ABCMdb

PMID: 12454269

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

Sentences

No. Mutations Sentence Comment

60 ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg When analyzing double mutants (HA819/ C1477R and HA819/1466), the comparison was carried out between each double and the relevant loss of function single mutant (C1477R and HA1466, respectively). Login to comment 139 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp 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 140 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Three point mutations in the region 580-600 had been reported in Tangier pedigrees (namely R587W, W590S, and Q597R). Login to comment 147 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp 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 148 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp 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 ABCA1 p.Gln597Arg 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 153 ABCA1 p.Gln597Arg 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 ABCA1 p.Gln597Arg Q597R and L693 are virtually absent from the cell surface while expressed normally. Login to comment 161 ABCA1 p.Gln597Arg Q597R and ⌬L693 are virtually absent from the cell surface while expressed normally. Login to comment 167 ABCA1 p.Cys1477Arg C1477R falls into a second class since it is detected at the plasma membrane but elicits a significantly reduced binding of apoA-I as compared with wild-type (33% 9, n 6, P 0.01 Table 1). Login to comment 168 ABCA1 p.Cys1477Arg C1477R falls into a second class since it is detected at the plasma membrane but elicits a significantly reduced binding of apoA-I as compared with wild-type (33% Ϯ 9, n ϭ 6, P Ͻ 0.01 Table 1). Login to comment 169 ABCA1 p.Trp590Ser ABCA1 p.Arg587Trp The last category is illustrated by R587W and W590S Tangier transporters, which are correctly targeted to the plasma membrane, but show a marked functional dissociation. Login to comment 170 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp The last category is illustrated by R587W and W590S Tangier transporters, which are correctly targeted to the plasma membrane, but show a marked functional dissociation. Login to comment 171 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp Indeed, these variants, while eliciting an apoA-I binding indistinguishable from wild-type ABCA1 (79% Ϯ 5, n ϭ 7, P Ͼ 0.05 and 126% Ϯ 18, n ϭ 6, P Ͼ 0.05 of wild type, respectively) fail to drive both flipping of PS (annexin V binding ϭ 39% Ϯ 11of wild type for R587W, n ϭ 5, P Ͻ 0.05 and 30% Ϯ 9 for W590S, n ϭ 7, P Ͻ 0.01) and membrane release of PL (27% Ϯ 16, n ϭ 3, P Ͻ 0.05 and 16% Ϯ 3, n ϭ 2, P Ͻ 0.01 of wild type, respectively, Table 3), thus indicating that apoA-I binding per se is insufficient for the generation of PL effluxes, which also requires PS flipping. Login to comment 172 ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg HA 819 acts as a suppressive mutations on Tangier C1477R Having observed that we could generate mutant transporters showing opposite behaviors with respect of apoA-I surface binding, we asked which effect could result from the introduction of a gain of function mutation on an hypomorphic mutant allele. Login to comment 173 ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg We thus grafted the HA819, which increases selectively apoA-I binding (151% Ϯ 11 of wild type, n ϭ 4, P Ͻ; 0.05), on HA1466 (apoA-I binding ϭ 27% Ϯ 7 of wild type, n ϭ 9, P Ͻ 0.001), and on C1477R (33% Ϯ 9 of wild type, n ϭ 6, P Ͻ 0.01). Login to comment 174 ABCA1 p.Cys1477Arg The latter showed similar levels of annexin V binding (52% Ϯ 10, n ϭ 7 for HA1466 P Ͼ 0.05 and 53% Ϯ 12, n ϭ 9, for C1477R, P Ͼ 0.05). Login to comment 176 ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg In both cases, the presence of an HA tag at position 819 partially and significantly rescued the binding of apoA-I but drove limited and nonsignificant modifications in the annexin V binding with respect to the original loss of function mutation (Table 3; apoA-I binding values reverted to 68% 6 for HA819/C1477R, n 4, P 0.05 and to 49% 7 for HA819/HA1466, n 6, P 0.05, whereas annexin V binding amounted to 57% 15, n 4, for HA819/ C1477R and 54% 12, n 6 for HA819/HA1466 P 0.05). Login to comment 177 ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg In both cases, the presence of an HA tag at position 819 partially and significantly rescued the binding of apoA-I but drove limited and nonsignificant modifications in the annexin V binding with respect to the original loss of function mutation (Table 3; apoA-I binding values reverted to 68% Ϯ 6 for HA819/C1477R, n ϭ 4, P Ͻ 0.05 and to 49% Ϯ 7 for HA819/HA1466, n ϭ 6, P Ͻ 0.05, whereas annexin V binding amounted to 57% Ϯ 15, n ϭ 4, for HA819/ C1477R and 54% Ϯ 12, n ϭ 6 for HA819/HA1466 P Ͼ 0.05). Login to comment 178 ABCA1 p.Cys1477Arg In both cases the ability to induce PL effluxes was also significantly increased (HA819/C1477R ϭ 142% Ϯ 2 4, n ϭ 2, P Ͻ 0.01 and HA819/HA1466 ϭ 108% Ϯ 28, n ϭ 4, P Ͻ 0.05). Login to comment 198 ABCA1 p.Gln597Arg 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 199 ABCA1 p.Gln597Arg 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 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp 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 205 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp 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 216 ABCA1 p.Trp590Ser This is the case of the Tangier variant W590S, whose behavior has also been documented by Fitzgerald et al. (22). Login to comment 217 ABCA1 p.Trp590Ser This is the case of the Tangier variant W590S, whose behavior has also been documented by Fitzgerald et al. (22). Login to comment 228 ABCA1 p.Cys1477Arg Since the presence of disulfide bridges connecting the two halves of the protein has been shown for ABCR and, by extension, suggested for ABCA1, C1477 may well be strategic for the structural configuration of extracellular domains (23); however, considering that the loss of apoA-I binding in C1477R can be rescued by a suppressive mutation in the loop between TM5 and TM6 unable in itself to restore a disulfide bridge, it seems more likely that the coexistence of two discrete modifications in the putative apoA-I docking domain can fine tune its spatial conformation. Login to comment 229 ABCA1 p.Cys1477Arg Since the presence of disulfide bridges connecting the two halves of the protein has been shown for ABCR and, by extension, suggested for ABCA1, C1477 may well be strategic for the structural configuration of extracellular domains (23); however, considering that the loss of apoA-I binding in C1477R can be rescued by a suppressive mutation in the loop between TM5 and TM6 unable in itself to restore a disulfide bridge, it seems more likely that the coexistence of two discrete modifications in the putative apoA-I docking domain can fine tune its spatial conformation. Login to comment