ABCMdb

PMID: 19344898

Maekawa M, Kikuchi J, Kotani K, Nagao K, Odgerel T, Ueda K, Kawano M, Furukawa Y, Sakurabayashi I
A novel missense mutation of ABCA1 in transmembrane alpha-helix in a Japanese patient with Tangier disease.
Atherosclerosis. 2009 Sep;206(1):216-22. Epub 2009 Feb 25., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCA1 p.Cys1660Arg ABCA1 p.Cys1660Arg The proband was homozygous for a point mutation of T4978C in exon 37, which results in the substitution of cysteine-1660 to arginine (C1660R) in the 8th transmembrane segment of ABCA1. Login to comment 35 ABCA1 p.Cys1660Arg ABCA1 p.Cys1660Arg Here we report a novel mutation of ABCA1 at T4978C, which causes substitution of cysteine-1660 to arginine (C1660R), in a Japanese patient with TD. Login to comment 47 ABCA1 p.Cys1660Arg ABCA1 p.Cys1660Arg Expression vector for mutant ABCA1 carrying C1660R (pcDNA3.1-C1660R ABCA1) was generated by PCR-based site-directed mutagenesis using pcDNA3.1-wild-type ABCA1 as a template. Login to comment 72 ABCA1 p.Cys1660Arg Expression and localization of the C1660R mutant. Login to comment 73 ABCA1 p.Cys1660Arg ABCA1 p.Cys1660Arg Expression and localization of ABCA1 protein were examined by confocal microscopy in (A) HEK293 cells transfected with empty pcDNA3.1 vector (mock), pcDNA3.1-wild-type ABCA1 (WT), and pcDNA3.1-C1660R ABCA1 (C1660R) and in (B) primary PB-MNC from the patient with TD (patient), her parents (father and mother), and a healthy volunteer (normal). Login to comment 87 ABCA1 p.Cys1660Arg As shown in Supplementary Fig. 1, the patient was found to be homozygous for a T to C transition at nucleotide 4978 (codon 1660, exon 37), which results in substitution of cysteine to arginine (C1660R). Login to comment 91 ABCA1 p.Cys1660Arg Apo A-I-binding ability of the C1660R mutant. Login to comment 98 ABCA1 p.Cys1660Arg ABCA1 p.Cys1660Arg (B) Alexa 488-conjugated apo A-I binding was analyzed in HEK293 cells transfected with empty pcDNA3.1 vector (mock), pcDNA3.1-wild-type ABCA1 (WT), and pcDNA3.1-C1660R mutant (C1660R) by flow cytometry. Login to comment 104 ABCA1 p.Cys1660Arg Apo A-I-mediated cholesterol efflux of the C1660R mutant. Login to comment 105 ABCA1 p.Cys1660Arg ABCA1 p.Cys1660Arg The contents of cholesterol (A) and phospholipids (B) in the medium of HEK293 cells transiently transfected with empty pcDNA3.1 vector (mock), pcDNA3.1-wild-type ABCA1 (WT), and pcDNA3.1-C1660R mutant (C1660R) were measured after 24-h incubation in the presence (black bars) or absence (white bars) of 10 ␮g/ml apo A-I. Login to comment 112 ABCA1 p.Arg219Lys 5 SNPs, InsG319, R219K, I680I, I883 M and T1472T, in this family (Supplementary Table 1). Login to comment 114 ABCA1 p.Cys1660Arg Expression and localization of the C1660R mutant Previous studies have shown that intracellular localization of ABCA1 protein is altered by specific mutations, whereas ABCA1 is normally localized on the plasma membrane [18,19]. Login to comment 117 ABCA1 p.Cys1660Arg First, we analyzed the expression of ABCA1 in HEK293 cells transfected with mock, wild-type ABCA1, and C1660R-mutant ABCA1 expression vectors using immunofluorescence confocal microscopy. Login to comment 118 ABCA1 p.Cys1660Arg As shown in Fig. 2A, both wild-type and C1660R-mutant ABCA1 transfectants expressed ABCA1 mainly on the cell surface, whereas ABCA1 was not detected in a mock transfectant because of the absence of endogenous ABCA1 expression in HEK293 cells. Login to comment 120 ABCA1 p.Cys1660Arg A strong signal was detected only on the cell surface in the patient as well as her parents and a healthy volunteer, suggesting that the C1660R mutation does not affect the localization of ABCA1 (Fig. 2B). Login to comment 122 ABCA1 p.Cys1660Arg ABCA1 p.Cys1660Arg Apo A-I-binding ability of the C1660R mutant Next, we hypothesized that the C1660R mutant lacked apo A-I binding due to disruption of the binding sites or alterations of the conformation necessary for interaction with apo A-I. Login to comment 134 ABCA1 p.Cys1660Arg ABCA1 p.Cys1660Arg Again, there was no significant difference in apo A-I binding between wild-type and C1660R-mutant transformants (Fig. 3B), although the expression level was slightly lower in C1660R-mutant ABCA1 (Fig. 3C and D). Login to comment 135 ABCA1 p.Cys1660Arg Overall, these results suggest that the C1660R mutant retains apo A-I-binding ability. Login to comment 137 ABCA1 p.Cys1660Arg ABCA1 p.Cys1660Arg Cellular lipid efflux of the C1660R mutant Finally, we tested whether C1660R-mutant ABCA1 could efflux intracellular lipids in an apo A-I-dependent manner. Login to comment 138 ABCA1 p.Cys1660Arg As shown in Fig. 4A, apo A-I-mediated cholesterol efflux was observed in HEK293 cells expressing wild-type ABCA1 but not in C1660R-mutant transformants. Login to comment 139 ABCA1 p.Cys1660Arg Apo A-I-mediated phospholipids efflux was also severely impaired in the C1660R mutant (Fig. 4B). Login to comment 141 ABCA1 p.Cys1660Arg These results suggest that the C1660R mutant has little or no activity of cellular lipid release. Login to comment 149 ABCA1 p.Cys1660Arg The C1660R mutation found in this study is the very first missense mutation in the transmembrane domain of ABCA1 (Fig. 5). Login to comment 150 ABCA1 p.Cys1660Arg Our analyses revealed that the expression, intracellular localization, and apo A-I-binding were not impaired, but cholesterol efflux was markedly reduced in the C1660R mutant. Login to comment 152 ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Ala255Thr The loss of lipid efflux without impaired apo A-I binding was also observed in A255T, W590S, and T929I mutations [18]. Login to comment 153 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Surface binding of apo A-I was intact in W590S mutant-transfected HeLa and HEK293 cells [18,26], and direct binding of apo A-I was enhanced in the W590S mutant compared with wild-type ABCA1 in chemical cross-linking studies [27,28]. Login to comment 155 ABCA1 p.Trp590Ser Rigot et al. [26] reported that the reduction of plasma membrane flipping might underlie the loss of lipid efflux in the W590S mutant. Login to comment 156 ABCA1 p.Cys1660Arg This may be the case with C1660R. Login to comment 157 ABCA1 p.Cys1660Arg Among five family members of the proband enrolled in the study, four (parents, grandmother, and brother) are heterozygous for C1660R mutation. Login to comment 161 ABCA1 p.Arg219Lys Among five SNPs identified in this family, previous epidemiological studies demonstrated that R219K and I883 M variants are associated with higher HDL-C levels [30,31], whereas InsG319 is negatively correlated with atherosclerosis independently of plasma HDL-C concentrations [32]. Login to comment 164 ABCA1 p.Arg219Lys For instance, the R219K variant is significantly associated with higher HDL-C levels in normal body weight individuals, but with lower HDL-C levels in overweight people [30]. Login to comment 165 ABCA1 p.Arg219Lys This may explain why HDL-C is low in the proband`s father despite the presence of homozygous R219K. Login to comment 168 ABCA1 p.Cys1477Arg To our knowledge, there is only one case with missense mutation resulting in the substitution of cysteine, i.e., C1477R [2]. Login to comment 169 ABCA1 p.Cys1477Arg ABCA1 p.Cys1660Arg The C1477R mutant can be expressed on the cell surface normally, but fails to bind to apo A-I unlike C1660R [18]. Login to comment 170 ABCA1 p.Cys1477Arg ABCA1 p.Cys1660Arg This difference may reflect the localization of the mutants: C1477R is in the extracellular loop and C1660R is in the transmembrane domain. Login to comment 171 ABCA1 p.Cys1660Arg Previous literature led to the following speculation regarding the mechanisms of defective cholesterol efflux of the C1660R mutant. Login to comment 173 ABCA1 p.Cys1660Arg Alternatively, the substitution of cysteine-1660 to arginine may directly or indirectly affect substrate recognition and/or conformational changes of ABCA1 associated with the ATPase cycle. Login to comment