individuals, and R587W in seven individuals (11). Login to comment
93 Carriers were recruited from five different families (four C1477R carriers, three M1091T carriers, three R587W carriers, four L996P carriers, and one Q978X carrier); family control subjects were siblings, cousins, or partners of a carrier. Login to comment
47 Subjects included in the present study were heterozygous carriers of the following extremely rare ABCA1 mutations: C1477R, M1091T, and R587W (4,7-10) and L996P and Q978X (C. Candini et al., manuscript in preparation). Login to comment
49 C1477R has been reported in nine heterozygous individuals, M1091T in four individuals, and R587W in seven individuals (11). Login to comment
95 Carriers were recruited from five different families (four C1477R carriers, three M1091T carriers, three R587W carriers, four L996P carriers, and one Q978X carrier); family control subjects were siblings, cousins, or partners of a carrier. Login to comment

[hide] Adenosine-triphosphate-binding cassette transporte... Trends Cardiovasc Med. 2010 Feb;20(2):41-9. Kang MH, Singaraja R, Hayden MR
Adenosine-triphosphate-binding cassette transporter-1 trafficking and function.
Trends Cardiovasc Med. 2010 Feb;20(2):41-9., [PMID:20656214]

Abstract [show]
Mutations in the adenosine-triphosphate-binding cassette transporter-1 (ABCA1) lead to Tangier disease, a genetic disorder characterized by an almost complete absence of plasma high-density lipoprotein cholesterol. Although the importance of ABCA1 localization to its cholesterol efflux function has been extensively characterized, the cellular itinerary of ABCA1 leading to the plasma membrane is not fully elucidated. This review will summarize the current knowledge of ABCA1 trafficking and its relationship to function. Understanding these crucial processes provides potential novel therapeutic targets to regulate high-density lipoprotein biogenesis through influencing pathways of ABCA1 trafficking.

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40 The importance of ABCA1 localization to the PM is observed in several mutations underlying TD (R587W, Q597R, ΔL693, M1091T), where mislocalization of ABCA1 away from the cell surface leads to a significant reduction in cholesterol efflux function (Singaraja et al. 2006). Login to comment
127 A number of clinically relevant mutations of ABCA1 (R587W, Q597R, ΔL693, N935H) acquire only the core and not the complex oligosaccharide chain and fail to exit the ER (Singaraja et al. 2006). Login to comment
128 A number of clinically relevant mutations of ABCA1 (R587W, Q597R, ƊL693, N935H) acquire only the core and not the complex oligosaccharide chain and fail to exit the ER (Singaraja et al. 2006). Login to comment

[hide] Severe HDL deficiency due to novel defects in the ... J Intern Med. 2009 Mar;265(3):359-72. Epub 2008 Oct 25. Pisciotta L, Bocchi L, Candini C, Sallo R, Zanotti I, Fasano T, Chakrapani A, Bates T, Bonardi R, Cantafora A, Ball S, Watts G, Bernini F, Calandra S, Bertolini S
Severe HDL deficiency due to novel defects in the ABCA1 transporter.
J Intern Med. 2009 Mar;265(3):359-72. Epub 2008 Oct 25., [PMID:19019193]

Abstract [show]
OBJECTIVES: The objective was the identification and functional characterization of mutations in the ABCA1 gene in four patients with severe HDL deficiency. SUBJECTS: Patients were referred to the clinic because of almost complete HDL deficiency. METHODS: The ABCA1 gene was sequenced directly. The analysis of the ABCA1 protein, ABCA1 mRNA and ABCA1-mediated cholesterol efflux was performed in cultured fibroblasts. Intracellular localization of ABCA1 mutants was investigated in transfected HEK293 cells. RESULTS: Two patients were homozygous for mutations in the coding region of the ABCA1 gene, resulting in an amino acid substitution (p.A1046D) and a truncated protein (p.I74YFsX76). The third patient was homozygous for a splice site mutation in intron 35 (c.4773 + 1g>a), resulting in an in-frame deletion of 25 amino acids (del p.D1567_K1591) in ABCA1. These patients had clinical manifestations of accumulation of cholesterol in the reticulo-endothelial system. The fourth patient, with preclinical atherosclerosis, was a compound heterozygote for two missense mutations (p.R587W/p.W1699C). ABCA1-mediated cholesterol efflux was abolished in fibroblasts from patients with p.A1046D and del p.D1567_K1591 mutants and in fibroblasts homozygous for p.R587W. A reduced ABCA1 protein content was observed in these cells, suggesting an increased intracellular degradation. The mutant p.W1699C was largely retained in the endoplasmic reticulum, when expressed in HEK293 cells. CONCLUSIONS: The homozygotes for mutations which abolish ABCA1 function showed overt signs of involvement of the reticulo-endothelial system. This was not the case in the compound heterozygote for missense mutations, suggesting that this patient retains some residual ABCA1 function that reduces cholesterol accumulation in the reticulo-endothelial system.

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16 The fourth patient, with preclinical atherosclerosis, was a compound heterozygote for two missense mutations (p.R587W/p.W1699C). Login to comment
17 ABCA1-mediated cholesterol efflux was abolished in fibroblasts from patients with p.A1046D and del p.D1567_K1591 mutants and in fibroblasts homozygous for p.R587W. Login to comment
98 p.D1567_K1591); M3 (p.I74YFsX76); M4 (p.W1699C); M5 (p.R587W); ND, not determined. Login to comment
147 The proband was found to be a compound heterozygote for the following mutations of the ABCA1 gene: (i) a C>T transition in exon 14 (c.1759 C>T, p.R587W), inherited from the mother; (ii) a G>T transversion in exon 37 (c.5097 G>T, p.W1699C), inherited from the father (Fig. S3). Login to comment
150 The sister of the proband`s mother (subject I.4) and her children (subjects II.4, II.6 and II.7) were carriers of the p.R587W mutation. Login to comment
157 The cholesterol efflux in fibroblasts of these probands was similar to that observed in fibroblasts of another patient with severe HDL deficiency (homozygous for the p.R587W mutation) we had previously identified [24] (Fig. 3). Login to comment
158 Enrichment of cholesterol in fibroblast plasma membrane We also measured the increase in cholesterol content in the fibroblast plasma membrane from the probands of families 1 and 2 and from the patient homozygous for p.R587W mutation, induced by the stimulation of ABCA1 expression with 9-cis-retinoic acid and 22-hydroxycholesterol. Login to comment
161 In the probands` fibroblasts and in the fibroblasts of the patient homozygous for the ABCA1 p.R587W mutation, the increase in membrane cholesterol varied considerably. Login to comment
162 In the p.A1046D (family 1) and homozygous p.R587W cells this increase was significant, although much lower than that of control cells, whilst in c.4773 + 1g>a mutant cells (family 2) no increase in membrane cholesterol was observed. Login to comment
168 Figure 5 also shows (lane 3) that the ABCA1 protein in fibroblasts from the previously reported patient [24] homozygous for p.R587W (the missense mutation found in the compound heterozygote of family 4) is clearly detect- Fig. 3 3 H-cholesterol efflux to Apo A-I in skin fibroblasts under basal conditions and following stimulation of ABCA1 gene expression with 22-hydroxycholesterol and 9-cis-retinoic acid (22OH / cRA). Login to comment
169 Ctrl, control cells; p.R587W, fibroblasts from a homozygous patient reported previously [24]; p.A1046D, fibroblasts from the proband of family 1; del p.D1567_K1591, fibroblasts of the proband of family 2. able, even though its content appears to be lower than that seen in control fibroblasts. Login to comment
181 Ctrl, control cells; p.R587W, fibroblasts from a homozygous patient reported previously [24]; p.A1046D, fibroblasts from the proband of family 1; del p.D1567_K1591, fibroblasts of the proband of family 2. Login to comment
215 The proband of family 4 was a compound heterozygote carrying two missense mutations (p.R587W and p.W1699C). Login to comment
216 The p.R587W mutation was first reported by Lawn et al. [33] in a compound heterozygote with HDL deficiency, in whom the second allele failed to produce detectable mRNA. Login to comment
218 Previous in vitro experiments in transfected cells expressing the p.R587W mutant (designed to define the alteration in the ABCA1 pathway) were somewhat inconsistent. Login to comment
219 Fitzgerald et al. [27] showed that the p.R587W mutant reached the cell surface just like its wild type counterpart, but showed a 50% reduction in the cross-linking efficiency to Apo A-I. Login to comment
220 Singaraja et al. [32] showed that the p.R587W mutation prevents the migration of the protein to the plasma membrane, causing a 75% decrease in ABCA1-mediated cholesterol efflux. Login to comment
221 In our patient homozygous for the p.R587W mutation [24] the ABCA1 protein content was lower than that observed in control fibroblasts (Fig. 5, lane 3), possibly suggesting an increased rate of intracellular degradation (an event expected if the mutant protein is retained, at least in part, in the endoplasmic reticulum) [32]. Login to comment

[hide] ABCA1 mutants reveal an interdependency between li... J Lipid Res. 2009 Feb;50(2):285-92. Epub 2008 Sep 5. Vaughan AM, Tang C, Oram JF
ABCA1 mutants reveal an interdependency between lipid export function, apoA-I binding activity, and Janus kinase 2 activation.
J Lipid Res. 2009 Feb;50(2):285-92. Epub 2008 Sep 5., [PMID:18776170]

Abstract [show]
ABCA1 exports cholesterol and phospholipids from cells by a multistep pathway that involves forming cell surface lipid domains, solubilizing these lipids by apolipoproteins, binding of apolipoproteins to ABCA1, and activating signaling processes. Here we used a mutational analysis approach to evaluate the relationship between these events. We prepared seven naturally occurring mutants and one artificial missense mutant of ABCA1 with varying degrees of impaired function, expressed them to similar levels as wild-type ABCA1 on the cell surface of BHK cells, and measured ABCA1-dependent lipid export, apolipoprotein A-I (apoA-I) binding, and signaling activities. Linear regression analyses showed that cholesterol and phospholipid efflux and cellular apoA-I binding correlated significantly with the ability of ABCA1 to form cell surface lipid domains. Lipid export and cellular apoA-I binding activities and formation of lipid domains also correlated with the amount of apoA-I that could be cross-linked to ABCA1. Moreover, each of these lipid export and apoA-I binding activities correlated with apoA-I-induced Janus kinase 2 (JAK2) activation. Thus, these missense mutations in ABCA1 impair lipid export, apoA-I binding, and apoA-I-stimulated JAK2 activities to similar extents, indicating that these processes are highly interactive components of a pathway that functions to export lipids from cells.

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49 the first extracellular loop (V399A, R587W, W590S, and Q597R), two were in the second extracellular loop (C1477R and I1517R), and one was in the Walker A motif of the first nucleotide binding domain (A937V, NBD1) (Fig. 1). Login to comment
96 Fitzgerald et al. (24) reported similar cell surface localizations for mutants R587W, W590S, Q597R, and C1477R. Login to comment
97 In contrast, Singaraja et al. (14) and Tanaka et al. (25) reported that ABCA1 with the R587W or Q597R mutations had impaired translocation to the plasma membrane in transfected cells. Login to comment
95 Fitzgerald et al. (24) reported similar cell surface localizations for mutants R587W, W590S, Q597R, and C1477R. Login to comment

[hide] Formation of cholesterol-enriched structures by ab... Genes Cells. 2008 Aug;13(8):889-904. Tanaka AR, Kano F, Yamamoto A, Ueda K, Murata M
Formation of cholesterol-enriched structures by aberrant intracellular accumulation of ATP-binding cassette transporter A1.
Genes Cells. 2008 Aug;13(8):889-904., [PMID:18782226]

Abstract [show]
ATP-binding cassette transporter A1 (ABCA1) is a key transporter associated with excess cellular lipid efflux. Here, we report that in HEK293 cells ABCA1 functions in intracellular compartments along the endocytic pathway. Inhibition of ABCA1-GFP degradation with proteasome inhibitors induced the internalization of ABCA1 and the formation of intracellular round-shaped structures, designated "A1 bodies". Importantly, cholesterol was selectively accumulated in A1 bodies, and this depended on the cholesterol efflux activity of ABCA1. Treatment with either lactacystin or acetylated LDL, which reduces proteasome activity, resulted in internalization of ABCA1 in mouse peritoneal macrophages. By performing array analysis on macrophages treated with these reagents, we identified Rab4 as a key protein involved in the internalization and aberrant accumulation of ABCA1 in HEK cells. Treatment of the cells with proteasome inhibitors inhibited the degradation of Rab4, and Rab4 over-expression induced the formation of small A1 bodies. Furthermore, A1 bodies formation was substantially inhibited by silencing of the endogenous Rab4 gene. Taken together, our findings suggest that the endocytic ABCA1 possesses cholesterol efflux activity, and thus the cellular control of post-endocytic sorting, retention or recycling of functional ABCA1 in the endocytic vesicles, which is in part regulated by Rab4, is important for cholesterol metabolism in living cells.

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14 Previously, we have reported that the defect in HDL assembly in two point mutants of ABCA1 (R587W, Q597R) responsible for familial HDL deficiency is due to the impaired localization to the plasma membrane (Tanaka et al. 2003). Login to comment

[hide] Lewis-Sumner syndrome and Tangier disease. Arch Neurol. 2008 Jul;65(7):968-70. Theaudin M, Couvert P, Fournier E, Bouige D, Bruckert E, Perrotte P, Vaschalde Y, Maisonobe T, Bonnefont-Rousselot D, Carrie A, Le Forestier N
Lewis-Sumner syndrome and Tangier disease.
Arch Neurol. 2008 Jul;65(7):968-70., [PMID:18625867]

Abstract [show]
OBJECTIVE: To report unusual electrophysiologic data in a patient with Tangier disease in an effort to better understand the pathophysiologic features of the peripheral nerve lesions in this disease. DESIGN: Case report. PATIENT: A 15-year-old girl had subacute onset of asymmetric neuropathy with persistent conduction block, resembling Lewis-Sumner syndrome. MAIN OUTCOME MEASURES: Electrophysiologic data in Tangier disease. RESULTS: After initially unsuccessful treatment with intravenously administered immunoglobulins, the finding of an abnormal lipid profile led to the diagnosis of Tangier disease due to the R587W mutation in the adenotriphosphate-binding cassette transporter-1 gene (ABCA1) (OMIM 9q22-q31). CONCLUSIONS: Conduction block, which is the electrophysiologic hallmark of focal demyelination, can be present in Tangier disease. It could be induced by focal nerve ischemia or by preferential lipid deposition in the paranodal regions of myelinated Schwann cells. The presence of a conduction block in Tangier disease may lead to a misdiagnosis of dysimmune neuropathy.

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4 Results: After initially unsuccessful treatment with intravenously administered immunoglobulins, the finding of an abnormal lipid profile led to the diagnosis of Tangier disease due to the R587W mutation in the ad- enotriphosphate-binding cassette transporter-1 gene (ABCA1) (OMIM 9q22-q31). Login to comment
34 A sequencing of the 50 exons of ABCA1 led to the identification of a homozygous substitution in exon 14 (c.1759C→T), R587W, which has already been reported in a family with Tangier disease.5 Both parents of the patient were heterozygous for this mutation. Login to comment
50 The abnormallipoproteinprofileledtothediagnosisofTangier disease and identification of the R587W homozygous mutation in exon 14 of ABCA1. Login to comment
64 The R587W homozygous mutation in exon 14 of ABC1 has been reported in only 1 Italian family.5 The proband had severe premature coronary heart disease and a mild clinical phenotype of Tangier disease without neuropathy andwithanormaltriglyceridelevel.Fromthiscase,Bertolini etal5 suggestthattheR587Wmutationmightaffectthefunc- tion of ABCA1, specifically in the intima of the arterial wall, and have a much lower effect on ABCA1 function in other locations such as the peripheral nerves. Login to comment
33 A sequencing of the 50 exons of ABCA1 led to the identification of a homozygous substitution in exon 14 (c.1759C࢐T), R587W, which has already been reported in a family with Tangier disease.5 Both parents of the patient were heterozygous for this mutation. Login to comment
47 The abnormallipoproteinprofileledtothediagnosisofTangier disease and identification of the R587W homozygous mutation in exon 14 of ABCA1. Login to comment
61 The R587W homozygous mutation in exon 14 of ABC1 has been reported in only 1 Italian family.5 The proband had severe premature coronary heart disease and a mild clinical phenotype of Tangier disease without neuropathy andwithanormaltriglyceridelevel.Fromthiscase,Bertolini etal5 suggestthattheR587Wmutationmightaffectthefunc- tion of ABCA1, specifically in the intima of the arterial wall, and have a much lower effect on ABCA1 function in other locations such as the peripheral nerves. Login to comment

[hide] The role of different regions of ATP-binding casse... Biochemistry. 2007 Aug 21;46(33):9388-98. Epub 2007 Jul 26. Mukhamedova N, Fu Y, Bukrinsky M, Remaley AT, Sviridov D
The role of different regions of ATP-binding cassette transporter A1 in cholesterol efflux.
Biochemistry. 2007 Aug 21;46(33):9388-98. Epub 2007 Jul 26., [PMID:17655203]

Abstract [show]
ABCA1 is a key element of cholesterol efflux, but the mechanism of ABCA1-dependent cholesterol efflux is still unclear. Monoclonal antibodies against ABCA1 were used to map functional domains of ABCA1. Two antibodies were directed against a fragment of the first extracellular loop of ABCA1, and the third antibody was directed against a fragment of the fourth extracellular loop. One antibody against the first loop inhibited cholesterol efflux from human macrophages without inhibiting apolipoprotein A-I (apoA-I) binding and internalization. Another antibody against the first loop inhibited apoA-I binding and internalization without inhibiting cholesterol efflux. The antibody against the fourth loop inhibited apoA-I binding to ABCA1 but enhanced cholesterol efflux from macrophages and reduced intracellular cholesterol content. This antibody also increased cholesterol efflux from HeLa cells transfected with ABCA1 but not from cells with DeltaPEST-ABCA1. The mechanism of the stimulating effect of this antibody on cholesterol efflux was found to be stabilization of ABCA1 leading to the increase in abundance of cell surface ABCA1. We conclude that a site on the first extracellular loop is required for cholesterol efflux, whereas a site on the fourth extracellular loop may be responsible for ABCA1 stability.

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269 Natural mutations found in the Tangier pedigree, R587W, W590S, Q597R, and S1506L, as well as generated mutant C1477R strongly inhibited cholesterol and phospholipid efflux (28, 35, 36) and, with the exception of W590S, also apoA-I binding (36). Login to comment

[hide] Specific mutations in ABCA1 have discrete effects ... Circ Res. 2006 Aug 18;99(4):389-97. Epub 2006 Jul 27. Singaraja RR, Visscher H, James ER, Chroni A, Coutinho JM, Brunham LR, Kang MH, Zannis VI, Chimini G, Hayden MR
Specific mutations in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes both in vivo and in vitro.
Circ Res. 2006 Aug 18;99(4):389-97. Epub 2006 Jul 27., [PMID:16873719]

Abstract [show]
Mutations in ATP-binding cassette transporter A1 (ABCA1) cause Tangier disease and familial hypoalphalipoproteinemia, resulting in low to absent plasma high-density lipoprotein cholesterol levels. However, wide variations in clinical lipid phenotypes are observed in patients with mutations in ABCA1. We hypothesized that the various lipid phenotypes would be the direct result of discrete and differing effects of the mutations on ABCA1 function. To determine whether there is a correlation between the mutations and the resulting phenotypes, we generated in vitro 15 missense mutations that have been described in patients with Tangier disease and familial hypoalphalipoproteinemia. Using localization of ABCA1, its ability to induce cell surface binding of apolipoprotein A-I, and its ability to elicit efflux of cholesterol and phospholipids to apolipoprotein A-I we determined that the phenotypes of patients correlate with the severity and nature of defects in ABCA1 function.

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44 In patients defined by missense mutations on both alleles, 2 clear groups were observed: those showing negligible plasma HDL-C (R587W, N935S, N1800H), and those with HDL-C levels that were Ϸ10% of HDL-C in controls (A255T). Login to comment
46 Indeed, patients heterozygous for the mutations R587W, Q597R, ⌬L693, N935S, A1046D, C1477R, and R2081W had between 47% and 69% of HDL-C levels of controls (Table). Login to comment
48 Six mutants, R587W, Q597R, ⌬L693, N935S, N1800H, and R2081W, showed no localization at the plasma membrane and instead accumulated intracellularly (Figure 2A), indicating that these mutations severely affect ABCA1 function by preventing its migration to the plasma membrane, thus diminishing its ability to efflux lipids and generate HDL. Login to comment
54 Of the mutants not localizing to the plasma membrane, R587W, Q597R, ⌬L693, and N935H are all EndoH sensitive, indicating that they do not exit the ER. Login to comment
58 R587W, Q597R, ⌬L693, S1506L, and R2081W showed significantly reduced cell surface ABCA1 expression, confirming our previous localization data. Login to comment
68 All 6 mutants showing no plasma membrane localization elicited significantly reduced cell surface ApoA-I binding (R587W, 33.0Ϯ8.9%, nϭ3, Pϭ0.006; Q597R, 17.4Ϯ14.0%, nϭ3, Pϭ0.009; ⌬L693, 32.6Ϯ10.6%, nϭ3, Pϭ0.008; N935S, 26.4Ϯ37.5%, nϭ3, Pϭ0.01; N1800H, 36.9Ϯ15.5%, nϭ3, Pϭ0.01; R2081W, 34.6Ϯ16.6%, nϭ3, Pϭ0.02) (Figure 3A), confirming that cell surface localization of ABCA1 is essential to elicit ApoA-I binding. Login to comment
78 Wild-type ABCA1 was localized intracellularly and at the plasma membrane. R587W, Q597R, ⌬L693, N935S, N1800H, and R2081W were only localized intracellularly. Login to comment
81 Wt ABCA1 shows both EndoH resistant and sensitive bands, indicating localization at the ER and plasma membrane. R587W, Q597R, ⌬L693, and N935S show only the lower EndoH sensitive band, indicating ER retention. Login to comment
95 Near-Complete Absence of Plasma HDL-C in Homozygotes for Mutations in ABCA1 Implies the Presence of Null Alleles of ABCA1 Patients homozygous for R587W mutations showed 6.3% of normal HDL-C levels, those with N935S showed 2.62%, and those with N1800H showed 3.4% of normal plasma HDL-C levels. Login to comment
158 P2150L has only been described in patients who also have the R587W variant (M.R.H., unpublished data, 2000). Login to comment
159 In our assays R587W clearly shows biochemical defects, implying that in these patients with R587W/P2150L, the defects in ABCA1 function are more appropriately ascribed to the R587W variant. In addition, TD has been diagnosed in patients homozygous for the R587W mutation,8,27 presumably without P2150L, making it more likely that P2150L is a nonfunctional variant. Login to comment
160 D1289N has been described as a variant in patients that are homozygous for R2081W.11 Biochemical characterization of R2081W results in defects in subcellular localization and lipid efflux, suggesting that D1289N is another variant. In addition, the bioinformatics analyses by PANTHER indicated that these were putative nonfunctional residues. Login to comment

[hide] Role of apoA-I, ABCA1, LCAT, and SR-BI in the biog... J Mol Med (Berl). 2006 Apr;84(4):276-94. Epub 2006 Feb 25. Zannis VI, Chroni A, Krieger M
Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL.
J Mol Med (Berl). 2006 Apr;84(4):276-94. Epub 2006 Feb 25., [PMID:16501936]

Abstract [show]
The concentration, composition, shape, and size of plasma high-density lipoprotein (HDL) are determined by numerous proteins that influence its biogenesis, remodeling, and catabolism. The discoveries of the HDL receptor (scavenger receptor class B type I, SR-BI) and the ABCA1 (ATP-binding cassette transporter A1) lipid transporter provided two missing links that were necessary to understand the biogenesis and some of the functions of HDL. Existing data indicate that functional interactions between apoA-I and ABCA1 are necessary for the initial lipidation of apoA-I. Through a series of intermediate steps, lipidated apoA-I proceeds to form discoidal HDL particles that can be converted to spherical particles by the action of lecithin:cholesterol acyltransferase (LCAT). Discoidal and spherical HDL can interact functionally with SR-BI and these interactions lead to selective lipid uptake and net efflux of cholesterol and thus remodel HDL. Defective apoA-I/ABCA1 interactions prevent lipidation of apoA-I that is necessary for the formation of HDL particles. In the same way, specific mutations in apoA-I or LCAT prevent the conversion of discoidal to spherical HDL particles. The interactions of lipid-bound apoA-I with SR-BI are affected in vitro by specific mutations in apoA-I or SR-BI. Furthermore, deficiency of SR-BI affects the lipid and apolipoprotein composition of HDL and is associated with increased susceptibility to atherosclerosis. Here we review the current status of the pathway of HDL biogenesis and mutations in apoA-I, ABCA1, and SR-BI that disrupt different steps of the pathway and may lead to dyslipidemia and atherosclerosis in mouse models. The phenotypes generated in experimental mouse models for apoA-I, ABCA1, LCAT, SR-BI, and other proteins of the HDL pathway may facilitate early diagnosis of similar phenotypes in the human population and provide guidance for proper treatment.

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147 In vitro analysis of the effects on apoA-I/ABCA1 interactions (cross-linking assay) by mutations in ABCA1 that are found in Tangier disease patients and diminish lipid efflux [71] showed that cross-linking was dramatically reduced to 5-10% of the WT control for three mutants (Gln597Arg, Cys1477Arg, and Ser1506Leu), reduced by 50% for the Arg587Trp mutant, and was remarkably increased to 125% of control for the Trp590Ser mutant [71]. Login to comment

[hide] Variations on a gene: rare and common variants in ... Annu Rev Nutr. 2006;26:105-29. Brunham LR, Singaraja RR, Hayden MR
Variations on a gene: rare and common variants in ABCA1 and their impact on HDL cholesterol levels and atherosclerosis.
Annu Rev Nutr. 2006;26:105-29., [PMID:16704350]

Abstract [show]
Cholesterol and its metabolites play a variety of essential roles in living systems. Virtually all animal cells require cholesterol, which they acquire through synthesis or uptake, but only the liver can degrade cholesterol. The ABCA1 gene product regulates the rate-controlling step in the removal of cellular cholesterol: the efflux of cellular cholesterol and phospholipids to an apolipoprotein acceptor. Mutations in ABCA1, as seen in Tangier disease, result in accumulation of cellular cholesterol, reduced plasma high-density lipoprotein cholesterol, and increased risk for coronary artery disease. To date, more than 100 coding variants have been identified in ABCA1, and these variants result in a broad spectrum of biochemical and clinical phenotypes. Here we review genetic variation in ABCA1 and its critical role in cholesterol metabolism and atherosclerosis in the general population.

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555 Since a complete loss of function allele would be expected to result in a 50% reduction in HDL levels, a greater than 50% reduction in HDL is most likely explained by a dominant negative allele, in which TABLE 3 Patient phenotypes associated with heterozygous ABCA1 mutations Mutation HDL (mmol/L) HDL (% of control) Number of patients M1091T 0.48 ± 0.5 30 ± 30 4 G1216V 0.50 40 1 R2144X 0.56 ± 0.2 41 ± 18 12 R282X 0.52 41 1 R909X 0.59 ± 0.3 42 ± 19 5 K776N 0.55 ± 0.1 47 ± 5 2 R587W 0.61 ± 0.1 47 ± 8 7 S364C 0.60 48 1 P1065S 0.80 51 1 c-ter deletion 0.75 53 1 N1800H - 56.5 33 P85L 0.72 ± 0.4 57 ± 33 5 Del693L 0.79 ± 0.2 57 ± 15 8 D1289N 0.80 ± 0.1 59 ± 12 4 R2081W 0.80 ± 0.1 59 ± 12 4 2203X 0.80 ± 0.2 59 ± 20 4 DelED1893,4 0.77 ± 0.2 59 ± 18 8 2145X 0.82 ± 0.1 59 ± 9 4 A1046D 0.70 ± 0.1 60 ± 8 2 Q597R 0.82 ± 0.1 60 ± 5 5 C1477R 0.82 ± 0.2 61 ± 15 9 IVS25 + 1G > C 0.78 ± 0.1 62 ± 12 4 D1099Y 0.83 ± 0.3 63 ± 21 5 1552X 1.00 64 1 F2009S 0.82 ± 0.2 64 ± 19 6 R587W 0.86 ± 0.1 65 ± 17 2 R1068H 0.90 ± 0.3 67 ± 26 9 N935S 1.00 ± 0.3 74 ± 16 7 T929I 1.01 ± 0.2 76 ± 7 8 1284X 1.11 ± 0.2 83 ± 14 5 A937V 1.15 ± 0.6 85 ± 28 2 R1680W 1.22 ± 0.2 87 ± 17 3 635X 1.24 ± 0.5 90 ± 32 7 W590S 1.32 ± 0.6 103 ± 46 15 the mutant protein actually interferes with the activity of the remaining wild-type protein. Login to comment

[hide] Accurate prediction of the functional significance... PLoS Genet. 2005 Dec;1(6):e83. Epub 2005 Dec 30. Brunham LR, Singaraja RR, Pape TD, Kejariwal A, Thomas PD, Hayden MR
Accurate prediction of the functional significance of single nucleotide polymorphisms and mutations in the ABCA1 gene.
PLoS Genet. 2005 Dec;1(6):e83. Epub 2005 Dec 30., [PMID:16429166]

Abstract [show]
The human genome contains an estimated 100,000 to 300,000 DNA variants that alter an amino acid in an encoded protein. However, our ability to predict which of these variants are functionally significant is limited. We used a bioinformatics approach to define the functional significance of genetic variation in the ABCA1 gene, a cholesterol transporter crucial for the metabolism of high density lipoprotein cholesterol. To predict the functional consequence of each coding single nucleotide polymorphism and mutation in this gene, we calculated a substitution position-specific evolutionary conservation score for each variant, which considers site-specific variation among evolutionarily related proteins. To test the bioinformatics predictions experimentally, we evaluated the biochemical consequence of these sequence variants by examining the ability of cell lines stably transfected with the ABCA1 alleles to elicit cholesterol efflux. Our bioinformatics approach correctly predicted the functional impact of greater than 94% of the naturally occurring variants we assessed. The bioinformatics predictions were significantly correlated with the degree of functional impairment of ABCA1 mutations (r2 = 0.62, p = 0.0008). These results have allowed us to define the impact of genetic variation on ABCA1 function and to suggest that the in silico evolutionary approach we used may be a useful tool in general for predicting the effects of DNA variation on gene function. In addition, our data suggest that considering patterns of positive selection, along with patterns of negative selection such as evolutionary conservation, may improve our ability to predict the functional effects of amino acid variation.

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48 This SNP has been reported to be associated with decreased HDL cholesterol and increased severity of atherosclerosis in Table 1. subPSEC Scores and Probability of Functional Impairment (Pdeleterious) for ABCA1 Mutations and SNPs Mutations SNPs Variant SubPSEC Pdeleterious Variant subPSEC Pdeleterious P85L À4.62 0.83 R219K À0.57 0.08 H160F À2.79 0.45 V399A À2.26 0.32 R230C À4.27 0.78 V771M À2.86 0.46 A255T À1.81 0.23 T774P À1.99 0.27 E284K À2.34 0.34 K776N À;3.53 0.63 Y482C À4.21 0.77 V825I À1.06 0.13 R587W À6.04 0.95 I883M À1.38 0.17 W590S À5.19 0.9 E1172D À1.96 0.26 W590L À4.48 0.82 R1587K À0.58 0.08 Q597R À7.15 0.98 S1731C À4.21 0.77 T929I À4.29 0.78 N935H À8.54 1 N935S À7.53 0.99 A937V À6.6 0.97 A1046D À7.52 0.99 M1091T À3.56 0.64 D1099Y À6.09 0.96 D1289N À2.48 0.37 L1379F À3.81 0.69 C1477R À5.44 0.92 S1506L À5.17 0.9 N1611D À5.69 0.94 R1680W À6.02 0.95 V1704D À3.21 0.55 N1800H À4.23 0.77 R1901S À5.06 0.89 F2009S À2.73 0.43 R2081W À8.08 0.99 P2150L À2.88 0.47 Q2196H À2.74 0.43 DOI: 10.1371/journal.pgen.0010083.t001 PLoS Genetics | www.plosgenetics.org December 2005 | Volume 1 | Issue 6 | e83 0740 Accurate Prediction of ABCA1 Variants Synopsis A major goal of human genetics research is to understand how genetic variation leads to differences in the function of genes. Login to comment
75 Cholesterol Efflux Values for 293 Cells Transfected with ABCA1 Variants and subPSEC and PolyPhen Predictions of the Functional Impact of these Variants Variant Variant Type subPSEC Cholesterol Efflux PolyPhen R2081W Mutation À8.08 21.1 6 21%* Probably damaging N935S Mutation À7.53 29.3 6 13%* Benign A1046D Mutation À7.52 16.8 6 7%* Possibly damaging Q597R Mutation À7.15 17.7 6 14%* Probably damaging R587W Mutation À6.04 31.7 6 33%* Probably damaging C1477R Mutation À5.44 20.5 6 10%* Probably damaging W590S Mutation À5.19 47.1 6 13%* Probably damaging S1506L Mutation À5.17 17.8 6 15%* Probably damaging T929I Mutation À4.29 69.9 6 11%* Possibly damaging N1800H Mutation À4.23 31.3 6 16%* Possibly damaging S1731C SNP À4.21 12.3 6 10%* Possibly damaging M1091T Mutation À3.56 6.9 6 20%* Probably damaging P2150L Mutation À2.88 88.4 6 21% Probably damaging V771M SNP À2.86 145.4 6 33% Benign D1289N Mutation À2.48 137.7 6 86% Benign I883M SNP À1.38 69.1 6 16%* Benign R219K SNP À0.57 103.7 6 21.05 Benign Wild-type - 0.0 100% - *p , 0.01 compared to wild-type ABCA1. Login to comment

[hide] ATP-binding cassette transporter A1: a cell choles... Physiol Rev. 2005 Oct;85(4):1343-72. Oram JF, Heinecke JW
ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease.
Physiol Rev. 2005 Oct;85(4):1343-72., [PMID:16183915]

Abstract [show]
Blood high-density lipoprotein (HDL) levels are inversely related to risk for cardiovascular disease, implying that factors associated with HDL metabolism are atheroprotective. One of these factors is ATP-binding cassette transporter A1 (ABCA1), a cell membrane protein that mediates the transport of cholesterol, phospholipids, and other metabolites from cells to lipid-depleted HDL apolipoproteins. ABCA1 transcription is highly induced by sterols, a major substrate for cellular export, and its expression and activity are regulated posttranscriptionally by diverse processes. Liver ABCA1 initiates formation of HDL particles, and macrophage ABCA1 protects arteries from developing atherosclerotic lesions. ABCA1 mutations can cause a severe HDL deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. Genetic manipulations of ABCA1 expression in mice also affect plasma HDL levels and atherogenesis. Metabolites elevated in individuals with the metabolic syndrome and diabetes destabilize ABCA1 protein and decrease cholesterol export from macrophages. Moreover, oxidative modifications of HDL found in patients with cardiovascular disease reduce the ability of apolipoproteins to remove cellular cholesterol by the ABCA1 pathway. These observations raise the possibility that an impaired ABCA1 pathway contributes to the enhanced atherogenesis associated with common inflammatory and metabolic disorders. The ABCA1 pathway has therefore become an important new therapeutic target for treating cardiovascular disease.

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412 Some studies have suggested that ABCA1 proteins with substitution mutations Q597R and R587W in the first extracellular loop do not localize to the plasma membrane (232, 263), but other studies have contradicted these findings (73, 150). Login to comment
411 Some studies have suggested that ABCA1 proteins with substitution mutations Q597R and R587W in the first extracellular loop do not localize to the plasma membrane (232, 263), but other studies have contradicted these findings (73, 150). Login to comment

[hide] ABC proteins: key molecules for lipid homeostasis. Med Mol Morphol. 2005 Mar;38(1):2-12. Takahashi K, Kimura Y, Nagata K, Yamamoto A, Matsuo M, Ueda K
ABC proteins: key molecules for lipid homeostasis.
Med Mol Morphol. 2005 Mar;38(1):2-12., [PMID:16158173]

Abstract [show]
Forty-nine ABC protein genes exist on human chromosomes. Eukaryotic ABC proteins were originally recognized as drug efflux pumps involved in the multidrug resistance of cancer cells. However, it is now realized that one of their major physiological roles is cellular lipid transport and homeostasis, and their dysfunction is often associated with human diseases. ABCA1 and ABCA7 mediate the apolipoprotein-dependent formation of a high-density lipoprotein-cholesterol complex. ABCA3 is indispensable for pulmonary surfactant secretion. ABCG5 and ABCG8 are involved in the secretion of plant sterols and cholesterol into bile. However, the primary substrates and mechanism of action of these ABC proteins have not been precisely defined. In this review article, we first describe the general structure and functions of eukaryotic ABC proteins. The current model of ABCA1 functionality is then explained based on studies on a topological model, subcellular localization, apoA-I dependence of HDL formation, functional defects of Tangier disease mutants, and ATP hydrolysis of purified ABCA1. ABCA1 is supposed to function as a transporter of lipids as well as a receptor for apoA-I. ABCA3 is likely involved in accumulating phospholipids and cholesterol in lamellar bodies and in generating multivesicular structures.

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90 Many mutations in patients with TD and FHA have been identified in ECD1 of ABCA1, and three mutations (R587W, W590S, Q597R) cluster amino acids 587 to 59746,48,72 (see Fig. 2). Login to comment
91 When these three TD mutations were introduced into ECD1 of ABCA1-GFP and the mutants were transiently or stably expressed in HEK293, R587W and Q597R appeared to be distributed mainly in the ER and not the plasma membrane. Login to comment

[hide] Effects of oxidized low density lipoprotein on the... J Huazhong Univ Sci Technolog Med Sci. 2005;25(2):113-6. Li Y, Bi H, Wu F, Zong Y, Wang Y, Qu S
Effects of oxidized low density lipoprotein on the expression and function of ABCA1 in macrophages.
J Huazhong Univ Sci Technolog Med Sci. 2005;25(2):113-6., [PMID:16116948]

Abstract [show]
In the present study, we examined the regulation of the expression and function of ABCA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-I for 24 h, RAW264.7 cells effluxed 37.65% cholesterol loaded by acetyl LDL (ac-LDL), and 9.78% cholesterol in ox-LDL group. The level of ABCA1 mRNA increased about three times either when cells were incubated with -100 microg /mL ac-LDL or with 100 microg /mL ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.

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102 The gene examination of Tangier patients showed that the gene mutation of R587W and Q597R could result in the localized dysfunction of ABCA1, accumulation of cholesterol in macrophages, and the occurrence of AS. Login to comment

[hide] Familial HDL deficiency due to ABCA1 gene mutation... Atherosclerosis. 2004 Feb;172(2):309-20. Pisciotta L, Hamilton-Craig I, Tarugi P, Bellocchio A, Fasano T, Alessandrini P, Bon GB, Siepi D, Mannarino E, Cattin L, Averna M, Cefalu AB, Cantafora A, Calandra S, Bertolini S
Familial HDL deficiency due to ABCA1 gene mutations with or without other genetic lipoprotein disorders.
Atherosclerosis. 2004 Feb;172(2):309-20., [PMID:15019541]

Abstract [show]
Mutations in ABCA1 have been shown to be the cause of Tangier disease (TD) and some forms of familial hypoalphalipoproteinemia (HA), two genetic disorders characterized by low plasma HDL levels. Here we report six subjects with low HDL, carrying seven ABCA1 mutations, six of which are previously unreported. Two mutations (R557X and H160FsX173) were predicted to generate short truncated proteins; two mutations (E284K and Y482C) were located in the first extracellular loop and two (R1901S and Q2196H) in the C-terminal cytoplasmic domain of ABCA1. Two subjects found to be compound heterozygotes for ABCA1 mutations did not have overt clinical manifestations of TD. Three subjects, all with premature coronary artery disease (pCAD), had a combination of genetic defects. Besides being heterozygotes for ABCA1 mutations, two of them were also carriers of the R3500Q substitution in apolipoprotein B and the third was a carrier of N291S substitution in lipoprotein lipase. By extending family studies we identified 17 heterozygotes for ABCA1 mutations. Plasma HDL-C and Apo A-I values in these subjects were 38.3 and 36.9% lower than in unaffected family members and similar to the values found in heterozygotes for Apo A-I gene mutations which prevent Apo A-I synthesis. This survey underlines the allelic heterogeneity of ABCA1 mutations and suggests that: (i) TD subjects, if asymptomatic, may be overlooked and (ii) there may be a selection bias in genotyping towards carriers of ABCA1 mutations who have pCAD possibly related to a combination of genetic and environmental cardiovascular risk factors.

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204 The E284K and the Y482C are located in the first extracellular loop of ABCA1, where they may interfere with the binding to Apo A-I and/or the membrane release of phospholipids, as it has been demonstrated for other mutations (R587W and Q597R) located in the same extracellular domain [40,41]. Login to comment
203 The E284K and the Y482C are located in the first extracellular loop of ABCA1, where they may interfere with the binding to Apo A-I and/or the membrane release of phospholipids, as it has been demonstrated for other mutations (R587W and Q597R) located in the same extracellular domain [40,41]. Login to comment

[hide] Posttranscriptional regulation of human ABCA7 and ... Biochem Biophys Res Commun. 2003 Nov 14;311(2):313-8. Ikeda Y, Abe-Dohmae S, Munehira Y, Aoki R, Kawamoto S, Furuya A, Shitara K, Amachi T, Kioka N, Matsuo M, Yokoyama S, Ueda K
Posttranscriptional regulation of human ABCA7 and its function for the apoA-I-dependent lipid release.
Biochem Biophys Res Commun. 2003 Nov 14;311(2):313-8., [PMID:14592415]

Abstract [show]
ABCA7 is expressed predominantly in myelo-lymphatic tissues or reticuloendothelial cells. Physiological role and function of this protein are not fully understood. We isolated the full-length cDNA (type I) and a splicing variant cDNA (type II) of human ABCA7, and developed monoclonal antibodies against extracellular domain (ECD)1 of ABCA7. RT-PCR experiments suggested that human ABCA7 gene produced the type II mRNA in a tissue-specific manner. Immunostaining revealed that the type I ABCA7, expressed in HEK293 cells, was localized to the plasma membrane and ECD1 was exposed to the extracellular space as was the case for ABCA1. HEK293 cells expressing type I ABCA7 showed apoA-I-dependent cholesterol and phospholipid release. In contrast, type II ABCA7 appeared to be localized mainly in endoplasmic reticulum and did not show apoA-I-dependent cholesterol and phospholipid release. Alternative splicing could be involved in the post-transcriptional regulation of the expression and function of human ABCA7.

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15 In fact, the three different mutants in ECD1 associated with TD, R587W, W590S, and Q597R, all had reduced apoA-I-mediated lipid release and subsequent HDL assembly when expressed in HEK293 [12-14]. Login to comment

[hide] Efflux and atherosclerosis: the clinical and bioch... Arterioscler Thromb Vasc Biol. 2003 Aug 1;23(8):1322-32. Epub 2003 May 22. Singaraja RR, Brunham LR, Visscher H, Kastelein JJ, Hayden MR
Efflux and atherosclerosis: the clinical and biochemical impact of variations in the ABCA1 gene.
Arterioscler Thromb Vasc Biol. 2003 Aug 1;23(8):1322-32. Epub 2003 May 22., [PMID:12763760]

Abstract [show]
Approximately 50 mutations and many single nucleotide polymorphisms have been described in the ABCA1 gene, with mutations leading to Tangier disease and familial hypoalphalipoproteinemia. Homozygotes and heterozygotes for mutations in ABCA1 display a wide range of phenotypes. Identification of ABCA1 as the molecular defect in these diseases has allowed for ascertainment based on genetic status and determination of genotype-phenotype correlations and has permitted us to identify mutations conferring a range of severity of cellular, biochemical, and clinical phenotypes. In this study we review how genetic variation at the ABCA1 locus affects its role in the maintenance of lipid homeostasis and the natural progression of atherosclerosis.

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76 Additional insights into how the mutations R587W, W590S, and Q597R that occur in the extracellular loops affect ABCA1 function have recently been described.58-60 Two studies have reported that ABCA1 containing the point mutation Q597R, which occurs in the first extracellular loop, does not localize to the plasma membrane.59,60 However, other studies have reported that this mutant is expressed at the plasma membrane but at reduced levels relative to wild-type ABCA1.58,44 R587W, another missense mutation in the first extracellular loop, also prevents the trafficking of ABCA1 to the plasma membrane, although results with this mutant have been variable.58-60 Both the R587W and Q597R mutants are resistant to PNGase digestion, indicating that they are not glycosylated, suggesting that ABCA1 harboring these mutations does not traverse the medial and trans Golgi network. Login to comment
83 TABLE 2. Conservation of Amino Acid Residues Mutated in Humans Mutation H. sapiens M. musculus G. gallus D. melanogaster C. elegans P85L P P P ⅐ ⅐ ⅐ P R230C R R R P G A255T A A S ⅐ ⅐ ⅐ ⅐ ⅐ ⅐ R587W R R R ⅐ ⅐ ⅐ ⅐ ⅐ ⅐ W590S W W W R Q Q597R Q Q Q Q Q ⌬L693 L L L L L T929I T T T T T N935S/H N N N N N A937V A A A A A A1046D A A A A A M1091T M M M M M D1099Y D D D D D D1289L/N D D D D D C1477R C C C ⅐ ⅐ ⅐ ⅐ ⅐ ⅐ S1506L S S S ⅐ ⅐ ⅐ ⅐ ⅐ ⅐ N1611D N N N N S R1680W R R R R R N1800H N N N A W F2009S F F F I M R2081W R R R R R P2150L P P P R N ⌬E1893 E E E D S ⌬D1894 D D D D D Twenty-three of 24 (95.83%) amino acids affected by mutations are conserved with G. gallus, reflecting the functional importance of these residues. Login to comment
68 Additional insights into how the mutations R587W, W590S, and Q597R that occur in the extracellular loops affect ABCA1 function have recently been described.58-60 Two studies have reported that ABCA1 containing the point mutation Q597R, which occurs in the first extracellular loop, does not localize to the plasma membrane.59,60 However, other studies have reported that this mutant is expressed at the plasma membrane but at reduced levels relative to wild-type ABCA1.58,44 R587W, another missense mutation in the first extracellular loop, also prevents the trafficking of ABCA1 to the plasma membrane, although results with this mutant have been variable.58-60 Both the R587W and Q597R mutants are resistant to PNGase digestion, indicating that they are not glycosylated, suggesting that ABCA1 harboring these mutations does not traverse the medial and trans Golgi network. Login to comment
75 TABLE 2. Conservation of Amino Acid Residues Mutated in Humans Mutation H. sapiens M. musculus G. gallus D. melanogaster C. elegans P85L P P P ዼ ዼ ዼ P R230C R R R P G A255T A A S ዼ ዼ ዼ ዼ ዼ ዼ R587W R R R ዼ ዼ ዼ ዼ ዼ ዼ W590S W W W R Q Q597R Q Q Q Q Q èc;L693 L L L L L T929I T T T T T N935S/H N N N N N A937V A A A A A A1046D A A A A A M1091T M M M M M D1099Y D D D D D D1289L/N D D D D D C1477R C C C ዼ ዼ ዼ ዼ ዼ ዼ S1506L S S S ዼ ዼ ዼ ዼ ዼ ዼ N1611D N N N N S R1680W R R R R R N1800H N N N A W F2009S F F F I M R2081W R R R R R P2150L P P P R N èc;E1893 E E E D S èc;D1894 D D D D D Twenty-three of 24 (95.83%) amino acids affected by mutations are conserved with G. gallus, reflecting the functional importance of these residues. Login to comment

[hide] Genetics of HDL regulation in humans. Curr Opin Lipidol. 2003 Jun;14(3):273-9. Miller M, Rhyne J, Hamlette S, Birnbaum J, Rodriguez A
Genetics of HDL regulation in humans.
Curr Opin Lipidol. 2003 Jun;14(3):273-9., [PMID:12840658]

Abstract [show]
PURPOSE OF REVIEW: To review gene regulation of HDL-cholesterol and discuss molecular abnormalities in HDL candidate genes that may lead to human pathologic states. RECENT FINDINGS: The inverse association between HDL-cholesterol and vascular disease, especially coronary heart disease, has long been recognized, but understanding gene regulation of HDL in humans gained considerable momentum following the identification of ABCA1 as playing a pivotal role in reverse cholesterol transport. Recent data suggest that potentially important targets for upregulating HDL in humans include upregulators of ABCA1 and APOA1 (e.g. peroxisome proliferator activated receptor and liver X receptor agonists) and downregulators of CETP (e.g. JTT-705). A host of other nuclear receptors under investigation in animal models may advance to human testing in the near future. SUMMARY: Disorders affecting HDL metabolism are complex because monogenic disorders causing low HDL do not necessarily correlate with premature vascular disease. To date, pathologic phenotypes have only been deduced among several HDL candidate genes. Understanding the genetic underpinnings associated with variant HDL and reverse cholesterol transport provides an exceptional opportunity to identify novel agents that may optimize this process and reduce vascular event rates beyond currently available LDL lowering therapies.

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66 TD 1591 T/C 11 V399A extracellular [68] TD 1979 (110bpAlu Ins) 12 truncated truncation [60] TD/FHA 2154 C/T 14 R587W extracellular [67,69] TD 2164 G/C 14 W590S extracellular [61] TD 2185 A/G 14 Q597R extracellular [59,67] TD 2219 G/del 14 truncated, 635X truncated [60,61] FHA 2472-2474 3bp del 15 Del L693 TM domain #3 [59] phosphorylation 2706 G/A 16 V771M extracellular [68] 2715 A/C 16 T774P extracellular [68] 2723 G/C 16 K776N extracellular [68] 2868 G/A 17 V825I TM domain #6 [67,68] TD/FHA 3044 A/G 18 I883M cytoplasmic [68] phosphorylat site FHA 3120 C/T 19 R909X truncation [63,71] TD 3181 C/T 19 T929I cytoplasmic [62] TD 3199 A/G 19 N935S Walker A [61] TD 3205 C/T 19 A937V Walker A [61] TD 3532 C/A 22 A1046D cytoplasmic, Walker A/B [70] FHA 3667 T/C 23 M1091T cytoplasmic [63] 3690 G/T 23 D1099Y cytoplasmic [9] TD 3738 2bp del 23 1145X truncation [66] FHA 3911 G/C 24 E1172D linker/cytoplasmic [68] FHA 4242 4bp del 27 1297X truncated [64] TD 4260 G/A 27 D1289N linker cytoplasm [64,65] TD 4824 T/C 31 C1477R extracellular [59] TD 4912 C/T 32 S1506L extracellular loop #2 [71] TD 5025 ins A 34 A1544S?1552X truncation [70] 5059 T/C 34 I1555T extracellular loop #2 [67] 5155 G/A 35 R1587K extracellular loop #2 [68] FHA 5226 A/G 36 N1611D extracellular loop #2 [75..] 5338 T/C 36 L1648P extracellular loop #2 [67] TD 5443 C/T 37 R1680W cytoplasmic [74.] Login to comment

[hide] Cellular phospholipid and cholesterol efflux in hi... Circulation. 2003 Mar 18;107(10):1366-71. Marcil M, Bissonnette R, Vincent J, Krimbou L, Genest J
Cellular phospholipid and cholesterol efflux in high-density lipoprotein deficiency.
Circulation. 2003 Mar 18;107(10):1366-71., [PMID:12642355]

Abstract [show]
BACKGROUND: Prospective studies have examined the relationship between coronary artery disease and low plasma levels of high-density lipoprotein cholesterol (HDL-C). METHODS AND RESULTS: We investigated the causes of hypoalphalipoproteinemia (HypoA; HDL-C <5th percentile) in 64 subjects (12 women and 52 men). Apolipoprotein AI-mediated cellular cholesterol and phospholipid efflux were measured in fibroblasts from HypoA subjects, 9 controls, 2 patients with Tangier disease, and 5 patients with hyperalphalipoproteinemia. A phospholipid efflux defect was defined as <70% of controls. Mean HDL-C was 0.49+/-0.21 mmol/L. Cholesterol and phospholipid efflux correlated strongly (r=0.72, P<0.001). Phospholipid efflux and HDL-C (r=0.64, P<0.001) correlated in HypoA subjects. However, phospholipid or cholesterol efflux was no longer a determinant of HDL-C levels at higher levels (> approximately 1.0 mmol/L) of HDL-C. In HypoA subjects, 4 cases of Tangier disease and 6 of familial HDL deficiency (heterozygous Tangier disease) were identified (10 of 64; 16%). In the remaining 54 subjects, mean lipid efflux was not significantly different from controls and subjects with hyperalphalipoproteinemia. A phospholipid efflux defect was identified in 7 additional HypoA subjects, and a cholesterol efflux defect was detected in 11 subjects. In 2 of these subjects, the ABCA1 gene was ruled out as the cause of the efflux defect, while in 3, the low HDL-C trait segregated with the ABCA1 gene locus. CONCLUSIONS: Lipidation of lipid-poor apolipoprotein AI may not be a major determinant of cholesterol accumulation within more mature HDL particles and increasing cholesterol or phospholipid efflux beyond normal levels may not lead to increase in plasma HDL-C levels. ABCA1 is essential in the initial steps of HDL formation but other plasma events are major modulators of HDL-C levels.

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85 Molecular Characterization of ABCA1 Gene in Study Subjects Cell Lines HDL-C, mmol/L Nucleotide Change Predicted Protein Alteration TD CTL-1 0.10 Exon 30 T4369C; exon 24 splice site G3C C1477R; part of the transcript deleted TD CTL-2 0.15 Exon 13 A1730G Q597R FHD-1 0.40 Exon 14 ⌬2017-9 ⌬L693 FHD-2 0.18 Exon 48 C6370T R2144X FHD-3 0.39 Exon 41 ⌬5618-23 ⌬ED1893,4 FHD-4 0.18 Exon 18 C2665T R909X FHD-5 0.10 Exon 23 T3667C M1091T FHD-6 0.57 Exon 49 C6844T P2150L, R587W TD-1 0.03 Exon 48 ⌬C6370; ND 2145X TD-2 0.07 ND ND TD-3 0.03 ND 2203X TD-4 0.09 Exon 19 C3181T; ND T929I; ND CTL indicates control. Login to comment
79 Molecular Characterization of ABCA1 Gene in Study Subjects Cell Lines HDL-C, mmol/L Nucleotide Change Predicted Protein Alteration TD CTL-1 0.10 Exon 30 T4369C; exon 24 splice site G3C C1477R; part of the transcript deleted TD CTL-2 0.15 Exon 13 A1730G Q597R FHD-1 0.40 Exon 14 èc;2017-9 èc;L693 FHD-2 0.18 Exon 48 C6370T R2144X FHD-3 0.39 Exon 41 èc;5618-23 èc;ED1893,4 FHD-4 0.18 Exon 18 C2665T R909X FHD-5 0.10 Exon 23 T3667C M1091T FHD-6 0.57 Exon 49 C6844T P2150L, R587W TD-1 0.03 Exon 48 èc;C6370; ND 2145X TD-2 0.07 ND ND TD-3 0.03 ND 2203X TD-4 0.09 Exon 19 C3181T; ND T929I; ND CTL indicates control. Login to comment

[hide] Effects of mutations of ABCA1 in the first extrace... J Biol Chem. 2003 Mar 7;278(10):8815-9. Epub 2002 Dec 31. Tanaka AR, Abe-Dohmae S, Ohnishi T, Aoki R, Morinaga G, Okuhira K, Ikeda Y, Kano F, Matsuo M, Kioka N, Amachi T, Murata M, Yokoyama S, Ueda K
Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.
J Biol Chem. 2003 Mar 7;278(10):8815-9. Epub 2002 Dec 31., [PMID:12509412]

Abstract [show]
ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha-(32)P]ATP was stimulated by adding ortho-vanadate in the presence of Mn(2+) as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.

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1 The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. Login to comment
5 R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Login to comment
8 These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis. Login to comment
20 Three TD mutants (R587W, W590S, Q597R), clustered in ECD1, were examined in the present report. Login to comment
24 On the other hand, the two mutants R587W and Q597R were only partially or scarcely localized to the PM, whereas W590S * This work was supported by Grant-in-aid for Scientific Research 10217205 on Priority Areas "ABC Proteins" from the Ministry of Education, Science, Sports, and Culture of Japan and by the Nakajima Foundation. Login to comment
44 DNA Construction-DNA fragments (XhoI-BclI) containing each missense TD mutation (R587W, W590S, or Q597R) were generated using the polymerase chain reaction method with R587W (XhoI) primer (5Ј-GTCCTCGAGCTGACCCCTTTGAGGACATGTGGTACGTC-3Ј), W590S (XhoI) primer (5Ј-GTCCTCGAGCTGACCCCTTTGAGGACAT- GCGGTACGTCTCGGGGGGCTTC-3Ј), or Q597 (XhoI) primer (5Ј-GT- CCTCGAGCTGACCCCTTTGAGGACATGCGGTACGTCTGGGGGGG- CTTCGCCTACTTGCGGGATGTGGTG-3Ј), where the mutated nucleotide is underlined, and BclI primer (5Ј-CGATGCCCTTGATGATCACA- GCCACTGAG-3Ј). Login to comment
47 In brief, 10 ␮g of membrane proteins from HEK293 cells stably expressing the wild-type, R587W, W590S, or Q597R ABCA1-GFP were treated with 500 units of Endo H or 0.3 units of PNGaseF for 1 h at 37 °C. Login to comment
64 Effects of ECD1 Mutations on Subcellular Localization of ABCA1-GFP-Many mutations in patients with TD and FHA have been identified in ECD1 of ABCA1, and three mutations (R587W, W590S, Q597R) cluster in the vicinity between amino acids 587 and 597 (20)(Fig. 2A). Login to comment
68 Confocal microscopic examination revealed that R587W and Q597R appeared to be localized mainly in the ER and not to the PM (Fig. 2B). Login to comment
70 Immunostaining with the antibody against ECD1 confirmed the proper orientation of W590S (Fig. 2C). Login to comment
71 Glycosylation of ABCA1-GFP-Glycosylation of the wild-type ABCA1-GFP and its mutants R587W, W590S, and Q597R was examined by the treatment with PNGaseF and Endo H (Fig. 3A). Login to comment
74 ABCA1 with TD mutations, R587W and Q597R ABCA1-GFP, was sensitive to Endo H to produce the deglycosylated form of ABCA1-GFP, whereas the wild-type ABCA1-GFP was little digested by Endo H. Login to comment
75 These results indicated that R587W and Q597R ABCA1-GFP did not contain complex oligosaccharides and supported the confocal microscopy observation, which suggested the localization of these two TD mutants in the ER or the cis-Golgi complex. Login to comment
84 The R587W mutation resulted in the apoA-I-mediated release of cholesterol and choline-phospholipids to 24 and 23% of the wild-type ABCA1-GFP, respectively. Login to comment
102 A, a putative secondary structure of ABCA1 and localization of Tangier Disease mutations R587W, W590S, and Q597R in ECD1. Login to comment
103 B, GFP fluorescence of HEK293 cells stably expressing the wild-type (WT) ABCA1-GFP and three TD mutants R587W, W590S, and Q597R ABCA1-GFP. Login to comment
107 The wild-type (WT), R587W, W590S, and Q597R ABCA1-GFP were treated with none (-), Endo H (H), or PNGaseF (F) and separated with 7% SDS-PAGE. Login to comment
111 Cholesterol (A) and choline-phospholipid (B) content in the medium in a 6-well plate containing HEK293 cells stably expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP were measured after a 24-h incubation in the presence (black bars) or absence (white bars) of 10 ␮g/ml apoA-I. The relative amount of cholesterol (C) and choline-phospholipid (D) in the medium in a 6-well plate containing HEK293 cells transiently expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP was measured after a 24-h incubation in the presence of 10 ␮g/ml apoA-I. The expression levels of mutants were normalized with the GFP fluorescence of cells. Login to comment
119 ABCA1-GFP with a R587W or Q597R mutation appeared to be impaired with intracellular trafficking and predominantly localized in the ER. Login to comment
122 R587W and Q597 ABCA1-GFP contained high mannose oligosaccharides, indicating that they do not reach the trans-Golgi complex. Login to comment
125 R587W and Q597R ABCA1-GFP appeared to be retained in the ER. Login to comment
127 This region (R587 to Q597) in ECD1 would be critical for proper folding of ABCA1 and would probably affect the intracellular translocation process, whereas the W590S mutation does not. Login to comment
128 Fitzgerald et al. (17) reported that R587W or Q597R mutation did not affect the PM localization but disrupted the direct interaction with ApoA-I. This supports a major conformational alteration of ECD1 by these mutations. Login to comment
154 Interestingly, clinical manifestations of these mutations are apparently different (20): R587W is associated with coronary heart disease, whereas W590S is associated with splenomegaly. Login to comment
155 In this study, we demonstrated that the defect of HDL assembly in R587W and Q597R is due to the impaired localization of ABCA1 to the PM. Login to comment
67 Confocal microscopic examination revealed that R587W and Q597R appeared to be localized mainly in the ER and not to the PM (Fig. 2B). Login to comment
73 ABCA1 with TD mutations, R587W and Q597R ABCA1-GFP, was sensitive to Endo H to produce the deglycosylated form of ABCA1-GFP, whereas the wild-type ABCA1-GFP was little digested by Endo H. Login to comment
83 The R587W mutation resulted in the apoA-I-mediated release of cholesterol and choline-phospholipids to 24 and 23% of the wild-type ABCA1-GFP, respectively. Login to comment
101 A, a putative secondary structure of ABCA1 and localization of Tangier Disease mutations R587W, W590S, and Q597R in ECD1. Login to comment
106 The wild-type (WT), R587W, W590S, and Q597R ABCA1-GFP were treated with none (afa;), Endo H (H), or PNGaseF (F) and separated with 7% SDS-PAGE. Login to comment
110 Cholesterol (A) and choline-phospholipid (B) content in the medium in a 6-well plate containing HEK293 cells stably expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP were measured after a 24-h incubation in the presence (black bars) or absence (white bars) of 10 òe;g/ml apoA-I. The relative amount of cholesterol (C) and choline-phospholipid (D) in the medium in a 6-well plate containing HEK293 cells transiently expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP was measured after a 24-h incubation in the presence of 10 òe;g/ml apoA-I. The expression levels of mutants were normalized with the GFP fluorescence of cells. Login to comment
118 ABCA1-GFP with a R587W or Q597R mutation appeared to be impaired with intracellular trafficking and predominantly localized in the ER. Login to comment
121 R587W and Q597 ABCA1-GFP contained high mannose oligosaccharides, indicating that they do not reach the trans-Golgi complex. Login to comment
124 R587W and Q597R ABCA1-GFP appeared to be retained in the ER. Login to comment
153 Interestingly, clinical manifestations of these mutations are apparently different (20): R587W is associated with coronary heart disease, whereas W590S is associated with splenomegaly. Login to comment

[hide] Abnormal splicing of ABCA1 pre-mRNA in Tangier dis... J Lipid Res. 2003 Feb;44(2):254-64. Epub 2002 Nov 4. Altilia S, Pisciotta L, Garuti R, Tarugi P, Cantafora A, Calabresi L, Tagliabue J, Maccari S, Bernini F, Zanotti I, Vergani C, Bertolini S, Calandra S
Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene.
J Lipid Res. 2003 Feb;44(2):254-64. Epub 2002 Nov 4., [PMID:12576507]

Abstract [show]
Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2-/mRNA), exon 4 (Ex4-/mRNA), or both these exons (Ex2-/Ex4-/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.

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158 The cholesterol efflux in proband`s fibroblasts was similar to that observed in another patient with TD (homozygous for R587W in ABCA1 gene) we have previously reported (13) (Fig. 2). Login to comment
179 Skin fibroblasts from a control subject, proband I.1 of Family 1 (IVS2 ϩ5GϾC/ R282X), and another TD patient (homozygous for R587W) reported previously (13) were labeled with [3H]cholesterol and then incubated in the presence or in the absence of 22-hydroxycholesterol and 9-cis- retinoic acid (22ch/RA). Login to comment
157 The cholesterol efflux in proband`s fibroblasts was similar to that observed in another patient with TD (homozygous for R587W in ABCA1 gene) we have previously reported (13) (Fig. 2). Login to comment
178 Skin fibroblasts from a control subject, proband I.1 of Family 1 (IVS2 5GC/ R282X), and another TD patient (homozygous for R587W) reported previously (13) were labeled with [3H]cholesterol and then incubated in the presence or in the absence of 22-hydroxycholesterol and 9-cis-retinoic acid (22ch/RA). Login to comment

[hide] Distinct sites on ABCA1 control distinct steps req... J Lipid Res. 2002 Dec;43(12):2077-86. Rigot V, Hamon Y, Chambenoit O, Alibert M, Duverger N, Chimini G
Distinct sites on ABCA1 control distinct steps required for cellular release of phospholipids.
J Lipid Res. 2002 Dec;43(12):2077-86., [PMID:12454269]

Abstract [show]
The loss of ABCA1 function leads to Tangier dyslipidemia in humans and to a Tangier-like phenotype in mice, by impairing the transformation of nascent apolipoproteins into mature HDL particles. Mechanistically this ensues from the inability of cells to release membrane lipids and cholesterol. Whereas the ability of ABCA1 to promote phospholipid effluxes, surface binding of apolipoproteins and outward flip of membrane lipids has been documented, the relationship between this series of ABCA1-dependent events is still elusive. Here we provide evidence that i) lipid effluxes require both flip of membrane lipids and binding of apolipoproteins to the cell surface, ii) apolipoprotein A-I binding depends on structural determinants on ABCA1, and iii) phospholipid effluxes can be modulated by engineered mutations on the structural determinants identified on ABCA1.

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140 Three point mutations in the region 580-600 had been reported in Tangier pedigrees (namely R587W, W590S, and Q597R). Login to comment
148 This was virtually complete in the case of W590S, Q597R, and ⌬L693, and reduced to one fourth for R587W and C1477R (Table 3). Login to comment
170 The last category is illustrated by R587W and W590S Tangier transporters, which are correctly targeted to the plasma membrane, but show a marked functional dissociation. Login to comment
171 Indeed, these variants, while eliciting an apoA-I binding indistinguishable from wild-type ABCA1 (79% Ϯ 5, n ϭ 7, P Ͼ 0.05 and 126% Ϯ 18, n ϭ 6, P Ͼ 0.05 of wild type, respectively) fail to drive both flipping of PS (annexin V binding ϭ 39% Ϯ 11of wild type for R587W, n ϭ 5, P Ͻ 0.05 and 30% Ϯ 9 for W590S, n ϭ 7, P Ͻ 0.01) and membrane release of PL (27% Ϯ 16, n ϭ 3, P Ͻ 0.05 and 16% Ϯ 3, n ϭ 2, P Ͻ 0.01 of wild type, respectively, Table 3), thus indicating that apoA-I binding per se is insufficient for the generation of PL effluxes, which also requires PS flipping. Login to comment
205 Morphological and functional evaluation of Tangier- associated ABCA1 variants Identity SL AnnV St ApoA-I St PL Efflux St % % % R587W PM 39 Ϯ 11(5) a 79 Ϯ 5 (7) a 27 Ϯ 16 (3) a W590S PM 30 Ϯ 9 (7) b 126 Ϯ 18 (6) ns 16 Ϯ 3 (2) b Q597R ER, PM 24 Ϯ 9 (4) b 15 Ϯ 8 (4) c 8 Ϯ 7 (2) b ⌬L693 ER 26 Ϯ 11 (5) a 12 Ϯ 6 (4) c nd C1477R PM 53 Ϯ 12 (9) ns 33 Ϯ 9 (6) b 12 Ϯ 2 (2) b HA819/1466 PM 54 Ϯ 12 (6) ns 49 Ϯ 7 (6) a 108 Ϯ 28 (4) b HA819/C1477R PM 57 Ϯ 15 (4) ns 68 Ϯ 6 (4) a 142 Ϯ 24 (2) b SL, subcellular localization as detected by confocal imaging and confirmed by surface biotynilation; AnnV and ApoA-I, binding of annexin V or apoA-I in cells successfully transfected with the test construct (GFP positive); St, statistical significance. Login to comment
139 We deliberately excluded mutations in the nucleotide binding folds, i.e., those expected to impair function by interference with the ATPase activity, and conversely selected the mutations located in the extracellular region defined by the topological model proposed in Fig. 4A. Three point mutations in the region 580-600 had been reported in Tangier pedigrees (namely R587W, W590S, and Q597R). Login to comment
147 This was virtually complete in the case of W590S, Q597R, and L693, and reduced to one fourth for R587W and C1477R (Table 3). Login to comment
169 The last category is illustrated by R587W and W590S Tangier transporters, which are correctly targeted to the plasma membrane, but show a marked functional dissociation. Login to comment
204 Morphological and functional evaluation of Tangier-associated ABCA1 variants Identity SL AnnV St ApoA-I St PL Efflux St % % % R587W PM 39 11(5) a 79 5 (7) a 27 16 (3) a W590S PM 30 9 (7) b 126 18 (6) ns 16 3 (2) b Q597R ER, PM 24 9 (4) b 15 8 (4) c 8 7 (2) b L693 ER 26 11 (5) a 12 6 (4) c nd C1477R PM 53 12 (9) ns 33 9 (6) b 12 2 (2) b HA819/1466 PM 54 12 (6) ns 49 7 (6) a 108 28 (4) b HA819/C1477R PM 57 15 (4) ns 68 6 (4) a 142 24 (2) b SL, subcellular localization as detected by confocal imaging and confirmed by surface biotynilation; AnnV and ApoA-I, binding of annexin V or apoA-I in cells successfully transfected with the test construct (GFP positive); St, statistical significance. Login to comment

[hide] Naturally occurring mutations in the largest extra... J Biol Chem. 2002 Sep 6;277(36):33178-87. Epub 2002 Jun 25. Fitzgerald ML, Morris AL, Rhee JS, Andersson LP, Mendez AJ, Freeman MW
Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I.
J Biol Chem. 2002 Sep 6;277(36):33178-87. Epub 2002 Jun 25., [PMID:12084722]

Abstract [show]
The ABCA1 transporter contains two large domains into which many of the genetic mutations in individuals with Tangier disease fall. To investigate the structural requirements for the cellular cholesterol efflux mediated by ABCA1, we have determined the topology of these two domains and generated transporters harboring five naturally occurring missense mutations in them. These mutants, unlike wild type ABCA1, produced little or no apoA-I-stimulated cholesterol efflux when transfected into 293 cells, establishing their causality in Tangier disease. Because all five mutant proteins were well expressed and detectable on the plasma membrane, their interaction with the ABCA1 ligand, apolipoprotein (apo) A-I, was measured using bifunctional cross-linking agents. Four of five mutants had a marked decline in cross-linking to apoA-I, whereas one (W590S) retained full cross-linking activity. Cross-linking of apoA-I was temperature-dependent, rapid in onset, and detectable with both lipid- and water-soluble cross-linking agents. These results suggest that apoA-I-stimulated cholesterol efflux cannot occur without a direct interaction between the apoprotein and critical residues in two extracellular loops of ABCA1. The behavior of the W590S mutant indicates that although binding of apoA-I by ABCA1 may be necessary, it is not sufficient for stimulation of cholesterol efflux.

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39 DNA Constructs-Five missense mutants of ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) were generated using overlap polymerase chain reaction methods, as described previously (17). Login to comment
70 Missense Mutations in Two Putative Extracellular Loops of ABCA1 Ablate Efflux Activity-Five missense mutations (R587W, W590S, Q597R, C1477R, and S1506L) were introduced into a wild type ABCA1 cDNA using PCR mutagenesis techniques. Login to comment
71 Three of these mutations (R587W, W590S, and Q597R) fall in a tight cluster within the large N-terminal loop at a point near the putative second transmembrane domain shown in Fig. 3. Login to comment
116 The cells were transfected with either empty vector (mock), wild type ABCA1 (WT), or ABCA1 constructs carrying the indicated point mutations (R587W, W590S, Q597R, C1477R, and S1506L). Login to comment
119 Absolute apoA-I and medium efflux values, respectively, are as follows: mock, 1.59 Ϯ 0.04% versus 1.21 Ϯ 0.39%; WT, 3.92 Ϯ 0.13% versus 1.9 Ϯ 0.08%; R587W, 1.78 Ϯ 0.11% versus 1.61 Ϯ 0.24%; W590S, 1.92 Ϯ 0.24% versus 1.63 Ϯ 0.08%; Q597R, 1.5 Ϯ 0.14% versus 1.49 Ϯ 0.03%; C1477R, 1.67 Ϯ 0.18% versus 1.52 Ϯ 0.15%; and S1506L, 1.66 Ϯ 0.28% versus 1.6 Ϯ 0.13%. Login to comment
199 The R587W mutant showed an intermediate phenotype, with cross-linking efficiency reduced ϳ50%. Login to comment
202 DISCUSSION In this study, we have established that several naturally occurring missense mutations in ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) located in the two largest loop domains of the protein (comprising amino acids ϳ44-640 and ϳ1371-1649, respectively) are, in fact, loss-of-function mutations. Login to comment
212 Although three of the mutations (Q597R, C1477R, and S1506L) showed no appreciable cross-linking to apoA-I, the R587W mutant had an intermediate activity, and the W590S mutant retained full, if not enhanced, cross-linking to the apoprotein. Login to comment
37 DNA Constructs-Five missense mutants of ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) were generated using overlap polymerase chain reaction methods, as described previously (17). Login to comment
67 Missense Mutations in Two Putative Extracellular Loops of ABCA1 Ablate Efflux Activity-Five missense mutations (R587W, W590S, Q597R, C1477R, and S1506L) were introduced into a wild type ABCA1 cDNA using PCR mutagenesis techniques. Login to comment
68 Three of these mutations (R587W, W590S, and Q597R) fall in a tight cluster within the large N-terminal loop at a point near the putative second transmembrane domain shown in Fig. 3. Login to comment
112 The cells were transfected with either empty vector (mock), wild type ABCA1 (WT), or ABCA1 constructs carrying the indicated point mutations (R587W, W590S, Q597R, C1477R, and S1506L). Login to comment
115 Absolute apoA-I and medium efflux values, respectively, are as follows: mock, 1.59 afe; 0.04% versus 1.21 afe; 0.39%; WT, 3.92 afe; 0.13% versus 1.9 afe; 0.08%; R587W, 1.78 afe; 0.11% versus 1.61 afe; 0.24%; W590S, 1.92 afe; 0.24% versus 1.63 afe; 0.08%; Q597R, 1.5 afe; 0.14% versus 1.49 afe; 0.03%; C1477R, 1.67 afe; 0.18% versus 1.52 afe; 0.15%; and S1506L, 1.66 afe; 0.28% versus 1.6 afe; 0.13%. Login to comment
191 The R587W mutant showed an intermediate phenotype, with cross-linking efficiency reduced b03;50%. Login to comment
194 DISCUSSION In this study, we have established that several naturally occurring missense mutations in ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) located in the two largest loop domains of the protein (comprising amino acids b03;44-640 and b03;1371-1649, respectively) are, in fact, loss-of-function mutations. Login to comment
204 Although three of the mutations (Q597R, C1477R, and S1506L) showed no appreciable cross-linking to apoA-I, the R587W mutant had an intermediate activity, and the W590S mutant retained full, if not enhanced, cross-linking to the apoprotein. Login to comment

[hide] A point mutation in ABC1 gene in a patient with se... Atherosclerosis. 2001 Feb 15;154(3):599-605. Bertolini S, Pisciotta L, Seri M, Cusano R, Cantafora A, Calabresi L, Franceschini G, Ravazzolo R, Calandra S
A point mutation in ABC1 gene in a patient with severe premature coronary heart disease and mild clinical phenotype of Tangier disease.
Atherosclerosis. 2001 Feb 15;154(3):599-605., [PMID:11257260]

Abstract [show]
The proband is a 50 year-old woman born from a consanguineous marriage. She has been suffering from angina pectoris since the age of 38 and underwent coronary bypass surgery for three-vessel disease at 48. The presence of low plasma levels of total cholesterol and high density lipoprotein (HDL) cholesterol (2.4 and 0.1 mmol/l) and apo AI (<15 mg/dl), associated with corneal lesions and a mild splenomegaly suggested the diagnosis of Tangier disease. However, none of the other features of Tangier disease, including hepatomegaly, anemia and peripheral neuropathy, were present. The analysis of the dinucleotide microsatellites located in chromosome 9q31 region demonstrated that the proband was homozygous for the alleles of D9S53, D9S1784 and D9S1832. The mother and son of the proband, both with low levels of HDL cholesterol, shared one of the proband's haplotypes, whereas neither of these haplotypes was present in the normolipidemic proband's sister. The sequence of ATP-binding cassette transporter 1 (ABC1-1) cDNA obtained by reverse transcription-PCR (RT-PCR) of total RNA isolated from cultured fibroblasts showed that the proband was homozygous for a C>T transition in exon 13, which caused a tryptophane for arginine substitution (R527W). This mutation was confirmed by direct sequencing of exon 13 amplified from genomic DNA. It can be easily screened, as the nucleotide change introduces a restriction site for the enzyme Afl III. R527W substitution occurs in a highly conserved region of the NH2 cytoplasmic domain of ABC1 protein. R527W co-segregates with the low HDL phenotype in the family and was not found in 200 chromosomes from normolipidemic individuals.

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142 Thus the correct location of the mutation reported in our paper is in exon 14 (instead of 13) and the amino acid substitution is R587W (instead of R527W). Login to comment
141 Thus the correct location of the mutation reported in our paper is in exon 14 (instead of 13) and the amino acid substitution is R587W (instead of R527W). Login to comment

[hide] Characterization and classification of ATP-binding... J Biol Chem. 2006 Nov 10;281(45):34503-14. Epub 2006 Sep 7. Matsumura Y, Ban N, Ueda K, Inagaki N
Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency.
J Biol Chem. 2006 Nov 10;281(45):34503-14. Epub 2006 Sep 7., [PMID:16959783]

Abstract [show]
The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency.

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219 For example, R587W and Q597R mutations of ABCA1, which are found in Tangier disease patients with high density lipoprotein deficiency, appear to be impaired in intracellular trafficking and localized predominantly to the ER (36). Login to comment
218 For example, R587W and Q597R mutations of ABCA1, which are found in Tangier disease patients with high density lipoprotein deficiency, appear to be impaired in intracellular trafficking and localized predominantly to the ER (36). Login to comment

[hide] ABCA4 mutations causing mislocalization are found ... Hum Mol Genet. 2005 Oct 1;14(19):2769-78. Epub 2005 Aug 15. Wiszniewski W, Zaremba CM, Yatsenko AN, Jamrich M, Wensel TG, Lewis RA, Lupski JR
ABCA4 mutations causing mislocalization are found frequently in patients with severe retinal dystrophies.
Hum Mol Genet. 2005 Oct 1;14(19):2769-78. Epub 2005 Aug 15., [PMID:16103129]

Abstract [show]
ABCA4, also called ABCR, is a retinal-specific member of the ATP-binding cassette (ABC) family that functions in photoreceptor outer segments as a flipase of all-trans retinal. Homozygous and compound heterozygous ABCA4 mutations are associated with various autosomal recessive retinal dystrophies, whereas heterozygous ABCA4 mutations have been associated with dominant susceptibility to age-related macular degeneration in both humans and mice. We analyzed a cohort of 29 arRP families for the mutations in ABCA4 with a commercial microarray, ABCR-400 in addition to direct sequencing and segregation analysis, and identified both mutant alleles in two families (7%): compound heterozygosity for missense (R602W) and nonsense (R408X) alleles and homozygosity for a complex [L541P; A1038V] allele. The missense mutations were analyzed functionally in the photoreceptors of Xenopus laevis tadpoles, which revealed mislocalization of ABCA4 protein. These mutations cause retention of ABCA4 in the photoreceptor inner segment, likely by impairing correct folding, resulting in the total absence of physiologic protein function. Patients with different retinal dystrophies harboring two misfolding alleles exhibit early age-of-onset (AO) (5-12 years) of retinal disease. Our data suggest that a class of ABCA4 mutants may be an important determinant of the AO of disease.

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156 Similar misfolding effects were observed for the analogous R587W mutation found in ABCA1 in patients with Tangier disease. Login to comment
157 Experiments in which COS7 cells were transfected with a mutant ABCA1 construct, R587W, demonstrated the retention of the misfolded protein in the ER (39). Login to comment

[hide] ABCA1 mutation carriers with low high-density lipo... Eur Heart J. 2013 Jan;34(4):286-91. doi: 10.1093/eurheartj/ehs376. Epub 2012 Nov 7. Bochem AE, van Wijk DF, Holleboom AG, Duivenvoorden R, Motazacker MM, Dallinga-Thie GM, de Groot E, Kastelein JJ, Nederveen AJ, Hovingh GK, Stroes ES
ABCA1 mutation carriers with low high-density lipoprotein cholesterol are characterized by a larger atherosclerotic burden.
Eur Heart J. 2013 Jan;34(4):286-91. doi: 10.1093/eurheartj/ehs376. Epub 2012 Nov 7., [PMID:23136402]

Abstract [show]
AIMS: Low HDL-C is a potent risk factor for cardiovascular disease (CVD). Yet, mutations in ABCA1, a major determinant of circulating HDL-C levels, were previously not associated with CVD risk in cohort studies. To study the consequences of low plasma levels of high-density lipoprotein cholesterol (HDL-C) due to ATP-binding cassette transporter A1 (ABCA1) dysfunction for atherosclerotic vascular disease in the carotid arteries. METHODS AND RESULTS: We performed 3.0 Tesla magnetic resonance imaging (MRI) measurements of the carotid arteries in 36 carriers of high impact functional ABCA1 mutations and 36 normolipidemic controls. Carriers presented with 42% lower HDL-C levels (P < 0.001), a larger mean wall area (18.6 +/- 6.0 vs. 15.8 +/- 4.3 mm(2); P = 0.02), a larger mean wall thickness (0.82 +/- 0.21 vs. 0.70 +/- 0.14 mm; P = 0.005), and a higher normalized wall index (0.37 +/- 0.06 vs. 0.33 +/- 0.04; P = 0.005) compared with controls, retaining significance after adjustment for smoking, alcohol consumption, systolic blood pressure, diabetes, body mass index, history of CVD, LDL-C, and statin use (P = 0.002). CONCLUSION: Carriers of loss of function ABCA1 mutations display a larger atherosclerotic burden compared with age and sex-matched controls, implying a higher risk for CVD. Further studies are needed to elucidate the full function of ABCA1 in the protection against atherosclerosis. These data support the development of strategies to up-regulate ABCA1 in patients with established CVD.

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69 Subjects were carriers of the following mutations: c.6401+2T.C, p.Ser930Phe, p.Ser824Leu, p.Arg587Trp, p.Thr929Ile, p.Asn935Ser, c.3535+1G.C, p.Asp571Gly, p.Asn1800his, p.Leu1056Pro, p.Gln1038Ter, c.1195-1G.C, p.Arg579Gln, and p.Phe1760Valfs*21. Login to comment
78 Five of these mutations have already been shown to have a significant impact on ABCA1 function (p.Asn1800his,27 p.Thr929Ile,27 p.Arg587Trp,28,29 p.Leu1056Pro,21 and p.Phe1760Valfs*21.30 ). Login to comment

[hide] Protein kinase C controls vesicular transport and ... J Biol Chem. 2013 Feb 15;288(7):5186-97. doi: 10.1074/jbc.M112.428961. Epub 2013 Jan 3. Karunakaran D, Kockx M, Owen DM, Burnett JR, Jessup W, Kritharides L
Protein kinase C controls vesicular transport and secretion of apolipoprotein E from primary human macrophages.
J Biol Chem. 2013 Feb 15;288(7):5186-97. doi: 10.1074/jbc.M112.428961. Epub 2013 Jan 3., [PMID:23288845]

Abstract [show]
Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCalpha/beta and not PKCdelta, -epsilon, -theta, or -iota/zeta isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages.

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131 HMDMs were isolated from three separate Tangier disease subjects, who have documented extremely low HDL cholesterol, with homozygous and compound heterozygous mutations in the ABCA1 gene (Patient 1, homozygous for c.4121Cb0e;T (R1270X) in exon 27 (27, 62); Patients 2 and 3, c.1759Cb0e;T (p.Arg587Trp) in exon 14 and c.4957_4961del (p.Val1653CysfsX48) in exon 37 (63)). Login to comment

[hide] ABCA1 mediates unfolding of apolipoprotein AI N te... Arterioscler Thromb Vasc Biol. 2013 Jun;33(6):1197-205. doi: 10.1161/ATVBAHA.112.301195. Epub 2013 Apr 4. Wang S, Gulshan K, Brubaker G, Hazen SL, Smith JD
ABCA1 mediates unfolding of apolipoprotein AI N terminus on the cell surface before lipidation and release of nascent high-density lipoprotein.
Arterioscler Thromb Vasc Biol. 2013 Jun;33(6):1197-205. doi: 10.1161/ATVBAHA.112.301195. Epub 2013 Apr 4., [PMID:23559627]

Abstract [show]
OBJECTIVE: To gain insight into the mechanism by which ABCA1 generates nascent high-density lipoprotein. APPROACH AND RESULTS: HEK293 cells were stably transfected with ABCA1 vectors, encoding wild type, and the W590S and C1477R Tangier disease mutation isoforms, along with the K939M ATP-binding domain mutant. Apolipoprotein AI (ApoAI) binding, plasma membrane remodeling, cholesterol efflux, apoAI cell surface unfolding, and apoAI cell surface lipidation were determined, the latter 2 measured using novel fluorescent apoAI indicators. The W590S isoform had decreased plasma membrane remodeling and lipid efflux activities, and the C1477R isoform had decreased apoAI binding, and lipid efflux activities, whereas the K939M isoform did not bind apoAI, remodel the membrane, or efflux cholesterol. However, all ABCA1 isoforms led to apoAI unfolding at the cell surface, which was higher for the isoforms that increased apoAI binding. ApoAI lipidation was not detected on ABCA1-expressing cells, only in the conditioned medium, consistent with rapid release of nascent high-density lipoprotein from ABCA1-expressing cells. CONCLUSIONS: We identified a third activity of ABCA1, the ability to unfold the N terminus of apoAI on the cell surface. Our results support a model in which unfolded apoAI on the cell surface is an intermediate in its lipidation and that, once apoAI is lipidated, it forms an unstable structure that is rapidly released from the cells to generate high-density lipoprotein.

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114 Fitzgerald et al17 examined 5 Tangier disease mutations that mapped to the 2 large extracellular domains, and reported that only the W590S mutation in the first extracellular domain was still competent to mediate apoAI cross-linking, whereas other mutations in the first (R587W and Q597R) and second (C1477R and S1506L) extracellular domains could not mediate apoAI cross-linking. Login to comment
115 Although the flag-tagged R587W and Q597R variants were reported to be expressed on the plasma membrane in transfected cells,17 2 other independent groups reported that these 2 variants have impaired processing and decreased cell surface expression5,8,18 ; but all agree that the W590S is expressed on the plasma membrane similarly to the WT isoform and can mediate apoAI binding. Login to comment

[hide] HDL does not influence the polarization of human m... Int J Cardiol. 2014 Mar 1;172(1):179-84. doi: 10.1016/j.ijcard.2013.12.168. Epub 2014 Jan 8. Colin S, Fanchon M, Belloy L, Bochem AE, Copin C, Derudas B, Stroes ES, Hovingh GK, Kuivenhoven JA, Dallinga-Thie GM, Staels B, Chinetti-Gbaguidi G
HDL does not influence the polarization of human monocytes toward an alternative phenotype.
Int J Cardiol. 2014 Mar 1;172(1):179-84. doi: 10.1016/j.ijcard.2013.12.168. Epub 2014 Jan 8., [PMID:24456889]

Abstract [show]
BACKGROUND: Macrophages are crucial cells in the pathogenesis of atherosclerosis. Macrophages are plastic cells which can switch from a classical pro-inflammatory M1 to an alternative anti-inflammatory M2 macrophage phenotype, depending on the environmental stimuli. Because high-density lipoprotein (HDL) cholesterol levels are inversely correlated to cardiovascular disease and since HDL displays anti-inflammatory properties, we investigated whether HDL can affect alternative macrophage differentiation of primary human monocytes in the presence of interleukin (IL)-4, a M2 macrophage polarization driver, in vitro and ex vivo. METHODS AND RESULTS: M2 macrophages are highly responsive to HDL stimulation, since the expression of pentraxin 3 (PTX3), a well known HDL target gene, is induced by HDL more strongly in M2 macrophages than in control unpolarized resting macrophages (RM). As expected, the expression of M2 markers, such as Mannose Receptor (MR), CD200 Receptor (CD200R), Coagulation factor XIII A1 (F13A1), IL-1 receptor antagonist (IL-1RA) and IL10, was induced in IL-4 polarized M2 macrophages compared to RM. However, incubation with HDL added in vitro did not modulate the gene expression of M2 macrophage polarization markers. Moreover, monocytes isolated from subjects with genetically low HDL levels, carrying ABCA1 or LCAT mutations, differentiated ex vivo into M2 macrophages without any difference in the alternative macrophage marker expression profile. CONCLUSIONS: These in vitro and ex vivo results indicate that, contrary to mouse macrophages, HDL does not influence macrophage M2 polarization of human monocyte-derived macrophages. Thus, the anti-inflammatory properties of HDL in humans are probably not related to the enhancement of the M2 macrophage phenotype.

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39 We included 6 subjects who carried heterozygous mutations in ABCA1: p.Arg587Trp, p.Val618Asp, p.Ser140Ter, p.Pro85Leu, p.Cys1941Arg/c.6402 + 2TNG, 3 subjects with a homozygous mutation in LCAT: p.Thr147Leu/p.Val333Met, p.Thr147Leu and 3 subjects with a heterozygous mutation in LCAT: p.Pro34Gln [19,20]. Login to comment

[hide] Increased Systemic and Plaque Inflammation in ABCA... Arterioscler Thromb Vasc Biol. 2015 Jul;35(7):1663-9. doi: 10.1161/ATVBAHA.114.304959. Epub 2015 Feb 19. Bochem AE, van der Valk FM, Tolani S, Stroes ES, Westerterp M, Tall AR
Increased Systemic and Plaque Inflammation in ABCA1 Mutation Carriers With Attenuation by Statins.
Arterioscler Thromb Vasc Biol. 2015 Jul;35(7):1663-9. doi: 10.1161/ATVBAHA.114.304959. Epub 2015 Feb 19., [PMID:26109739]

Abstract [show]
OBJECTIVE: We previously demonstrated that subjects with functional ATP-binding cassette (ABC) A1 mutations have increased atherosclerosis, which has been attributed to the role of ABCA1 in reverse cholesterol transport. More recently, a proinflammatory effect of Abca1 deficiency was shown in mice, potentially contributing to atherogenesis. In this study, we investigated whether ABCA1 deficiency was associated with proinflammatory changes in humans. APPROACH AND RESULTS: Thirty-one heterozygous, 5 homozygous ABCA1 mutation carriers, and 21 matched controls were studied. (18)Fluorodeoxyglucose positron emission tomography with computed tomographic scanning was performed in a subset of carriers and controls to assess arterial wall inflammation (target:background ratio). Heterozygous ABCA1 mutation carriers had a 20% higher target:background ratio than in controls (target:background ratio; P=0.008). In carriers using statins (n=7), target:background ratio was 21% reduced than in nonstatin users (n=7; P=0.03). We then measured plasma cytokine levels. Tumor necrosis factor alpha, monocyte chemoattractant protein-1, and interleukin-6 levels were increased in heterozygous and homozygous ABCA1 mutation carriers. We isolated monocytes from carriers and controls and measured inflammatory gene expression. Only TNFalpha mRNA was increased in monocytes from heterozygous ABCA1 mutation carriers. Additional studies in THP-1 macrophages showed that both ABCA1 deficiency and lipoprotein-deficient plasma from ABCA1 mutation carriers increased inflammatory gene expression. CONCLUSIONS: Our data suggest a proinflammatory state in ABCA1 mutation carriers as reflected by an increased positron emission tomography-MRI signal in nonstatin using subjects, and increased circulating cytokines. The increased inflammation in ABCA1 mutation carriers seems to be attenuated by statins.

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28 Homozygous and compoundheterozygoussubjectshadTangierDisease.Subjects were carriers of the following mutations: p.Leu1056Pro, c.3535+1G>C, c.6401+2T>C, p.Asn1800his, p.Ser930Phe, p.Phe1760Valfs*21, p.Ser824Leu, p.Gln1038Ter, p.Thr929Ile, p.Arg587Trp, p.Asn935Ser, and p.Arg579Gln. Login to comment