ABCMdb

PMID: 23559627

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

Sentences

No. Mutations Sentence Comment

1 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg One recent model from Phillips proposes that ABCA1 shuttles phospholipids from the inner to extracellular face of the plasma membrane, resulting in membrane bulges with high curvature that are sufficient to allow apoAI penetration and nHDL formation.4 Mutations in ABCA1 lead to Tangier disease and familial hypoalphalipoproteinemia, characterized by very low levels of plasma HDL-cholesterol.5 Functional studies of certain Tangier disease mutations that are properly trafficked to the plasma membrane demonstrate that ABCA1 seems to have at least 2 distinct activities6 : apoAI binding and plasma membrane remodeling, the latter demonstrated through phosphatidylserine (PS) translocation to the outer leaflet of the plasma membrane.7 The W590S mutation in the first extracellular domain of ABCA1 is defective in PS translocation, but competent for apoAI binding.5,7,8 In contrast, the C1477R mutation in the second extracellular domain of ABCA1 is defective in apoAI binding, but competent for PS translocation.7 Further demonstration of the ability of ABCA1, and the deficit of W590S isoform, to remodel the plasma membrane was provided by Nagao et al,8 who used sodium taurocholate (NaTC) as a weak detergent extracellular lipid acceptor; wild type (WT) but not W590S ABAC1 mediates increased lipid efflux to NaTC. Login to comment 3 ABCA1 p.Lys939Met Although not discovered in a Tangier disease subject, the K939M mutation in the first ATP-binding domain has been shown to be defective in phosphatidylserine translocation, apoAI binding, and cholesterol efflux.9-11 In addition to nHDL, apoAI can spontaneously form reconstituted HDL (rHDL) particles in vitro when incubated with dimyristoylphosphatidylcholine (DMPC) dispersions or liposomes but not when incubated with the physiologically (c) 2013 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.112.301195 Objective-To gain insight into the mechanism by which ABCA1 generates nascent high-density lipoprotein. Login to comment 4 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met Approach and Results-HEK293 cells were stably transfected with ABCA1 vectors, encoding wild type, and the W590S and C1477R Tangier disease mutation isoforms, along with the K939M ATP-binding domain mutant. Login to comment 6 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met The W590S isoform had decreased plasma membrane remodeling and lipid efflux activities, and the C1477R isoform had decreased apoAI binding, and lipid efflux activities, whereas the K939M isoform did not bind apoAI, remodel the membrane, or efflux cholesterol. Login to comment 24 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met HEK293 cells were stably transfected with different murine ABCA1-green fluorescent protein (GFP) fusion vectors encoding WT, K939M, W590S, and C1477R isoforms, the latter 2 identified as Tangier disease-causing mutations in the first and second large extracellular domains, respectively.6 Several clonally derived cell lines from each construction were screened by confocal fluorescence microscopy and flow cytometry to select lines for further study with equivalent ABCA1 expression. Login to comment 25 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met As previously described,5,9 WT, W590S, C1477R, and K939M ABCA1-GFP fusions were processed correctly in cells and expressed on the plasma membrane (Figure 1A). Login to comment 28 ABCA1 p.Trp590Ser We confirmed the previously identified apoAI-binding activity of the WT and W590S-ABCA1 isoforms, with both having ࣈ6-fold higher apoAI binding than the control cells (P<0.001 by ANOVA posttest). Login to comment 29 ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met In contrast, the C1477R and K939M isoform-expressing cells had no significant increase in apoAI binding compared with control cells (by ANOVA posttest). Login to comment 31 ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met We were able to take advantage of this nonspecific apoAI binding to the control and the C1477R and K939M isoform-expressing cells in cell studies described below. Login to comment 32 ABCA1 p.Cys1477Arg ABCA1 has been shown to possess PS floppase activity, resulting in plasma membrane remodeling that has been postulated to facilitate HDL assembly.7 Using flow cytometry to measure cell surface PS assayed by Cy5-AnnexinV binding, we showed that WT and C1477R-ABCA1 isoforms increased cell surface PS by ࣈ2.2-fold compared with control cells (P<0.001 by ANOVA posttest). Login to comment 33 ABCA1 p.Trp590Ser ABCA1 p.Lys939Met The W590S cells had a small 1.26-fold increase in cell surface PS (P<0.05 versus control by ANOVA posttest), whereas the K939M-ABCA1 isoform had no cell surface PS increase (Figure 2B). Login to comment 34 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg Thus, our results confirmed previous findings by Singaraja et al5 and Nagao et al8 that the W590S mutation in ABCA1`s first extracellular domain greatly diminishes PS floppase activity indicative of a defect in plasma membrane remodeling, whereas the C1477R mutation in its second large extracellular domain abolishes apoAI binding. Login to comment 36 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg In the absence of any acceptor, WT ABCA1 increased basal 3 H cholesterol efflux by 32% (P<0.05 by ANOVA posttest versus HEK), which has previously been shown to be attributable to increased microparticle release.14 The C1477R-ABCA1 isoform also had increased basal cholesterol efflux activity (34% increase; P<0.05); however, efflux from the W590S-ABCA1 isoform cell line was not significantly different from the control HEK cells. Login to comment 39 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met A, Confocal microscopy of wild type (WT), W590S, C1477R, and K939M ABCA1-GFP isoforms shows cell surface expression. Login to comment 40 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met B, Similar expression levels of WT, W590S, C1477R, and K939M ABCA1-GFP cell lines shown by flow cytometry (n=3; mean&#b1;SD; different numbers above the bars show P<0.001, by ANOVA posttest). Login to comment 42 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg Interestingly, both Tangier disease mutations supported partial efflux to apoAI with 4.27and 4.02-fold increases above the control HEK cells for the W590S and C1477R isoforms, respectively. Login to comment 43 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg All 4 cell lines have significantly different efflux to apoAI (P<0.001 by ANOVA posttest), except for efflux from the W590S and C1477R cells that were not different from each other. Login to comment 44 ABCA1 p.Lys939Met In a separate study, we found that the K939M cells had efflux to apoAI similar to the nontransfected control HEK cells, thus they had no detectable ABCA1 activity as had been previously determined.5 In the presence of the weak detergent NaTC, the control HEK cells increased cholesterol efflux to 2.62% (a 5.36-fold increase versus absence of acceptor). Login to comment 46 ABCA1 p.Trp590Ser The W590S-ABCA1 isoform, which can mediate binding of apoAI, yielded a similar cholesterol efflux as the HEK cells at 2.57%. Login to comment 47 ABCA1 p.Cys1477Arg However, the C1477R-ABCA1 isoform yielded 5.04% cholesterol efflux, more similar to the WT ABCA1 isoform. Login to comment 48 ABCA1 p.Cys1477Arg Thus, both in the absence of acceptor or in the presence of NaTC, cholesterol efflux followed PS floppase activity (highest for WT and C1477R isoforms), and we confirmed the use of NaTC acceptor as an independent indicator of ABCA1-mediated membrane remodeling. Login to comment 49 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg Thus, our findings show that ABCA1 mutations that disrupt either plasma membrane remodeling (W590S) or apoAI binding (C1477R) are still competent to mediate cholesterol efflux to apoAI, albeit at reduced efficiency. Login to comment 62 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met A, Alexa647-labeled apoAI binding to nontransfected cells (HEK) and cells stably transfected with wild type (WT), W590S, C1477R, and K939M ABCA1-green fluorescent protein (GFP) vectors assayed by flow cytometry (n=3; mean&#b1;SD; P<0.0005 for HEK in the presence or absence of labeled apoAI by t test; for the 5 cell types in the presence of labeled apoAI different numbers above the bars show P<0.001; by ANOVA posttest). Login to comment 64 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg C, Cholesterol efflux from control HEK cells and WT, W590S, and C1477R ABCA1-GFP transfected cells in the absence of exogenous acceptors or in the presence of 5 bc;g/mL apoAI or 2 mmol/L sodium taurocholate (NaTC; n=3; mean&#b1;SD; different numbers above the bars show P<0.05, by ANOVA posttest). Login to comment 69 ABCA1 p.Cys1477Arg ABCA1 p.Lys939Met Here, we took advantage of the high sensitivity of flow cytometry and our observation that even control HEK cells and cells expressing the apoAI-binding defective C1477R and K939M-ABCA1 isoforms bound apoAI nonspecifically, enabling us to determine the Bodipy-TMR/Alexa647 ratio of each cell. Login to comment 71 ABCA1 p.Trp590Ser However, on incubation of this indicator with the WT and W590S ABCA1-expressing cells, there was a rightward shift to a higher cellular Bodipy-TMR/Alexa647 ratio peak of 1.3 to 1.4 (1.86-2.0-fold increase). Login to comment 72 ABCA1 p.Lys939Met The C1447R and K939M apoAI-binding deficient mutants displayed intermediate activity with Bodipy-TMR/Alexa647 ratio peaks of 1.0 (1.43-fold increase versus control HEK cells). Login to comment 74 ABCA1 p.Trp590Ser The partially active W590S isoform has 2 activities: apoAI binding and apoAI unfolding. Login to comment 75 ABCA1 p.Cys1477Arg The other partially active C1477R isoform has 2 activities: apoAI unfolding (albeit reduced) and plasma membrane remodeling. Login to comment 76 ABCA1 p.Lys939Met Finally, the defective K939M isoform retains only one activity: apoAI unfolding (also reduced). Login to comment 84 ABCA1 p.Lys939Met Medium recovered from control HEK cells and the defective K939M-ABAC1 isoform Figure 3. Login to comment 92 ABCA1 p.Trp590Ser The W590S and C1447R mutant isoforms had intermediate ratios ࣈ1.4 and 1.2-fold higher than that observed from the control HEK cells (P<0.001 and P<0.05, respectively, versus control cells by ANOVA posttest). Login to comment 94 ABCA1 p.Trp590Ser We attribute the intermediate fluorescence ratio levels in the conditioned media from the W590S and C1447R transfected cells to be a result of fewer lipidated particles released into the conditioned media, rather than to less unfolding of apoAI per particle. Login to comment 113 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg We chose to further study 2 specific mutations in the first (W590S) and second (C1477R) extracellular domains, respectively, based on their previously identified distinct activities. Login to comment 114 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu 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 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp 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 116 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg Our findings reproduce that the W590S isoform has plasma membrane localization and WT levels of apoAI-binding activity; and that it has partial cholesterol efflux activity to extracellular apoAI compared with the WT isoform, as previously demonstrated.5,7,8,18 We chose to study the C1477R mutation in the second extracellular domain because it is processed correctly to the plasma membrane and has defective apoAI binding,5,7,17 but it retains its PS translocase activity and partial efflux activity,7 all findings that we confirmed in the current study, where we found ࣈ50% cholesterol efflux activity to apoAI. Login to comment 117 ABCA1 p.Trp590Ser Nagao et al8 first demonstrated the use of NaTC as a nonpeptide acceptor of cellular lipids, and that WT ABCA1 increased lipid efflux to this weak detergent, and that the W590S mutation abolished this activity. Login to comment 118 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg Here, we compared the acceptor activity of NaTC for cells expressing WT, W590S, and C1477R-ABCA1 isoforms, and found that the C1477R mutant has equivalent efflux to NaTC as the WT isoform, whereas the W590S mutant has no detectable efflux to NaTC above nontransfected cells. Login to comment 119 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg The NaTC efflux activities of these ABCA1 isoforms is similar to the PS translocase activity, and thus both of these assays are evidence that the WT and C1477R isoforms can remodel the plasma membrane, whereas the W590S isoform is mostly deficient in this activity. Login to comment 130 ABCA1 p.Trp590Ser Through the use of an N-terminal apoAI unfolding indicator, we observed what we estimate to be ࣈ75% unfolding of apoAI on the surface of HEK cells expressing either WT or W590S-ABCA1 isoforms, both of which have full apoAI-binding activity (Figure 4A). Login to comment 131 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The slight rightward shift of the W590S isoform may be as a result of defective membrane remodeling in the W590S-expressing cells, leading to the unfolded apoAI having fewer opportunities to form rHDL and be released from the cell. Login to comment 132 ABCA1 p.Lys939Met Interestingly, the 2 ABCA1 isoforms with impaired apoAI binding, C1447R and K939M, also displayed some, albeit reduced, apoAI unfolding activity compared with nontransfected HEK cells (Figure 4A). Login to comment 133 ABCA1 p.Lys939Met Because the K939M isoform is also deficient in plasma membrane remodeling, this partial unfolding activity cannot be attributed to this membrane remodeling. Login to comment 138 ABCA1 p.Trp590Ser The presence of the high-affinity apoAI-binding site in WT and W590S-ABCA1 isoforms would promote apoAI proximity to the low-affinity binding site and therefore increase apoAI unfolding. Login to comment 149 ABCA1 p.Trp590Ser A model for the mechanism of ABCA1 action from Phillips states that phospholipid translocation produces membrane protuberances that in themselves because of their small radius and surface packing are sufficient to spontaneously interact with apoAI and form nHDL.4 However, the observation that the W590S-ABCA1 isoform is not competent for phospholipid translocation and membrane remodeling but is still able to mediate HDL assembly, albeit at reduced efficiency, does not support this model. Login to comment 152 ABCA1 p.Lys939Met It is of interest that both the apoAI binding and the plasma membrane remodeling activities are disrupted in the K939M isoform without totally abolishing this apoAI unfolding activity. Login to comment 158 ABCA1 p.Lys939Met The levels of apoAI in the Int and U states are increased by specific binding to ABCA1, but these apoAI states are still present in cells expressing the C1447C and K939M-ABCA1 isoforms that lack high-affinity apoAI binding. Login to comment 161 ABCA1 p.Trp590Ser We propose the steps through formation of the LC state may be reversible, and there is experimental evidence that W590S ABCA1-expressing cells bind and release nonlipidated apoAI from the cell,30 and we show here that the apoAI bound by this mutant isoform is also in the U state. Login to comment