ABCMdb

PMID: 15280376

Denis M, Haidar B, Marcil M, Bouvier M, Krimbou L, Genest J
Characterization of oligomeric human ATP binding cassette transporter A1. Potential implications for determining the structure of nascent high density lipoprotein particles.
J Biol Chem. 2004 Oct 1;279(40):41529-36. Epub 2004 Jul 26., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCA1 p.Gln597Arg Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. Login to comment 9 ABCA1 p.Gln597Arg Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. Login to comment 37 ABCA1 p.Gln597Arg EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from three normal control subjects and one patient with Tangier disease (homozygous for Q597R at the ABCA1 gene). Login to comment 39 ABCA1 p.Gln597Arg EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from three normal control subjects and one patient with Tangier disease (homozygous for Q597R at the ABCA1 gene). Login to comment 58 ABCA1 p.Gln597Arg The presence of labeled 125 I-apoA-I/ABCA1 complexes was directly detected by autoradiography by using XAR-2 Kodak film. Isolation of Nascent LpA-I Particles and Cross-linking with DSP- 125 I-apoA-I (10 òe;g/ml) was incubated in DMEM for 24 h at 37 &#b0;C with either stimulated normal or Q597R cells. Login to comment 60 ABCA1 p.Gln597Arg 15 òe;g of isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells was adjusted to 10 mM sodium phosphate, 140 mM NaCl, pH 7.4, with a final protein concentration of 1 òe;g/òe;l. Immediately before an experiment, 1 mg of DSP was dissolved in 1000 òe;l of Me2SO to a final concentration of 1 òe;g/òe;l. The dissolved DSP was added to the reaction mixture for 10 DSP to 1 apoA-I molar ratio on ice. Login to comment 61 ABCA1 p.Gln597Arg Isolation of Nascent LpA-I Particles and Cross-linking with DSP- 125 I-apoA-I (10 ␮g/ml) was incubated in DMEM for 24 h at 37 °C with either stimulated normal or Q597R cells. Login to comment 63 ABCA1 p.Gln597Arg 15 ␮g of isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells was adjusted to 10 mM sodium phosphate, 140 mM NaCl, pH 7.4, with a final protein concentration of 1 ␮g/␮l. Immediately before an experiment, 1 mg of DSP was dissolved in 1000 ␮l of Me2SO to a final concentration of 1 ␮g/␮l. The dissolved DSP was added to the reaction mixture for 10 DSP to 1 apoA-I molar ratio on ice. Login to comment 110 ABCA1 p.Gln597Arg To determine the relationship between oligomeric ABCA1 complex and the structural properties of nascent apoA-I-containing particles in our cell culture model, stimulated cells either from normal or from Tangier disease (Q597R) subjects in 100-mm diameter dishes were incubated with 10 òe;g/ml 125 I-apoA-I in 6 ml of DMEM for 24 h at 37 &#b0;C. Login to comment 114 ABCA1 p.Gln597Arg To determine the relationship between oligomeric ABCA1 complex and the structural properties of nascent apoA-I-containing particles in our cell culture model, stimulated cells either from normal or from Tangier disease (Q597R) subjects in 100-mm diameter dishes were incubated with 10 ␮g/ml 125 I- apoA-I in 6 ml of DMEM for 24 h at 37 °C. Login to comment 120 ABCA1 p.Gln597Arg In contrast, lipid-free apoA-I incubated with stimulated ABCA1 mutant (Q597R) cells was unable to form such particles (third gel), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (first gel). Login to comment 123 ABCA1 p.Gln597Arg In contrast, lipid-free apoA-I incubated with stimulated ABCA1 mutant (Q597R) cells was unable to form such particles (third gel), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (first gel). Login to comment 145 ABCA1 p.Gln597Arg Either lipid-free apoA-I incubated without cells or lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells were used as controls. Login to comment 147 ABCA1 p.Gln597Arg In contrast, both lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells and lipid-free apoA-I incubated without cells remained in the monomeric form following cross-linking. Login to comment 150 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg Either lipid-free apoA-I incubated without cells or lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells were used as controls. Login to comment 152 ABCA1 p.Gln597Arg In contrast, both lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells and lipid-free apoA-I incubated without cells remained in the monomeric form following cross-linking. Login to comment 154 ABCA1 p.Gln597Arg Lower panel, isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells were treated with DSP as described under "Experimental Procedures." Login to comment 155 ABCA1 p.Gln597Arg Upper panels, 125 I-apoA-I (10 ␮g/ml) was incubated in DMEM for 24 h at 37 °C without cells (first gel) or with both stimulated normal and Q597R cells (second and third gels, respectively) for 24 h at 37 °C. Login to comment 159 ABCA1 p.Gln597Arg Lower panel, isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells were treated with DSP as described under "Experimental Procedures." Login to comment 175 ABCA1 p.Gln597Arg We demonstrate that lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells remained in the monomeric form (Fig. 5, lower panel). Login to comment 177 ABCA1 p.Cys1477Arg Interestingly, we found that pre-beta- LpE3 contains four and eight molecules of apoE3 per particle, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) remained in the monomeric form (data not shown). Login to comment 180 ABCA1 p.Gln597Arg We demonstrate that lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells remained in the monomeric form (Fig. 5, lower panel). Login to comment 182 ABCA1 p.Cys1477Arg Interestingly, we found that pre-beta- LpE3 contains four and eight molecules of apoE3 per particle, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) remained in the monomeric form (data not shown). Login to comment 194 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg Although ABCA1 mutants Q597R and C1477R were found to oligomerize normally (data not shown) and localized to the plasma membrane, they showed the total absence of binding to apoA-I (21, 23). Login to comment 195 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg These results indicate that the apoA-I lipidation defect observed in either Q597R or C1477R ABCA1 mutants is not caused by impaired oligomerization of ABCA1. Login to comment 196 ABCA1 p.Cys1477Arg Furthermore, C1477R, a naturally occurring mutant of ABCA1 in which cysteine 1477 within the second large extracellular loop is replaced with arginine (10), was found to dimerize normally. Login to comment 199 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg Although ABCA1 mutants Q597R and C1477R were found to oligomerize normally (data not shown) and localized to the plasma membrane, they showed the total absence of binding to apoA-I (21, 23). Login to comment 200 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg These results indicate that the apoA-I lipidation defect observed in either Q597R or C1477R ABCA1 mutants is not caused by impaired oligomerization of ABCA1. Login to comment 201 ABCA1 p.Cys1477Arg Furthermore, C1477R, a naturally occurring mutant of ABCA1 in which cysteine 1477 within the second large extracellular loop is replaced with arginine (10), was found to dimerize normally. Login to comment