ABCMdb

PMID: 19202195

Nagao K, Zhao Y, Takahashi K, Kimura Y, Ueda K
Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation.
J Lipid Res. 2009 Jun;50(6):1165-72. Epub 2009 Feb 8., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCA1 p.Trp590Ser Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation Kohjiro Nagao,* Yu Zhao,* Kei Takahashi,* Yasuhisa Kimura,* and Kazumitsu Ueda1,*,† * Laboratory of Cellular Biochemistry,* Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan; and Institute for Integrated Cell-Material Sciences,† Kyoto University, Kyoto 606-8502, Japan Abstract ABCA1 plays a major role in HDL metabolism. Login to comment 5 ABCA1 p.Trp590Ser The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. Login to comment 6 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. Login to comment 7 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser However, the W590S mutation did not impair apoA-I binding and, conversely, retarded the dissociation of apoA-I from ABCA1. Login to comment 8 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading, facilitates apoA-I dissociation from ABCA1. Login to comment 9 ABCA1 p.Trp590Ser NaTC is a good tool for analyzing ABCA1-mediated lipid efflux and allows dissection of the steps of HDL formation by ABCA1.- Nagao, K., Y. Zhao, K. Takahashi, Y. Kimura, and K. Ueda. Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation. Login to comment 43 ABCA1 p.Trp590Ser ABCA1 p.Lys939Met ABCA1 p.Lys1952Met Plasmids The expression vectors for wild-type ABCA1, ABCA1-W590S, and ABCA1-K939M,K1952M (MM), fused to GFP at their C termini, were generated as described previously (3, 18). Login to comment 45 ABCA1 p.Trp590Ser ABCA1 p.Lys939Met ABCA1 p.Lys1952Met Cell culture Human embryonic kidney (HEK293) cells and WI-38 fibroblasts were grown in a humidified incubator (5% CO2) at 37°C in DMEM supplemented with 10% heat-inactivated FBS. Plasmids The expression vectors for wild-type ABCA1, ABCA1-W590S, and ABCA1-K939M,K1952M (MM), fused to GFP at their C termini, were generated as described previously (3, 18). Login to comment 96 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The W590S Tangier mutation impairs NaTC-dependent lipid efflux but not apoA-I binding It has been reported that ABCA1 carrying the Tangier disease mutation W590S in the first extra cellular domain is correctly targeted to the plasma membrane and interacts with apoA-I but fails to mediate normal apoA-I-dependent lipid efflux (3, 26-28). Login to comment 97 ABCA1 p.Trp590Ser We compared NaTC-dependent lipid efflux from HEK293 cells stably expressing wild-type or ABCA1-W590S fused to GFP at the C terminus. Login to comment 98 ABCA1 p.Trp590Ser Western blotting (Fig. 4A) and fluorescence microscopy (see supplementary Fig. II) suggested that the expression and surface localization of the wild type and ABCA1-W590S were similar. Login to comment 99 ABCA1 p.Trp590Ser Both NaTC-dependent and apoA-I-dependent cholesterol and phospholipid efflux were greatly decreased by the W590S mutation (Fig. 4B, C). Login to comment 100 ABCA1 p.Trp590Ser As previously reported, apoA-I bound to the cell surface expressing ABCA1-W590S as efficiently as to that expressing wild-type ABCA1 (see supplementary Fig. II). Login to comment 101 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The W590S Tangier mutation impairs NaTC-dependent lipid efflux but not apoA-I binding It has been reported that ABCA1 carrying the Tangier disease mutation W590S in the first extra cellular domain is correctly targeted to the plasma membrane and interacts with apoA-I but fails to mediate normal apoA-I-dependent lipid efflux (3, 26-28). Login to comment 102 ABCA1 p.Trp590Ser We compared NaTC-dependent lipid efflux from HEK293 cells stably expressing wild-type or ABCA1-W590S fused to GFP at the C terminus. Login to comment 103 ABCA1 p.Trp590Ser Western blotting (Fig. 4A) and fluorescence microscopy (see supplementary Fig. II) suggested that the expression and surface localization of the wild type and ABCA1-W590S were similar. Login to comment 104 ABCA1 p.Trp590Ser Both NaTC-dependent and apoA-I-dependent cholesterol and phospholipid efflux were greatly decreased by the W590S mutation (Fig. 4B, C). Login to comment 105 ABCA1 p.Trp590Ser As previously reported, apoA-I bound to the cell surface expressing ABCA1-W590S as efficiently as to that expressing wild-type ABCA1 (see supplementary Fig. II). Login to comment 114 ABCA1 p.Trp590Ser 1168 Journal of Lipid Research Volume 50, 2009 at SEMMELWEIS EGYETEM NET KORELET, on December 3, 2015 www.jlr.org Downloaded from .DC1.html are mediated by ABCA1 in an ATP-dependent manner, are in fact separable and that the W590S mutation impairs only the latter step. Login to comment 119 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser 1168 Journal of Lipid Research Volume 50, 2009 atHealthScienceLibraryCB#7585,onSeptember24,2012www.jlr.orgDownloadedfrom 0.DC1.html are mediated by ABCA1 in an ATP-dependent manner, are in fact separable and that the W590S mutation impairs only the latter step. Login to comment 122 ABCA1 p.Trp590Ser The kinetics of apoA-I binding to cells expressing ABCA1-W590S was quite similar to that to cells expressing wild-type ABCA1. Login to comment 124 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The W590S mutation also impaired cholesterol secretion at 27°C. Login to comment 125 ABCA1 p.Trp590Ser These results suggest that the W590S mutation retards apoA-I dissociation from cells. Login to comment 127 ABCA1 p.Trp590Ser The kinetics of apoA-I binding to cells expressing ABCA1-W590S was quite similar to that to cells expressing wild-type ABCA1. Login to comment 129 ABCA1 p.Trp590Ser In contrast, ,40% of apoA-I dissociated from cells expressing ABCA1-W590S in 2 min, and ?60% of apoA-I dissociated in 4 min. Login to comment 130 ABCA1 p.Trp590Ser These results suggest that the W590S mutation retards apoA-I dissociation from cells. Login to comment 133 ABCA1 p.Trp590Ser The W590S mutation impairs NaTC-dependent lipid efflux by ABCA1. Login to comment 136 ABCA1 p.Trp590Ser The efflux of cellular free cholesterol (B) and choline phospholipids (C) was analyzed. HEK/ABCA1-GFP cells and HEK/ ABCA1-W590S-GFP cells were incubated for 24 h in DMEM containing 0.02% BSA (open bars), 0.02% BSA and 1 mM NaTC (filled bars), or 0.02% BSA and 5 mg/ml apoA-I (hatched bars). Login to comment 138 ABCA1 p.Trp590Ser The W590S mutation impairs NaTC-dependent lipid efflux by ABCA1. Login to comment 139 ABCA1 p.Trp590Ser HEK/ABCA1-GFP, HEK/ABCA1-MM-GFP, or HEK/ABCA1-W590S-GFP cells were incubated with 5 mg/ml Alexa546-labeled apoA-I for the indicated times at 27&#b0;C. Login to comment 141 ABCA1 p.Trp590Ser The efflux of cellular free cholesterol (B) and choline phospholipids (C) was analyzed. HEK/ABCA1-GFP cells and HEK/ ABCA1-W590S-GFP cells were incubated for 24 h in DMEM containing 0.02% BSA (open bars), 0.02% BSA and 1 mM NaTC (filled bars), or 0.02% BSA and 5 mg/ml apoA-I (hatched bars). Login to comment 144 ABCA1 p.Trp590Ser HEK/ABCA1-GFP, HEK/ABCA1-MM-GFP, or HEK/ABCA1-W590S-GFP cells were incubated with 5 mg/ml Alexa546-labeled apoA-I for the indicated times at 27°C. Login to comment 149 ABCA1 p.Trp590Ser Alternatively, binding of significant amounts of apoA-I on the cell surface may cause spontaneous cholesterol loading on apoA-I. Indeed, greater cholesterol efflux was observed in the presence of apoA-I from cells expressing ABCA1-W590S, which allows apoA-I binding, than in the presence of NaTC, and this effect was strongly suppressed at 27&#b0;C (Fig. 4; see supplementary Fig. VI). Login to comment 154 ABCA1 p.Trp590Ser There may be other proteins (32, 33) in WI-38 cells that mediate cholesterol efflux in the presence of apoA-I. Alternatively, binding of significant amounts of apoA-I on the cell surface may cause spontaneous cholesterol loading on apoA-I. Indeed, greater cholesterol efflux was observed in the presence of apoA-I from cells expressing ABCA1-W590S, which allows apoA-I binding, than in the presence of NaTC, and this effect was strongly suppressed at 27°C (Fig. 4; see supplementary Fig. VI). Login to comment 156 ABCA1 p.Trp590Ser The Tangier mutation W590S, which impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. Login to comment 157 ABCA1 p.Trp590Ser It has been reported that ABCA1-W590S interacts in a normal manner with ATP (3). Login to comment 158 ABCA1 p.Trp590Ser The W590S mutation may abolish the coupling of ATP-induced conformational changes of ABCA1 with lipid translocation. Login to comment 159 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The kinetics of apoA-I binding to cells expressing ABCA1-W590S were similar to those of binding to cells expressing wild-type ABCA1, consistent with a previous report showing that the W590S mutation does not impair apoA-I binding (8, 26-28). Login to comment 160 ABCA1 p.Trp590Ser Thus, the W590S mutation would not affect the conformational changes of ABCA1 Fig. 7. Login to comment 161 ABCA1 p.Trp590Ser The Tangier mutation W590S, which impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. Login to comment 162 ABCA1 p.Trp590Ser It has been reported that ABCA1-W590S interacts in a normal manner with ATP (3). Login to comment 163 ABCA1 p.Trp590Ser The W590S mutation may abolish the coupling of ATP-induced conformational changes of ABCA1 with lipid translocation. Login to comment 164 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The kinetics of apoA-I binding to cells expressing ABCA1-W590S were similar to those of binding to cells expressing wild-type ABCA1, consistent with a previous report showing that the W590S mutation does not impair apoA-I binding (8, 26-28). Login to comment 165 ABCA1 p.Trp590Ser Thus, the W590S mutation would not affect the conformational changes of ABCA1 Fig. 7. Login to comment 168 ABCA1 p.Trp590Ser Time-dependent apoA-I dissociation from cells expressing ABCA1 or ABCA1-W590S. Login to comment 169 ABCA1 p.Trp590Ser HEK/ABCA1-GFP and HEK/ABCA1-W590S-GFP cells were incubated with 5 mg/ml Alexa546-labeled apoA-I for 15 min at 37&#b0;C and further incubated with 25 mg/ml nonlabeled apoA-I for the indicated times at 27&#b0;C. Login to comment 173 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Time-dependent apoA-I dissociation from cells expressing ABCA1 or ABCA1-W590S. Login to comment 174 ABCA1 p.Trp590Ser HEK/ABCA1-GFP and HEK/ABCA1-W590S-GFP cells were incubated with 5 mg/ml Alexa546-labeled apoA-I for 15 min at 37°C and further incubated with 25 mg/ml nonlabeled apoA-I for the indicated times at 27°C. Login to comment 178 ABCA1 p.Trp590Ser The W590S mutation retarded the dissociation of apoA-I from ABCA1. Login to comment 181 ABCA1 p.Trp590Ser In addition, ABCA1-W590S was reported not to be stabilized by apoA-I (29). Login to comment 184 ABCA1 p.Trp590Ser Our results suggest that the W590S mutation impaired NaTC-dependent lipid efflux but not apoA-I binding. Login to comment 186 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser In addition, ABCA1-W590S was reported not to be stabilized by apoA-I (29). Login to comment 189 ABCA1 p.Trp590Ser Our results suggest that the W590S mutation impaired NaTC-dependent lipid efflux but not apoA-I binding. Login to comment 191 ABCA1 p.Trp590Ser These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading facilitates apoA-I dissociation from ABCA1. Login to comment