ABCMdb

PMID: 16873719

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., [PubMed]

Sentences

No. Mutations Sentence Comment

43 ABCA1 p.Trp590Ser ABCA1 p.Met1091Thr ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr In ABCA1 heterozygotes, 3 distinct phenotypic groups emerged, one in which HDL-C levels were Ϸ50% of those of age-and sex-matched controls, one in which HDL-C levels were at least 70% of controls (A255T, W590S, T929I), and one in which HDL-C levels were significantly below the expected 50% of the levels for controls (30.4% of controls) (M1091T) (Table). Login to comment 44 ABCA1 p.Asn1800His ABCA1 p.Arg587Trp ABCA1 p.Asn935Ser ABCA1 p.Ala255Thr 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 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Asn935Ser ABCA1 p.Asn935Ser ABCA1 p.Ala1046Asp ABCA1 p.Ala1046Asp ABCA1 p.Arg2081Trp ABCA1 p.Arg2081Trp 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 ABCA1 p.Gln597Arg ABCA1 p.Asn1800His ABCA1 p.Asn1800His ABCA1 p.Arg587Trp ABCA1 p.Asn935Ser ABCA1 p.Asn935Ser ABCA1 p.Arg2081Trp ABCA1 p.Arg2081Trp 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 49 ABCA1 p.Cys1477Arg ABCA1 p.Asp1289Asn ABCA1 p.Pro2150Leu In contrast, C1477R, D1289N, and P2150L showed similar distribution to wild-type ABCA1, indicating that these mutants cause defects despite their normal localization at the plasma membrane. Login to comment 50 ABCA1 p.Ser1506Leu ABCA1 p.Ala1046Asp Two mutants, A1046D and S1506L, showed an intermediate phenotype where plasma membrane localization was reduced (Figure 2A). Login to comment 54 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Asn935His ABCA1 p.Asn935His 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 55 ABCA1 p.Cys1477Arg ABCA1 p.Asp1289Asn ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp R2081W, along with the mutants localizing to the plasma membrane by fluorescence, D1289N, C1477R, and P2150L, showed both EndoH resistant and sensitive bands, indicating that they are distributed at the ER and at the Golgi, thus confirming the GFP localization data. Login to comment 56 ABCA1 p.Ser1506Leu ABCA1 p.Ser1506Leu S1506L, which appeared to have partial plasma membrane localization, showed a digestion pattern essentially similar to wild-type ABCA1, supporting the notion that a portion of S1506L is localized at the plasma membrane. Login to comment 58 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu ABCA1 p.Ser1506Leu ABCA1 p.Arg2081Trp ABCA1 p.Arg2081Trp R587W, Q597R, ⌬L693, S1506L, and R2081W showed significantly reduced cell surface ABCA1 expression, confirming our previous localization data. Login to comment 59 ABCA1 p.Cys1477Arg ABCA1 p.Asp1289Asn ABCA1 p.Pro2150Leu By contrast, D1289N, C1477R, and P2150L were at the plasma membrane in similar quantities as wild-type ABCA1(Figure 2C), also confirming our previous localization data. Login to comment 60 ABCA1 p.Ala1046Asp Mutant A1046D showed an intermediate phenotype with some ABCA1 localized at the plasma membrane (Figure 2C). Login to comment 67 ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp ABCA1 p.Arg2081Trp Comparison of Patient HDL-C Levels to Those of Age-and Sex-Matched Controls from the Lipid Research Clinic (LRC) Database ABCA1 Heterozygotes Ͼ70% of Normal HDL-C Levels (Allele With Residual Function) 45% to 70% of Normal HDL-C Levels (Allele With Loss of Function) Ϸ30% of Normal HDL-c Levels (Allele with Dominant Negative Function) Mutation % of LRC Controls Mutation % of LRC Controls Mutation % of LRC Controls A255T6 75.94 R587W8 65.20 M1091T13 30.40 W590S3 82.62 P2150L/R587W8 47.48 T929I9 75.57 Q597R8 60.38 ⌬L6931 57.05 N935S3,12 69.79 A1046D5 59.90 C1477R1 60.99 R2081W/D1289N11 58.64 ABCA1 Homozygotes Ͼ10% of Normal HDL-C Levels (Alleles With Residual Activity) Ͻ10% of Normal HDL-C Levels (Alleles With no Activity) Mutation % of LRC Controls Mutation % of LRC Controls A255T6 13.33 R587W8 6.25 N935S3,12 2.62 N1800H7 3.38 ABCA1 mutants that localize to the plasma membrane but still lack ApoA-I binding may have specifically disrupted binding sites or alterations in the conformation necessary for interaction with ApoA-I. Login to comment 68 ABCA1 p.Gln597Arg ABCA1 p.Asn1800His ABCA1 p.Asn1800His ABCA1 p.Arg587Trp ABCA1 p.Asn935Ser ABCA1 p.Asn935Ser ABCA1 p.Arg2081Trp ABCA1 p.Arg2081Trp 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 69 ABCA1 p.Asp1289Asn ABCA1 p.Asp1289Asn ABCA1 p.Pro2150Leu ABCA1 p.Pro2150Leu Of the 3 mutants that showed normal plasma membrane localization, D1289N and P2150L showed ApoA-I binding similar to wild-type (D1289N, 100.5Ϯ26.3%, nϭ3; P2150L, 80.0Ϯ10.8%, nϭ2). Login to comment 70 ABCA1 p.Cys1477Arg Interestingly however, C1477R, which localized to the plasma membrane elicited almost no ApoA-I binding (19.04Ϯ3.9%, nϭ3, Pϭ0.0008) (Figure 3A), indicating that although plasma membrane localization of ABCA1 is essential, it is not sufficient for ApoA-I binding. Login to comment 71 ABCA1 p.Ser1506Leu ABCA1 p.Ala1046Asp The A1046D and S1506L mutants displayed a partial ability to induce cell surface ApoA-I binding (56.3Ϯ16.4%, nϭ3, Pϭ0.02 and 61.0Ϯ12.7%, nϭ3, Pϭ0.004, respectively; Figure 3A), suggesting that in these mutants, some of the protein is localized at the plasma membrane. Login to comment 78 ABCA1 p.Gln597Arg ABCA1 p.Asn1800His ABCA1 p.Asn1800His ABCA1 p.Arg587Trp ABCA1 p.Asn935Ser ABCA1 p.Asn935Ser ABCA1 p.Arg2081Trp ABCA1 p.Arg2081Trp 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 79 ABCA1 p.Cys1477Arg ABCA1 p.Ser1506Leu ABCA1 p.Ala1046Asp ABCA1 p.Pro2150Leu ABCA1 p.Asp1289Leu A1046D and S1506L showed reduced localization at the plasma membrane, and C1477R, D1289L, and P2150L showed localization at the plasma membrane and intracellularly. Login to comment 81 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Asn935Ser ABCA1 p.Asn935Ser 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 82 ABCA1 p.Cys1477Arg ABCA1 p.Ser1506Leu ABCA1 p.Arg2081Trp C1477R, S1506L, and R2081W show both EndoH sensitive and resistant bands indicating localization at both the ER and the plasma membrane. Login to comment 84 ABCA1 p.Cys1477Arg ABCA1 p.Asp1289Asn ABCA1 p.Pro2150Leu D1289N, C1477R, and P2150L showed normal ABCA1 localization at the membrane. Login to comment 85 ABCA1 p.Ala1046Asp A1046D showed intermediate plasma membrane localization and the other missense mutations showed little plasma membrane localization. Login to comment 87 ABCA1 p.Cys1477Arg Of the 3 mutants showing plasma membrane localization, C1477R showed significantly reduced lipid efflux. Login to comment 88 ABCA1 p.Cys1477Arg Because C1477R showed diminished cell surface ApoA-I binding, this finding indicates that ApoA-I binding is required for ABCA1-mediated lipid efflux. Login to comment 89 ABCA1 p.Asp1289Asn ABCA1 p.Pro2150Leu However, D1289N and P2150L showed normal cholesterol efflux and close to normal phosphocholine efflux. Login to comment 92 ABCA1 p.Met1091Thr Indeed the loss of function mutations showed an intermediate phenotype compared with the mutants with partial function, and the putative dominant negative mutant M1091T showed the most severe phenotype. Login to comment 95 ABCA1 p.Asn1800His ABCA1 p.Arg587Trp ABCA1 p.Asn935Ser 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 97 ABCA1 p.Met1091Thr <50% of Normal HDL-C Levels in ABCA1 Heterozygotes Imply a Dominant-Negative Function for the Mutant Allele Of the ABCA1 heterozygotes described thus far, only 1 missense mutation, M1091T, results in HDL-C levels that are Figure 3. Login to comment 100 ABCA1 p.Pro2150Leu ABCA1 p.Asp1289Leu All loss of function mutants showed significantly lowered ApoA-I binding with the exception of D1289L and P2150L, which showed normal ApoA-I binding. Login to comment 102 ABCA1 p.Asp1289Leu All except D1289L showed reduced choline efflux. Login to comment 103 ABCA1 p.Pro2150Leu ABCA1 p.Asp1289Leu C, All mutants with the exception of D1289L and P2150L showed significant defects in cholesterol efflux. Login to comment 107 ABCA1 p.Met1091Thr A significant reduction in biotinylated M1091T was observed, thus corroborating the intracellular localization and EndoH data (Figure 4C). Login to comment 109 ABCA1 p.Met1091Thr Patients heterozygous for this mutation have only 36% of normal relative efflux levels.4 These findings, along with the Ϸ30% of normal HDL-C levels observed in patients harboring this mutation, led us to hypothesize that M1091T acted in a dominant-negative manner and prevented the functioning of the normal ABCA1 allele. Login to comment 111 ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr Three mutations fit this criteria, with patients harboring A255T showing 76%, W590S showing 83%, and T929I showing 76% of normal HDL-C levels. Login to comment 112 ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr Intracellular Localization All 3 mutants A255T, W590S, and T929I, were localized by immunofluorescence to the plasma membrane and to intracellular regions in a manner indistinguishable from wild-type ABCA1 (Figure 5A). Login to comment 115 ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr ApoA-I Binding All mutants showed normal ApoA-I binding compared with wild-type ABCA1 (A255T, 98.0Ϯ10.2%, nϭ4; W590S, 94.9Ϯ26.7%, nϭ3; T929I, 83.6Ϯ14.5, nϭ3) (Figure 5D). Login to comment 116 ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr Cholesterol and Phosphocholine Efflux All 3 mutants displayed defects in both cholesterol (Figure 5E) and phosphocholine (Figure 5F) efflux (A255T, cholesterol 49.2Ϯ7.7%, nϭ5, Pϭ0.0001, choline 41.5Ϯ22.5%, nϭ8, Pϭ0.0002; W590S, cholesterol 47.1Ϯ13.1%, nϭ5, Pϭ0.0008, choline 44.7Ϯ21.1%, nϭ3, PϽ0.05; and T929I, Figure 4. Login to comment 118 ABCA1 p.Met1091Thr A, The M1091T mutant was retained in an intracellular compartment and did not reach the plasma membrane. Login to comment 119 ABCA1 p.Met1091Thr B, Endo H digestion of M1091T showed that it was localized to the ER. Login to comment 120 ABCA1 p.Met1091Thr C, Cell surface biotinylation of M1091T confirmed its lack of localization at the plasma membrane compared with wild-type ABCA1. Login to comment 121 ABCA1 p.Met1091Thr D, The M1091T mutant showed a significant reduction in its ability to induce ApoA-I binding. Login to comment 122 ABCA1 p.Met1091Thr A significant reduction in the ability of M1091T to promote efflux of both phosphocholine (E) and cholesterol efflux (F) to ApoA-I acceptors was observed. Login to comment 125 ABCA1 p.Trp590Ser Mutant W590S has been previously shown to induce reduced annexin V binding, which is a measure of plasma membrane flipping.18 Plasma membrane flipping is observed in wild-type ABCA1-expressing cells and may be a critical factor for efflux to occur. Login to comment 126 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr ABCA1 p.Ala255Thr Recent work has shown that W590S associates normally with ApoA-I. However, the ApoA-I released from wild-type ABCA1 was bound to lipids, whereas the ApoA-I released from W590S was lipid-free, indicating a defect in the lipidation of ApoA-I.24 When the ability of the ABCA1 mutants to promote ␣HDL formation was assessed (Figure 5G), wild-type ABCA1 was able to form ␣HDL of 10.4 to 12.2 nM diameter, whereas A255T and W590S formed only lipid-free ApoA-I and T929I formed lipid-free and ApoA-I of 7.1 to Ϸ9-nM diameter. Login to comment 127 ABCA1 p.Ala255Thr Increased Plasma HDL-C Levels (>10th Percentile) in Homozygotes Confirm the Retention of Partial Activity by Mutant Alleles Data generated thus far would predict that mutant alleles that retain partial activity in patients homozygous for mutations in ABCA1 would confer higher plasma HDL-C levels than of those in whom both mutant alleles lack complete function. We had previously hypothesized and confirmed in heterozygous FHA patients that the mutant A255T allele retained partial activity. Login to comment 128 ABCA1 p.Ala255Thr Similarly, patients with TD who were homozygous for A255T show 13.3% of age-and sex-matched control HDL-C levels. Login to comment 132 ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr Heterozygous patients with the mutations A255T, W590S and T929I show Ͼ70% of normal HDL-C levels, and therefore are hypothesized to have mutant alleles that partially retain function. Login to comment 135 ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr C, Cell surface biotinylation revealed normal levels of A255T, W590S, and T929I at the plasma membrane. Login to comment 138 ABCA1 p.Thr929Ile G, All 3 mutants, despite their ability to bind lipid-free ApoA-I, either failed completely to lipidate ApoA-I or, in the case of the T929I mutant, produced lipidated species with abnormal size. Login to comment 148 ABCA1 p.Cys1477Arg C1477R, however, did reach the plasma membrane, similar to wild-type ABCA1. Login to comment 150 ABCA1 p.Cys1477Arg The defect in the C1477R mutant suggests that although plasma membrane localization is critical for lipid efflux, clearly other mechanisms must result in the failure to recruit ApoA-I. Login to comment 151 ABCA1 p.Cys1477Arg C1477R is localized within the second large extracellular loop in ABCA1, and studies in the related transporter ABCA4 revealed that its 2 large extracellular loops interact with each other through disulfide bonds.26 Because ABCA4 and ABCA1 are closely related, it is possible that the disrupted cysteine in the second large extracellular loop in ABCA1 may also prevent interaction between the 2 large extracellular loops, which could be essential for cell surface ApoA-I binding and lipid efflux. Login to comment 152 ABCA1 p.Ser1506Leu ABCA1 p.Ala1046Asp Both A1046D and S1506L were partially localized at the plasma membrane and showed significantly reduced ApoA-I binding. Login to comment 153 ABCA1 p.Ser1506Leu ABCA1 p.Ser1506Leu The data for S1506L agree with previous data.25 The S1506L mutation also occurs in the second large extracellular portion of ABCA1 and may potentially destroy a domain of interaction for the extracellular loops necessary for ApoA-I binding. Login to comment 154 ABCA1 p.Ala1046Asp The mutation A1046D, however, occurs in the intracellular domain of ABCA1 and is localized between the first Walker A and B motifs. Login to comment 156 ABCA1 p.Asp1289Asn ABCA1 p.Pro2150Leu Both P2150L and D1289N are functionally undistinguishable from wild-type ABCA1. Login to comment 158 ABCA1 p.Arg587Trp ABCA1 p.Pro2150Leu P2150L has only been described in patients who also have the R587W variant (M.R.H., unpublished data, 2000). Login to comment 159 ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp ABCA1 p.Pro2150Leu ABCA1 p.Pro2150Leu ABCA1 p.Pro2150Leu 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 ABCA1 p.Arg587Trp ABCA1 p.Asp1289Asn ABCA1 p.Asp1289Asn ABCA1 p.Pro2150Leu ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp ABCA1 p.Arg2081Trp 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 161 ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Asp1289Asn ABCA1 p.Asp1289Asn ABCA1 p.Ala255Thr ABCA1 p.Arg2081Trp ABCA1 p.Arg2081Trp All 3 missense mutations (A255T, W590S, and T929I) that showed residual function were localized to the plasma membrane and induced cell surface ApoA-I binding at levels similar to wild-type ABCA1. Login to comment 162 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Our findings for the mutant W590S therefore agree with previous findings that W590S does localize normally and shows normal ApoA-I binding.18,19,25 These data imply that although mutant ABCA1 localizes to the plasma membrane and induces ApoA-I binding, these events are insufficient for normal lipid efflux to occur. Login to comment 163 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Thr929Ile ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr ABCA1 p.Ala255Thr ABCA1 p.Ala255Thr Previous studies showed that W590S, which has defective lipid efflux, cross-links efficiently to ApoA-I ,and its rate of dissociation from ApoA-I was similar to wild-type ABCA1.24 However, the ApoA-I released from wild-type ABCA1 was bound to lipids, whereas the ApoA-I released from W590S was lipid-free.24 Mutants A255T, W590S, and T929I were normal in their binding to lipid-free ApoA-I. However, A255T and W590S failed completely to lipidate ApoA-I, and T929I produced lipidated species with abnormal size. Login to comment 164 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Met1091Thr One mutant, M1091T, when present in heterozygous patients, results in significantly lower than 50% of normal HDL-C and efflux levels. Login to comment 165 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr ABCA1 p.Ala255Thr Previous studies showed that W590S, which has defective lipid efflux, cross-links efficiently to ApoA-I ,and its rate of dissociation from ApoA-I was similar to wild-type ABCA1.24 However, the ApoA-I released from wild-type ABCA1 was bound to lipids, whereas the ApoA-I released from W590S was lipid-free.24 Mutants A255T, W590S, and T929I were normal in their binding to lipid-free ApoA-I. However, A255T and W590S failed completely to lipidate ApoA-I, and T929I produced lipidated species with abnormal size. Login to comment 166 ABCA1 p.Met1091Thr One mutant, M1091T, when present in heterozygous patients, results in significantly lower than 50% of normal HDL-C and efflux levels. Login to comment 169 ABCA1 p.Met1091Thr Thus M1091T may act in a dominant-negative manner by an as of yet unknown mechanism. Login to comment 171 ABCA1 p.Met1091Thr Thus M1091T may act in a dominant-negative manner by an as of yet unknown mechanism. Login to comment