ABCMdb

ABCA1 p.Gln597Arg

None has been submitted yet.

Publications

PMID: 21567408 [PubMed] Nakagawa H et al: "Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts."

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

PMID: 22981231 [PubMed] Smith B et al: "Anticancer Activity of the Cholesterol Exporter ABCA1 Gene."

No. Sentence Comment

124 To this end, we utilized two missense mutations of human ABCA1 proteins (Q597R and C1477R) linked to TD and familial hypoalphalipoproteinemia (FHA), which were previously characterized to have diminished cholesterol efflux activity (Singaraja et al., 2006). Login to comment

PMID: 21875686 [PubMed] Tietjen I et al: "Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT."

No. Sentence Comment

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

PMID: 22190590 [PubMed] Ji A et al: "Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI."

No. Sentence Comment

61 The generation of such nascent HDL particles is dependent on ABCA1 because cells lacking ABCA1 or expressing an inactive ABCA1 mutant (Q597R) were unable to form such particles (14-17). Login to comment

PMID: 21846716 [PubMed] Iatan I et al: "Membrane microdomains modulate oligomeric ABCA1 function: impact on apoAI-mediated lipid removal and phosphatidylcholine biosynthesis."

No. Sentence Comment

64 Fibroblasts obtained from patients with Tangier disease (TD; homozygous for Q597R at the ABCA1 gene) were used as a negative control throughout the study as previously described (9, 17). Login to comment
95 ABCA1 mutant (Q597R) associated with TD was used as a negative control for binding specificity. Login to comment
125 Normal and ABCA1 mutant (Q597R) human fibroblasts (B-D) and THP-1 cells (E-G) stimulated or not with 22OH/9CRA were incubated with 10 ␮g/ml [ 125 I]apoA-I for 45 min at 37°C. After washing to remove unbound [ 125 I] apoA-I, cross-linking with DSP was performed as described in Experimental Procedures. Login to comment
151 An ABCA1 mutant (Q597R) used as a negative control for binding specificity did not show significant [125 I]apoA-I association with any sucrose fraction (Fig. 1B). Login to comment
212 An ABCA1 mutant (Q597R) that has been shown to have no significant apoA-I binding was used as a negative control. Login to comment
227 Incubation with apoA-I did not upregulate mRNA expression of CCT␣ or CK␣ in ABCA1 mutant (Q597R) fibroblasts (Fig. 5D). Login to comment
252 (A, B) Normal and ABCA1 mutant (Q597R) fibroblasts were stimulated with 22OH/9CRA and incubated or not with 20 ␮g/ml apoA-I for 16 h. Cells were then pulsed with 10 ␮Ci/ml methyl[ 3 H]choline for 1 h, and the distribution of radioactivity among metabolites (Cho, Pcho, and CDP-cho) and PtdCho was determined as described in Experimental Procedures. Values shown are means ± SD of triplicate measures. Login to comment
254 (C, D) Stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated or not with 20 ␮g/ml apoA-I for 16 h, followed by mRNA extraction using the RNeasy mini RNA extraction kit (Qiagen). Login to comment
270 (Top) Normal and ABCA1 mutant (Q597R) fibroblasts were stimulated with 22OH/9CRA and incubated in the presence of 10 ␮g/ml of [ 125 I]apoA-I for 45 min at 37°C. After washing to remove unbound [ 125 I]apoAI, DMEM was added, and plates were immediately incubated at 37°C or 4°C. Login to comment
65 Fibroblasts obtained from patients with Tangier disease (TD; homozygous for Q597R at the ABCA1 gene) were used as a negative control throughout the study as previously described (9, 17). Login to comment
96 ABCA1 mutant (Q597R) associated with TD was used as a negative control for binding specificity. Login to comment
128 Normal and ABCA1 mutant (Q597R) human fibroblasts (B-D) and THP-1 cells (E-G) stimulated or not with 22OH/9CRA were incubated with 10 g/ml [ 125 I]apoA-I for 45 min at 37&#b0;C. After washing to remove unbound [ 125 I] apoA-I, cross-linking with DSP was performed as described in Experimental Procedures. Login to comment
155 An ABCA1 mutant (Q597R) used as a negative control for binding specificity did not show significant [125 I]apoA-I association with any sucrose fraction (Fig. 1B). Login to comment
216 An ABCA1 mutant (Q597R) that has been shown to have no significant apoA-I binding was used as a negative control. Login to comment
232 Incubation with apoA-I did not upregulate mRNA expression of CCT or CK in ABCA1 mutant (Q597R) fibroblasts (Fig. 5D). Login to comment
257 (A, B) Normal and ABCA1 mutant (Q597R) fibroblasts were stimulated with 22OH/9CRA and incubated or not with 20 g/ml apoA-I for 16 h. Cells were then pulsed with 10 Ci/ml methyl[ 3 H]choline for 1 h, and the distribution of radioactivity among metabolites (Cho, Pcho, and CDP-cho) and PtdCho was determined as described in Experimental Procedures. Values shown are means &#b1; SD of triplicate measures. Login to comment
259 (C, D) Stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated or not with 20 g/ml apoA-I for 16 h, followed by mRNA extraction using the RNeasy mini RNA extraction kit (Qiagen). Login to comment
275 (Top) Normal and ABCA1 mutant (Q597R) fibroblasts were stimulated with 22OH/9CRA and incubated in the presence of 10 g/ml of [ 125 I]apoA-I for 45 min at 37&#b0;C. After washing to remove unbound [ 125 I]apoAI, DMEM was added, and plates were immediately incubated at 37&#b0;C or 4&#b0;C. Login to comment

PMID: 20656214 [PubMed] Kang MH et al: "Adenosine-triphosphate-binding cassette transporter-1 trafficking and function."

No. Sentence Comment

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

PMID: 18776170 [PubMed] Vaughan AM et al: "ABCA1 mutants reveal an interdependency between lipid export function, apoA-I binding activity, and Janus kinase 2 activation."

No. Sentence Comment

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
54 The rank order of severity in impaired apoA-I-mediated cholesterol efflux was Q597R.A937V.R587W.C1477R.V399A.I1517R. Login to comment
93 The two most severe mutations, which reduced apoA-I-mediated lipid efflux to less than 20% of normal, were located in the first extracellular loop (Q597R) and the ATP binding site (A937V). 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
99 Tanaka et al. (26) showed that endoplasmic reticulum stress can promote translocation of the Q597R ABCA1 mutant to the plasma membrane, where it still has impaired function, indicating that decreased trafficking alone cannot explain the dysfunctional effects of this mutation. Login to comment
92 The two most severe mutations, which reduced apoA-I-mediated lipid efflux to less than 20% of normal, were located in the first extracellular loop (Q597R) and the ATP binding site (A937V). Login to comment
95 Fitzgerald et al. (24) reported similar cell surface localizations for mutants R587W, W590S, Q597R, and C1477R. Login to comment
98 Tanaka et al. (26) showed that endoplasmic reticulum stress can promote translocation of the Q597R ABCA1 mutant to the plasma membrane, where it still has impaired function, indicating that decreased trafficking alone cannot explain the dysfunctional effects of this mutation. Login to comment

PMID: 18706283 [PubMed] Iatan I et al: "Effect of ABCA1 mutations on risk for myocardial infarction."

No. Sentence Comment

119 Genetic variation in ABCA1 and risk of myocardial infarction Study* / year Population screened HDL-C, mmol/L ABCA1 variants Conclusions Clee et al. [28] / 2000 Within 11 TD families: Del L693, R2144X † , Del E,D1893,94 † , R909X, M1091T † , P2150L † , ivs25+1G→C, Del C6825→2145X, CTC6952- 4TT→2203X, C1477R, Q597R, T929I ABCA1 heterozygous patients had a 40%-45% decrease in HDL-C and a greater than threefold increased risk of CHD versus unaffected individuals. Login to comment

PMID: 18782226 [PubMed] Tanaka AR et al: "Formation of cholesterol-enriched structures by aberrant intracellular accumulation of ATP-binding cassette transporter A1."

No. Sentence Comment

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

PMID: 18385134 [PubMed] Denis M et al: "ATP-binding cassette A1-mediated lipidation of apolipoprotein A-I occurs at the plasma membrane and not in the endocytic compartments."

No. Sentence Comment

55 Primary human skin fibroblasts of normal and Tangier disease patients (Q597R) were provided by Dr. Genest (McGill University, Montreal, Canada). Login to comment

PMID: 18343215 [PubMed] Tanaka AR et al: "The ABCA1 Q597R mutant undergoes trafficking from the ER upon ER stress."

No. Sentence Comment

0 The ABCA1 Q597R mutant undergoes trafficking from the ER upon ER stress Arowu R. Tanaka a , Fumi Kano a , Kazumitsu Ueda b , Masayuki Murata a,* a Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan b Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan a r t i c l e i n f o Article history: Received 4 March 2008 Available online 14 March 2008 Keywords: ABCA1 CFTR ABC protein Tangier disease Cholesterol COPII ER stress ATF6 Thapsigargin DTT a b s t r a c t Mutations in ATP binding cassette transporter 1 (ABCA1), a membrane protein associated with cellular cholesterol efflux, cause Tangier disease (TD). Login to comment
1 Previously, we showed that an ABCA1 Q597R mutant (QR) identified in TD is retained in the endoplasmic reticulum. Login to comment
17 We previously found that the Q597R (QR) mutation in ABCA1, which has been identified in cases of TD, disrupts both the trafficking of ABCA1 from the ER and its ability to facilitate apoA-I induced cholesterol efflux [17]. Login to comment
25 The anti-ABCA1 antibody and the expression vectors encoding wild-type (WT) ABCA1 or the Q597R mutant, each fused with GFP, were generated as described previously [17]. Login to comment
30 HEK293 cells stably expressing WT or Q597R mutant ABCA1-GFP (HEK-WT or HEK-QR) were selected and maintained in Dulbecco`s Modified Eagle`s Medium (DMEM) supplemented with 10% fetal calf 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. Login to comment
49 Results Thapsigargin rescues the ABCA1-QR trafficking defect, but not its cholesterol efflux activity We have previously compared the intracellular localization of the wild-type ABCA1-GFP fusion protein with that of the Q597R mutant fused to GFP. Login to comment
102 (A) Fluorescence microscopy of cells transfected with WT ABCA1-GFP and ABCA1-Q597R-GFP. Login to comment
103 ABCA1-Q597R-GFP is retained in the ER. Login to comment
142 W590S mutation and the Q597R mutation are within extracellular domain 1 (the ER-lumenal domain) of ABCA1, which is a mutational hotspot in TD. Login to comment
101 (A) Fluorescence microscopy of cells transfected with WT ABCA1-GFP and ABCA1-Q597R-GFP. Login to comment
141 W590S mutation and the Q597R mutation are within extracellular domain 1 (the ER-lumenal domain) of ABCA1, which is a mutational hotspot in TD. Login to comment

PMID: 18218626 [PubMed] Hassan HH et al: "Quantitative analysis of ABCA1-dependent compartmentalization and trafficking of apolipoprotein A-I: implications for determining cellular kinetics of nascent high density lipoprotein biogenesis."

No. Sentence Comment

7 Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). Login to comment
35 EXPERIMENTAL PROCEDURES Patient Selection-For this study, we selected fibroblasts from three normal control subjects and two patients with TD (homozygous for Q597R at the ABCA1 gene and compound heterozygous for C1477R as described previously (12)). Login to comment
71 Cellular Compartmentalization and Trafficking of ApoA-I/ABCA1 APRIL 25, 2008•VOLUME 283•NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11165 Dissociation of 125 I-ApoA-I from Intact Cells-BHK-ABCA1, normal human fibroblasts, or fibroblasts with ABCA1 mutations (Q597R and C1477R) from Tangier disease subjects were used. Login to comment
87 To examine the specificity of the biotinylation reaction, a 30-fold excess of unlabeled apoA-I, absence of ABCA1 stimulation with 22OH/9CRA, and ABCA1 mutant fibroblasts (Q597R) were used as controls. Login to comment
88 As shown in Fig. 1B, the presence of excess unlabeled apoA-I, the absence of stimulation with 22OH/9CRA, or utilization of an ABCA1 mutant (Q597R) 0.25 m g 0.5 m g 1 m g 2 m g 3 m g 0.0 0.1 0.2 0.3 0.4 PM ICC 125 I-apoA-ICellDistribut (ng/µgcellprotein) Biotin mg/mL ion A B 0.25 m g 0.5 m g 1 m g 2 m g 3 m g 0.0 0.1 0.2 0.3 0.4 PM ICC 125 I-apoA-ICellDistribut (ng/µgcellprotein) Biotin mg/mL ion C ontrol Excess apoA -I -22O H /9C R A A B C A 1(Q 597R ) 0.0 0.1 0.2 0.3 0.4 PM ICC 125 I-apoA-ICellDistribution (ng/µgcellprotein) Hsp-70 ICCPM Tubulin 150- 70- KDa 50- C Hsp-70 ICCPM Tubulin 150- 70- KDa 50- Hsp-70 ICCPM Tubulin 150- 70- KDa 50- VLA-2 FIGURE 1. Login to comment
94 B, normal fibroblasts stimulated with 22OH/9CRA in the presence or absence of 30-fold excess of unlabeled apoA-I, unstimulated normal fibroblasts, and ABCA1 mutant (Q597R) were incubated with 10 ␮g/ml 125 I-apoA-I for 45 min at 37 °C. Surface proteins were biotinylated with0.5mg/mlsulfo-NHS-biotin,and125 I-apoA-IassociatedwiththePMandICCs was determined as described in A. Login to comment
97 We have previously reported that the Q597R- ABCA1 mutant does not bind apoA-I but is expressed normally and localizes to the cell surface (19). Login to comment
174 Similar results were obtained with normal fibroblasts but not TD mutants (Q597R and C1477R). Login to comment
175 The ratio of ABCA1 over activin receptor type II in 22OH/9CRA-stimulated normal fibroblasts showed increased internalization of ABCA1 (Fig. 5F), whereas ABCA1 mutant fibroblasts (Q597R) associated with TD showed no significant internalization in the presence of apoA-I. A representative experiment of internalized ABCA1 in normal and Q597R fibroblasts is shown (Fig. 5, D and E). Login to comment
199 In light of this, we hypothesized that PM-associated apoA-I could be released by treatment with phosphatidylcholine-specific phospholipase C (PC- BHK-ABCA1 (+ ApoA-I) A Endocytosed ABCA1 - 250 kDa - 150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa B C Endocytosed Integrin α4 Endocytosed ABCA1 Endocytosed Integrin α4 Endocytosed ABCA1 Endocytosed ActRII - 250 kDa - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 BHK-ABCA1(Basal) ABCA1 (Q597R) (+ ApoA-I)E F Normal Fibroblasts (+ ApoA-I) D Total ABCA1 - 250 kDa Total ABCA1 - 250 kDa - 250 kDa Total ABCA1 Total ABCA1 - 250 kDa 0 20 40 60 80 100 120 0 3 6 9 12 Normal Q597R Time of Incubation (min) RatioofEndocytosedABCA1ActRII 0 20 40 0 3 6 9 12 + ApoA-I Basal 60 80 100 120 Time of Incubation (min) RatioofEndocytosedABCA1/Intα4 FIGURE 5. Login to comment
202 Confluent normal fibroblasts (D) and ABCA1 mutant (Q597R) fibroblasts (E) were stimulated with 22OH/9CRA. Login to comment
36 EXPERIMENTAL PROCEDURES Patient Selection-For this study, we selected fibroblasts from three normal control subjects and two patients with TD (homozygous for Q597R at the ABCA1 gene and compound heterozygous for C1477R as described previously (12)). Login to comment
73 Cellular Compartmentalization and Trafficking of ApoA-I/ABCA1 APRIL 25, 2008ߦVOLUME 283ߦNUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11165 at SEMMELWEIS UNIV OF MEDICINE on December 3, Dissociation of 125 I-ApoA-I from Intact Cells-BHK-ABCA1, normal human fibroblasts, or fibroblasts with ABCA1 mutations (Q597R and C1477R) from Tangier disease subjects were used. Login to comment
90 To examine the specificity of the biotinylation reaction, a 30-fold excess of unlabeled apoA-I, absence of ABCA1 stimulation with 22OH/9CRA, and ABCA1 mutant fibroblasts (Q597R) were used as controls. Login to comment
91 As shown in Fig. 1B, the presence of excess unlabeled apoA-I, the absence of stimulation with 22OH/9CRA, or utilization of an ABCA1 mutant (Q597R) 0 . Login to comment
101 B, normal fibroblasts stimulated with 22OH/9CRA in the presence or absence of 30-fold excess of unlabeled apoA-I, unstimulated normal fibroblasts, and ABCA1 mutant (Q597R) were incubated with 10 òe;g/ml 125 I-apoA-I for 45 min at 37 &#b0;C. Surface proteins were biotinylated with0.5mg/mlsulfo-NHS-biotin,and125 I-apoA-IassociatedwiththePMandICCs was determined as described in A. Login to comment
104 We have previously reported that the Q597R- ABCA1 mutant does not bind apoA-I but is expressed normally and localizes to the cell surface (19). Login to comment
182 Similar results were obtained with normal fibroblasts but not TD mutants (Q597R and C1477R). Login to comment
183 The ratio of ABCA1 over activin receptor type II in 22OH/9CRA-stimulated normal fibroblasts showed increased internalization of ABCA1 (Fig. 5F), whereas ABCA1 mutant fibroblasts (Q597R) associated with TD showed no significant internalization in the presence of apoA-I. Login to comment
184 A representative experiment of internalized ABCA1 in normal and Q597R fibroblasts is shown (Fig. 5, D and E). Login to comment
208 In light of this, we hypothesized that PM-associated apoA-I could be released by treatment with phosphatidylcholine-specific phospholipase C (PC- BHK-ABCA1 (+ ApoA-I) A Endocytosed ABCA1 - 250 kDa - 150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa B C Endocytosed Integrin b1;4 Endocytosed ABCA1 Endocytosed Integrin b1;4 Endocytosed ABCA1 Endocytosed ActRII - 250 kDa - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 BHK-ABCA1(Basal) ABCA1 (Q597R) (+ ApoA-I) E F Normal Fibroblasts (+ ApoA-I) D Total ABCA1 - 250 kDa Total ABCA1 - 250 kDa - 250 kDa Total ABCA1 Total ABCA1 - 250 kDa 0 20 40 60 80 100 120 0 3 6 9 12 Normal Q597R Time of Incubation (min) Ratio of Endocytosed ABCA1ActRII 0 20 40 0 3 6 9 12 + ApoA-I Basal 60 80 100 120 Time of Incubation (min) Ratio of Endocytosed ABCA1/Int b1; 4 FIGURE 5. Login to comment
211 Confluent normal fibroblasts (D) and ABCA1 mutant (Q597R) fibroblasts (E) were stimulated with 22OH/9CRA. Login to comment

PMID: 17656736 [PubMed] Hassan HH et al: "Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis."

No. Sentence Comment

4 Glyburide drastically reduced 125I-apoA-I binding to the HCBS, whereas 125I- apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Login to comment
35 MATERIALS AND METHODS Patient selection For the present study, we selected fibroblasts from three normal control subjects and one patient with Tangier disease (homozygous for Q597R at the ABCA1 gene), as described previously (14). Login to comment
103 To test the specificity of the cross-liking and immunoprecipitation methods, the presence of a 30-fold excess of unlabeled apoA-I during the incubation of 125 I-apoA-I with 22OH/9CRA-stimulated fibroblasts, the absence of 22OH/9CRA treatment, or ABCA1 mutant fibroblasts (Q597R) drastically reduced the larger fraction of 125 I- apoA-I nonassociated with ABCA1 (Fig. 1B). Login to comment
106 The observation that the presence of ABCA1 mutant Q597R abolished the association of 125 I-apoA-I to both ABCA1 associated and nonassociated Fig. 1. Login to comment
108 A: Confluent unstimulated, 22-(R)-hydroxycholesterol/9-cis-retinoic acid (22OH/9CRA)-stimulated fibroblasts and ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I in the presence or absence of a 30-fold excess of unlabeled apoA-I (+ Excess) for 45 min at 37jC. Login to comment
134 Affinity binding of apoA-I to the putative non-ABCA1 binding site To better characterize the new binding site for apoA-I, intact normal fibroblasts treated or not with 22OH/9CRA or ABCA1 mutant Q597R were incubated with increasing concentrations of 125I-apoA-I for 45 min at 37jC. Login to comment
137 At the same time, ABCA1 mutant fibroblasts (Q597R) showed no significant binding to both sites (Fig. 3D). Login to comment
159 Stimulated intact normal or ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I for 45 min at 37jC. Login to comment
162 Furthermore, no significant apoA-I binding was detected in both PM and ICC fractions in ABCA1 mutant Q597R, used as a negative control in the present experiment (Fig. 5A). Login to comment
185 Confluent normal (A-C) and ABCA1 mutant (Q597R) (D) fibroblasts were incubated with increasing concentrations of 125 I-apoA-I for 45 min at 37jC in the presence (A, B, D) or absence (C) of 22OH/9CRA or 300 mM glyburide (B). Login to comment
215 In parallel experiments, we documented that apoA-I-mediated cholesterol efflux, as well as the formation of nascent LpA-I particles, after phospholipase treatments were absent in ABCA1 mutant (Q597R) fibroblasts under conditions similar to those used in the present experiments (data not shown). Login to comment
216 On the other hand, there is no evidence for lipid-free 125 I-apoA-I aggregation, as assessed by the complete removal of lipid-free 125 I-apoA-I incubated with ABCA1 mutant (Q597R) fibroblasts, under treatment or not with phospholipases by filtration with a 50,000 molecular weight cut off filter system (23). Login to comment
227 This is consistent with the results showing that overexpressing ABCA1 in BHK cells promoted the formation of the HCBS, whereas the inhibition of ABCA1 activity by glyburide or the presence of ABCA1 mutant fibroblasts (Q597R) drastically reduced the binding of apoA-I to the HCBS (Figs. 2, 3). Login to comment
244 A: Confluent stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I for 45 min at 37jC. Login to comment
265 For example: 1) inhibition of ABCA1 activity by glyburide decreased the binding of apoA-I to both ABCA1 and the HCBS; 2) rLpA-I exhibited reduced affinity for ABCA1 and also for the HCBS (Fig. 4A); and 3) ABCA1 mutants (Q597R and C1447R) that failed to bind apoA-I also failed to mediate the formation of the HCBS. Login to comment
319 Furthermore, the presence of ABCA1 mutant Q597R abolished the association of apoA-I with both compartments, consistent with the idea that ABCA1 seems to be required for the association of apoA-I with both the PM and ICCs. Login to comment
186 Confluent normal (A-C) and ABCA1 mutant (Q597R) (D) fibroblasts were incubated with increasing concentrations of 125 I-apoA-I for 45 min at 37jC in the presence (A, B, D) or absence (C) of 22OH/9CRA or 300 mM glyburide (B). Login to comment
217 On the other hand, there is no evidence for lipid-free 125 I-apoA-I aggregation, as assessed by the complete removal of lipid-free 125 I-apoA-I incubated with ABCA1 mutant (Q597R) fibroblasts, under treatment or not with phospholipases by filtration with a 50,000 molecular weight cut off filter system (23). Login to comment
228 This is consistent with the results showing that overexpressing ABCA1 in BHK cells promoted the formation of the HCBS, whereas the inhibition of ABCA1 activity by glyburide or the presence of ABCA1 mutant fibroblasts (Q597R) drastically reduced the binding of apoA-I to the HCBS (Figs. 2, 3). Login to comment
245 A: Confluent stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I for 45 min at 37jC. Login to comment
266 For example: 1) inhibition of ABCA1 activity by glyburide decreased the binding of apoA-I to both ABCA1 and the HCBS; 2) rLpA-I exhibited reduced affinity for ABCA1 and also for the HCBS (Fig. 4A); and 3) ABCA1 mutants (Q597R and C1447R) that failed to bind apoA-I also failed to mediate the formation of the HCBS. Login to comment
320 Furthermore, the presence of ABCA1 mutant Q597R abolished the association of apoA-I with both compartments, consistent with the idea that ABCA1 seems to be required for the association of apoA-I with both the PM and ICCs. Login to comment

PMID: 17655203 [PubMed] Mukhamedova N et al: "The role of different regions of ATP-binding cassette transporter A1 in cholesterol efflux."

No. Sentence Comment

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

PMID: 16873719 [PubMed] Singaraja RR et al: "Specific mutations in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes both in vivo and in vitro."

No. Sentence 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

PMID: 16709568 [PubMed] Trompier D et al: "Transition from dimers to higher oligomeric forms occurs during the ATPase cycle of the ABCA1 transporter."

No. Sentence Comment

121 The first took advantage of the Q597R and ⌬L693 mutations, previously shown to be massively retained in the endoplasmic reticulum (3), whereas the second set comprised mutants that, although correctly expressed and trafficked to the plasma membrane, showed reduced ability to elicit the functional phenotypes associated with ABCA1 expression in transfected cells. Login to comment
122 The analysis of lysates from cells transfected with EGFP-tagged Q597R or ⌬L693 mutants either alone or in combination with wild type ungrafted ABCA1 transporter indicated that three molecular species of dimers are detectable (Fig. 3A). Login to comment
176 The mixtures contained equimolar amounts of ungrafted ABCA1 and of either Q597R or ⌬L693 variants grafted with EGFP (Q597RG or ⌬L693G). Login to comment
177 For comparison, the banding profile of ungrafted (A1WT) and EYFP-grafted (A1Y) ABCA1 is shown. Detection was performed as indicated by either the 891.3 or the anti-GFP mAb. MW, molecular weight markers. B and C, confocal analysis of cells transfected with equimolar mixtures of wild type ABCA1 grafted with ECFP (A1C) and either Q597R or ⌬L693 grafted with EGFP indicate that the presence of wild type transporter in the dimers fails to rescue the trafficking defect of both variants. Login to comment
120 The first took advantage of the Q597R and èc;L693 mutations, previously shown to be massively retained in the endoplasmic reticulum (3), whereas the second set comprised mutants that, although correctly expressed and trafficked to the plasma membrane, showed reduced ability to elicit the functional phenotypes associated with ABCA1 expression in transfected cells. Login to comment
175 The mixtures contained equimolar amounts of ungrafted ABCA1 and of either Q597R or èc;L693 variants grafted with EGFP (Q597RG or èc;L693G). Login to comment

PMID: 16680030 [PubMed] Krimbou L et al: "New insights into the biogenesis of human high-density lipoproteins."

No. Sentence Comment

63 We have previously suggested that the high content in phosphatidylinositol [41] or the high number of apoA-I molecules per particle [43] (Fig. 2b, lower panels) may contribute to the increase in the net negative charge of nascent LpA-I and consequently cause 260 Lipid metabolism Table 1 Formation and speciation of nascent apolipoprotein (apo)A-I-containing particles in various cell lines Cell type Pre-b1-LpA-I a-LpA-I Macrophages monocyte-derived þ þ THP-1 þ þ Hepatocytes HepG2 (apoA-I endo, exo) þ þ primary mouse (apoA-I endo) þ þ CaCo-2 (apoA-I endo, exo) - þ Fibroblasts normal - þ Tangier (Q597R) - - Tangier (C1477R) - - HUVEC - - CHO - þ CHO overexpressing ABCA1 - þ The presence of pre-b1-LpA-I and a-LpA-I generated by different cell lines was determined by two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis based on our previous study [28]. Login to comment

PMID: 16501936 [PubMed] Zannis VI et al: "Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL."

No. Sentence Comment

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

PMID: 16704350 [PubMed] Brunham LR et al: "Variations on a gene: rare and common variants in ABCA1 and their impact on HDL cholesterol levels and atherosclerosis."

No. Sentence Comment

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

PMID: 16429166 [PubMed] Brunham LR et al: "Accurate prediction of the functional significance of single nucleotide polymorphisms and mutations in the ABCA1 gene."

No. Sentence Comment

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 À3.53 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

PMID: 16183915 [PubMed] Oram JF et al: "ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease."

No. Sentence Comment

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

PMID: 15897603 [PubMed] Krimbou L et al: "Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines."

No. Sentence Comment

2 Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only ␣-nascent apolipoprotein A-I-containing particles (␣-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Login to comment
26 MATERIALS AND METHODS Patient selection For the present study, we selected fibroblasts from three normal control subjects and two patients with TD (TD1, homozygous for Q597R at the ABCA1 gene; and TD2, compound heterozygous carrying the mutations C1477R and the splice site G→C in exon 24), as described previously (13, 14). Login to comment
75 However, the incomplete removal of lipid-free apoA-I from the medium may result in an artifactual presence of prebeta1-LpA-I. To ensure the quality of the complete removal of lipid-free apoA-I, the filtration of the medium from either HepG2 cells or macrophages found to form prebeta1-LpA-I was carefully controlled for each experiment by the filtration of the medium from normal fibroblasts, ABCA1 mutant Q597R fibroblasts, or lipid-free 125I-apoA-I incubated without cells. Login to comment
78 As shown in Fig. 1 (upper panels), incubation of stimulated normal fibroblasts, CaCo-2 cells, or CHO-overexpressing ABCA1 (CHO-ABCA1) with 10 ␮g/ml exogenously added 125I-apoA-I for 6 h at 37ЊC followed by the removal of lipid-free apoA-I as described above, and then separation of samples by 2D-PAGGE, generated only ␣-LpA-I with a particle size ranging from 8 to 20 nm, whereas lipid-free apoA-I incubated with either HUVEC or ABCA1 mutant (Q597R) was unable to form such particles. Login to comment
83 Furthermore, inactivation of ABCA1 with glyburide in fibroblasts, CaCo-2, or CHO-ABCA1 led to the formation of smaller ␣-LpA-I having a diameter of ‫8ف‬ nm; these particles were not formed in the presence of ABCA1 mutant Q597R cells. Login to comment
101 Normal fibroblasts, CaCo-2, human umbilical vein endothelial cells (HUVEC), HepG2, or ABCA1 mutant (Q597R) cells in 100 mm diameter dishes were loaded with 20 ␮g/ml cholesterol for 24 h and then stimulated with 2.5 ␮g/ml 22(R)-hydroxycholesterol and 10 ␮M 9-cis-retinoic acid (22OH/9CRA) for 20 h. THP-1 cells and human monocyte-derived macrophages in 100 mm diameter dishes were loaded with 20 ␮g/ml cholesterol for 24 h and then stimulated with 0.3 mM cAMP for 12 h. CHO-overexpressing ABCA1 cells (CHO-ABCA1) were used as a control. Login to comment
105 beled with [14C]cholesterol or [32P]orthophosphate as described in Materials and Methods and then incubated with 10 ␮g/ml lipid-free apoA-I for 24 h at 37ЊC. Radiolabeled ABCA1 mutant Q597R cells were used as a control in the present experiment. Login to comment
107 At the same time, we have not been able to detect any ␣-LpA-I formation during incubation with ABCA1 mutant Q597R (Fig. 4). Login to comment
128 In contrast, lipid-free apoA-I was unable to form such particles in the presence of HUVEC and ABCA1 mutant Q597R cells (Fig. 1), consistent with previous studies showing that lipid-free apoA-I did not promote cholesterol efflux from endothelial cells in which the expression of ABCA1 protein is very low and seems not to be sensitive to treatment with cholesterol or 22-hydroxycholesterol (24, 28). Login to comment
151 This is also strongly supported by our results showing that glyburide did not affect the formation of prebeta1-LpA-I but had a dramatic effect on ␣-LpA-I, providing strong support for the Fig. 4. Characterization of lipidated apoA-I-containing particles generated during lipid-free apoA-I incubation with normal fibroblasts and ABCA1 mutant (Q597R). Login to comment
152 Normal fibroblasts or ABCA1 mutant (Q597R) were labeled with either [14C]cholesterol or [32P]orthophosphate as described in Materials and Methods and incubated with 10 ␮g/ml apoA-I for 24 h. Media were recovered and concentrated with a size-exclusion filter with a MWCO of 10,000 (without the removal of lipid-free apoA-I). Login to comment
1 Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only -nascent apolipoprotein A-I-containing particles (-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Login to comment
77 In separate experiments (see below), we show that both the centrifugal filter and the dialysis membrane retained native plasma pre1-LpA-I having an apparent molecular mass of 67 kDa. As shown in Fig. 1 (upper panels), incubation of stimulated normal fibroblasts, CaCo-2 cells, or CHO-overexpressing ABCA1 (CHO-ABCA1) with 10 g/ml exogenously added 125I-apoA-I for 6 h at 37C followed by the removal of lipid-free apoA-I as described above, and then separation of samples by 2D-PAGGE, generated only -LpA-I with a particle size ranging from 8 to 20 nm, whereas lipid-free apoA-I incubated with either HUVEC or ABCA1 mutant (Q597R) was unable to form such particles. Login to comment
82 Furthermore, inactivation of ABCA1 with glyburide in fibroblasts, CaCo-2, or CHO-ABCA1 led to the formation of smaller -LpA-I having a diameter of 8 nm; these particles were not formed in the presence of ABCA1 mutant Q597R cells. Login to comment
100 Normal fibroblasts, CaCo-2, human umbilical vein endothelial cells (HUVEC), HepG2, or ABCA1 mutant (Q597R) cells in 100 mm diameter dishes were loaded with 20 g/ml cholesterol for 24 h and then stimulated with 2.5 g/ml 22(R)-hydroxycholesterol and 10 M 9-cis-retinoic acid (22OH/9CRA) for 20 h. THP-1 cells and human monocyte-derived macrophages in 100 mm diameter dishes were loaded with 20 g/ml cholesterol for 24 h and then stimulated with 0.3 mM cAMP for 12 h. CHO-overexpressing ABCA1 cells (CHO-ABCA1) were used as a control. Login to comment
130 Here, we present evidence that incubation of exogenously added lipid-free apoA-I with various cell lines, including human fibroblasts, CaCo-2, and CHO-ABCA1, generates only -LpA-I. In contrast, lipid-free apoA-I was unable to form such particles in the presence of HUVEC and ABCA1 mutant Q597R cells (Fig. 1), consistent with previous studies showing that lipid-free apoA-I did not promote cholesterol efflux from endothelial cells in which the expression of ABCA1 protein is very low and seems not to be sensitive to treatment with cholesterol or 22-hydroxycholesterol (24, 28). Login to comment
153 Normal fibroblasts or ABCA1 mutant (Q597R) were labeled with either [14C]cholesterol or [32P]orthophosphate as described in Materials and Methods and incubated with 10 g/ml apoA-I for 24 h. Login to comment

PMID: 15654121 [PubMed] Hajj Hassan H et al: "Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release."

No. Sentence Comment

122 Furthermore, we have previously documented that the specific binding of 125I-apoA-I to unstimulated fibroblasts was very low and totally absent in ABCA1 mutant (Q597R) fibroblasts (18). Login to comment
123 Furthermore, we have previously documented that the specific binding of 125I-apoA-I to unstimulated fibroblasts was very low and totally absent in ABCA1 mutant (Q597R) fibroblasts (18). Login to comment

PMID: 16158173 [PubMed] Takahashi K et al: "ABC proteins: key molecules for lipid homeostasis."

No. Sentence Comment

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

PMID: 16116948 [PubMed] Li Y et al: "Effects of oxidized low density lipoprotein on the expression and function of ABCA1 in macrophages."

No. Sentence Comment

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

PMID: 15280376 [PubMed] Denis M et al: "Characterization of oligomeric human ATP binding cassette transporter A1. Potential implications for determining the structure of nascent high density lipoprotein particles."

No. Sentence Comment

9 Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. Login to comment
39 EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from three normal control subjects and one patient with Tangier disease (homozygous for Q597R at the ABCA1 gene). Login to comment
61 Isolation of Nascent LpA-I Particles and Cross-linking with DSP- 125 I-apoA-I (10 ␮g/ml) was incubated in DMEM for 24 h at 37 °C with either stimulated normal or Q597R cells. Login to comment
63 15 ␮g of isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells was adjusted to 10 mM sodium phosphate, 140 mM NaCl, pH 7.4, with a final protein concentration of 1 ␮g/␮l. Immediately before an experiment, 1 mg of DSP was dissolved in 1000 ␮l of Me2SO to a final concentration of 1 ␮g/␮l. The dissolved DSP was added to the reaction mixture for 10 DSP to 1 apoA-I molar ratio on ice. Login to comment
114 To determine the relationship between oligomeric ABCA1 complex and the structural properties of nascent apoA-I-containing particles in our cell culture model, stimulated cells either from normal or from Tangier disease (Q597R) subjects in 100-mm diameter dishes were incubated with 10 ␮g/ml 125 I- apoA-I in 6 ml of DMEM for 24 h at 37 °C. Login to comment
123 In contrast, lipid-free apoA-I incubated with stimulated ABCA1 mutant (Q597R) cells was unable to form such particles (third gel), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (first gel). Login to comment
150 Either lipid-free apoA-I incubated without cells or lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells were used as controls. Login to comment
152 In contrast, both lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells and lipid-free apoA-I incubated without cells remained in the monomeric form following cross-linking. Login to comment
155 Upper panels, 125 I-apoA-I (10 ␮g/ml) was incubated in DMEM for 24 h at 37 °C without cells (first gel) or with both stimulated normal and Q597R cells (second and third gels, respectively) for 24 h at 37 °C. Login to comment
159 Lower panel, isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells were treated with DSP as described under "Experimental Procedures." Login to comment
180 We demonstrate that lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells remained in the monomeric form (Fig. 5, lower panel). Login to comment
199 Although ABCA1 mutants Q597R and C1477R were found to oligomerize normally (data not shown) and localized to the plasma membrane, they showed the total absence of binding to apoA-I (21, 23). Login to comment
200 These results indicate that the apoA-I lipidation defect observed in either Q597R or C1477R ABCA1 mutants is not caused by impaired oligomerization of ABCA1. Login to comment
8 Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. Login to comment
37 EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from three normal control subjects and one patient with Tangier disease (homozygous for Q597R at the ABCA1 gene). Login to comment
58 The presence of labeled 125 I-apoA-I/ABCA1 complexes was directly detected by autoradiography by using XAR-2 Kodak film. Isolation of Nascent LpA-I Particles and Cross-linking with DSP- 125 I-apoA-I (10 òe;g/ml) was incubated in DMEM for 24 h at 37 &#b0;C with either stimulated normal or Q597R cells. Login to comment
60 15 òe;g of isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells was adjusted to 10 mM sodium phosphate, 140 mM NaCl, pH 7.4, with a final protein concentration of 1 òe;g/òe;l. Immediately before an experiment, 1 mg of DSP was dissolved in 1000 òe;l of Me2SO to a final concentration of 1 òe;g/òe;l. The dissolved DSP was added to the reaction mixture for 10 DSP to 1 apoA-I molar ratio on ice. Login to comment
110 To determine the relationship between oligomeric ABCA1 complex and the structural properties of nascent apoA-I-containing particles in our cell culture model, stimulated cells either from normal or from Tangier disease (Q597R) subjects in 100-mm diameter dishes were incubated with 10 òe;g/ml 125 I-apoA-I in 6 ml of DMEM for 24 h at 37 &#b0;C. Login to comment
120 In contrast, lipid-free apoA-I incubated with stimulated ABCA1 mutant (Q597R) cells was unable to form such particles (third gel), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (first gel). Login to comment
145 Either lipid-free apoA-I incubated without cells or lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells were used as controls. Login to comment
147 In contrast, both lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells and lipid-free apoA-I incubated without cells remained in the monomeric form following cross-linking. Login to comment
154 Lower panel, isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells were treated with DSP as described under "Experimental Procedures." Login to comment
175 We demonstrate that lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells remained in the monomeric form (Fig. 5, lower panel). Login to comment
194 Although ABCA1 mutants Q597R and C1477R were found to oligomerize normally (data not shown) and localized to the plasma membrane, they showed the total absence of binding to apoA-I (21, 23). Login to comment
195 These results indicate that the apoA-I lipidation defect observed in either Q597R or C1477R ABCA1 mutants is not caused by impaired oligomerization of ABCA1. Login to comment

PMID: 14660648 [PubMed] Denis M et al: "Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1)."

No. Sentence Comment

6 A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited ␣-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated ␣-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. Login to comment
38 EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from 3 normal control subjects and 1 patient with TD (homozygous for Q597R at the ABCA1 gene). Login to comment
62 ABCA1 mutant (Q597R) was used as a negative control. Login to comment
126 In addition, ABCA1 mutant (Q597R) that has been shown previously to not cross-link to apoA-I (13) was used as a negative control for the present experiment and showed no binding or cross-linking to ABCA1 (Fig. 3, A and B). Login to comment
129 Also, to rule out the possibility that treatment with phospholipases might induce membrane aggregation or affect ABCA1 protein structure, which may trap apoA-I and result in nonspecific cross-linking, we examined the effect of phospholipases treatment on the cross-linking of 125 I-apoA-I to ABCA1 mutant (Q597R). Login to comment
130 As shown in Fig. 3B (upper panel), 125 I-apoA-I did not cross-link to Q597R mutant whether treated with PC-PLC, SM-ase, or left intact. Login to comment
139 In addition, to ensure that cholesterol efflux was ABCA1-dependent in our cell culture system, apoA-I-mediated cholesterol efflux from ABCA1 mutant (Q597R) cells was also monitored. Login to comment
140 In order to investigate the nature of apoA-I-containing particles generated by ABCA1 activity, stimulated cells from either normal or from TD (Q597R) subjects in 100 mm diameter dishes were incubated with 10 ␮g/ml of 125 I-apoA-I in 8 ml of DMEM for 24 h at 37 °C. Login to comment
143 In contrast, lipid-free apoA-I incubated with stimulated mutant Q597R cells was unable to form such particles (panel C), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (panel A). Login to comment
196 ABCA1 mutant (Q597R) was used as a negative control. Login to comment
197 B, upper panel, intact stimulated normal or Q597R cells in 100-mm diameter dishes were incubated or not with PC-PLC or SM-ase as described above and then incubated with 10 ␮g/ml of 125 I-apoA-I for 1 h at 37 °C in the presence or absence of a 20-fold excess of unlabeled apoA-I (200 ␮g/ml). Login to comment
233 B, stimulated normal or Q597R cells were radiolabeled with [3 H]cholesterol and incubated with 10 ␮g/ml of apoA-I or 1 mg/ml of BSA at 37 °C for the indicated time points. Login to comment
242 125 I- apoA-I was incubated in DMEM/BSA for 24 h at 37 °C without cells (A) or with both stimulated normal and Q597R cells (B and C, respectively) for 24 h at 37 °C. Login to comment
253 to form larger particles during its incubation with Q597R mutant cells. Login to comment
37 EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from 3 normal control subjects and 1 patient with TD (homozygous for Q597R at the ABCA1 gene). Login to comment
61 ABCA1 mutant (Q597R) was used as a negative control. Login to comment
124 In addition, ABCA1 mutant (Q597R) that has been shown previously to not cross-link to apoA-I (13) was used as a negative control for the present experiment and showed no binding or cross-linking to ABCA1 (Fig. 3, A and B). Login to comment
127 Also, to rule out the possibility that treatment with phospholipases might induce membrane aggregation or affect ABCA1 protein structure, which may trap apoA-I and result in nonspecific cross-linking, we examined the effect of phospholipases treatment on the cross-linking of 125 I-apoA-I to ABCA1 mutant (Q597R). Login to comment
128 As shown in Fig. 3B (upper panel), 125 I-apoA-I did not cross-link to Q597R mutant whether treated with PC-PLC, SM-ase, or left intact. Login to comment
137 In addition, to ensure that cholesterol efflux was ABCA1-dependent in our cell culture system, apoA-I-mediated cholesterol efflux from ABCA1 mutant (Q597R) cells was also monitored. Login to comment
138 In order to investigate the nature of apoA-I-containing particles generated by ABCA1 activity, stimulated cells from either normal or from TD (Q597R) subjects in 100 mm diameter dishes were incubated with 10 òe;g/ml of 125 I-apoA-I in 8 ml of DMEM for 24 h at 37 &#b0;C. Login to comment
141 In contrast, lipid-free apoA-I incubated with stimulated mutant Q597R cells was unable to form such particles (panel C), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (panel A). Login to comment
194 ABCA1 mutant (Q597R) was used as a negative control. Login to comment
195 B, upper panel, intact stimulated normal or Q597R cells in 100-mm diameter dishes were incubated or not with PC-PLC or SM-ase as described above and then incubated with 10 òe;g/ml of 125 I-apoA-I for 1 h at 37 &#b0;C in the presence or absence of a 20-fold excess of unlabeled apoA-I (200 òe;g/ml). Login to comment
230 B, stimulated normal or Q597R cells were radiolabeled with [3 H]cholesterol and incubated with 10 òe;g/ml of apoA-I or 1 mg/ml of BSA at 37 &#b0;C for the indicated time points. Login to comment
239 125 I-apoA-I was incubated in DMEM/BSA for 24 h at 37 &#b0;C without cells (A) or with both stimulated normal and Q597R cells (B and C, respectively) for 24 h at 37 &#b0;C. Login to comment
249 Results shown are representative of two different independent experiments. Lipidation of ApoA-I by ABCA1 to form larger particles during its incubation with Q597R mutant cells. Login to comment

PMID: 15019541 [PubMed] Pisciotta L et al: "Familial HDL deficiency due to ABCA1 gene mutations with or without other genetic lipoprotein disorders."

No. Sentence Comment

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

PMID: 14592415 [PubMed] Ikeda Y et al: "Posttranscriptional regulation of human ABCA7 and its function for the apoA-I-dependent lipid release."

No. Sentence Comment

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

PMID: 12763760 [PubMed] Singaraja RR et al: "Efflux and atherosclerosis: the clinical and biochemical impact of variations in the ABCA1 gene."

No. Sentence Comment

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
79 This is indeed the case with mutant Q597R,58,60 which shows no ApoA-I binding. 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
71 This is indeed the case with mutant Q597R,58,60 which shows no ApoA-I binding. 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

PMID: 12840658 [PubMed] Miller M et al: "Genetics of HDL regulation in humans."

No. Sentence Comment

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

PMID: 12642355 [PubMed] Marcil M et al: "Cellular phospholipid and cholesterol efflux in high-density lipoprotein deficiency."

No. Sentence Comment

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

PMID: 12509412 [PubMed] Tanaka AR et al: "Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis."

No. Sentence Comment

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
85 The Q597R mutant almost completely lost this activity. 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
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
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
124 R587W and Q597R ABCA1-GFP appeared to be retained in the ER. Login to comment

PMID: 12454270 [PubMed] Haidar B et al: "cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts."

No. Sentence Comment

176 Molecular characterization of ABCA1 gene in study subjects Cell Lines HDL-C Nucleotide Change Predicted Protein Alteration mmol/l CTR1 1.63 - - CTR2 1.20 - - FHD1 0.27 Exon 14 ⌬2017-9 ⌬L693 FHD2 0.18 Exon 18 C2665T R909X FHD3 0.39 Exon 41 ⌬5618-23 ⌬ED1893,4 FHD4 0.18 Exon 48 C6370T R2144X FHD5 0.09 Exon 36 GG5277,8C fs 1628G, Q1636X TD1 Ͻ0.1 Exon 30 T4369C; Exon 24 splice site G→C C1477R; Part of the transcript deleted TD2 Ͻ0.1 Exon 13 A1730G Q597R TD3 Ͻ0.1 Exon 48 ⌬C6370; nd 2145X; nd FHD1-5 are heterozygous for the reported mutation; TD1,3 are compound heterozygous and TD2 is homozygous. Login to comment

PMID: 12454269 [PubMed] Rigot V et al: "Distinct sites on ABCA1 control distinct steps required for cellular release of phospholipids."

No. Sentence Comment

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
153 Two of the Tangier transporters (namely Q597R and ⌬L693) reach the plasma membrane very inefficiently, and as a consequence fail to elicit any significant function as measured by surface binding of either annexin V or Fig. 4. Login to comment
161 Q597R and ⌬L693 are virtually absent from the cell surface while expressed normally. Login to comment
199 Confocal images of ABCA1 mutant alleles expressed in HeLa cells (upper panel) indicate that in the case of Q597R the mutation affects intracellular trafficking and leads to retention into the ER. 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
152 Two of the Tangier transporters (namely Q597R and L693) reach the plasma membrane very inefficiently, and as a consequence fail to elicit any significant function as measured by surface binding of either annexin V or Fig. 4. Login to comment
160 Q597R and L693 are virtually absent from the cell surface while expressed normally. Login to comment
198 Confocal images of ABCA1 mutant alleles expressed in HeLa cells (upper panel) indicate that in the case of Q597R the mutation affects intracellular trafficking and leads to retention into the ER. 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

PMID: 12084722 [PubMed] Fitzgerald ML et al: "Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I."

No. Sentence Comment

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
198 Three of the mutants (Q597R, C1477R, and S1506L) had dramatic reductions in their cross-linking efficiency to apoA-I, relative to the wild type transporter (90% or greater reduction). 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
190 Three of the mutants (Q597R, C1477R, and S1506L) had dramatic reductions in their cross-linking efficiency to apoA-I, relative to the wild type transporter (90% or greater reduction). 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

PMID: 16959783 [PubMed] Matsumura Y et al: "Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency."

No. Sentence Comment

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

PMID: 23087442 [PubMed] Sorrenson B et al: "Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate."

No. Sentence Comment

161 Plasma membrane localization of the p.Q597R protein was promoted with thapsigargin, an inducer of ER stress, whereas 4-PBA is known to alleviate ER stress (14) by acting as a chaperone itself (13) as well as inducing the expression of endogenous chaperone proteins (15). Login to comment
195 Improved positioning of ABCA1 at the plasma membrane has been shown previously for a p.Q597R mutant under conditions of induced ER stress; however, in that instance, no improvement in efflux activity was seen (23). Login to comment
196 This is in contrast with our results where the majority of the mutants showed significant improvements in function, including the p.A594T mutant, which is of a similar location to p.Q597R. Login to comment

PMID: 23559627 [PubMed] Wang S et al: "ABCA1 mediates unfolding of apolipoprotein AI N terminus on the cell surface before lipidation and release of nascent high-density lipoprotein."

No. Sentence Comment

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