ABCMdb

PMID: 17656736

Hassan HH, Denis M, Lee DY, Iatan I, Nyholt D, Ruel I, Krimbou L, Genest J
Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis.
J Lipid Res. 2007 Nov;48(11):2428-42. Epub 2007 Jul 26., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCA1 p.Gln597Arg Glyburide drastically reduced 125I-apoA-I binding to the HCBS, whereas 125I- apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Login to comment 35 ABCA1 p.Gln597Arg MATERIALS AND METHODS 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), as described previously (14). Login to comment 103 ABCA1 p.Gln597Arg To test the specificity of the cross-liking and immunoprecipitation methods, the presence of a 30-fold excess of unlabeled apoA-I during the incubation of 125 I-apoA-I with 22OH/9CRA-stimulated fibroblasts, the absence of 22OH/9CRA treatment, or ABCA1 mutant fibroblasts (Q597R) drastically reduced the larger fraction of 125 I- apoA-I nonassociated with ABCA1 (Fig. 1B). Login to comment 106 ABCA1 p.Gln597Arg The observation that the presence of ABCA1 mutant Q597R abolished the association of 125 I-apoA-I to both ABCA1 associated and nonassociated Fig. 1. Login to comment 108 ABCA1 p.Gln597Arg A: Confluent unstimulated, 22-(R)-hydroxycholesterol/9-cis-retinoic acid (22OH/9CRA)-stimulated fibroblasts and ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I in the presence or absence of a 30-fold excess of unlabeled apoA-I (+ Excess) for 45 min at 37jC. Login to comment 134 ABCA1 p.Gln597Arg Affinity binding of apoA-I to the putative non-ABCA1 binding site To better characterize the new binding site for apoA-I, intact normal fibroblasts treated or not with 22OH/9CRA or ABCA1 mutant Q597R were incubated with increasing concentrations of 125I-apoA-I for 45 min at 37jC. Login to comment 137 ABCA1 p.Gln597Arg At the same time, ABCA1 mutant fibroblasts (Q597R) showed no significant binding to both sites (Fig. 3D). Login to comment 159 ABCA1 p.Gln597Arg Stimulated intact normal or ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I for 45 min at 37jC. Login to comment 162 ABCA1 p.Gln597Arg Furthermore, no significant apoA-I binding was detected in both PM and ICC fractions in ABCA1 mutant Q597R, used as a negative control in the present experiment (Fig. 5A). Login to comment 185 ABCA1 p.Gln597Arg Confluent normal (A-C) and ABCA1 mutant (Q597R) (D) fibroblasts were incubated with increasing concentrations of 125 I-apoA-I for 45 min at 37jC in the presence (A, B, D) or absence (C) of 22OH/9CRA or 300 mM glyburide (B). Login to comment 186 ABCA1 p.Gln597Arg Confluent normal (A-C) and ABCA1 mutant (Q597R) (D) fibroblasts were incubated with increasing concentrations of 125 I-apoA-I for 45 min at 37jC in the presence (A, B, D) or absence (C) of 22OH/9CRA or 300 mM glyburide (B). Login to comment 215 ABCA1 p.Gln597Arg In parallel experiments, we documented that apoA-I-mediated cholesterol efflux, as well as the formation of nascent LpA-I particles, after phospholipase treatments were absent in ABCA1 mutant (Q597R) fibroblasts under conditions similar to those used in the present experiments (data not shown). Login to comment 216 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg On the other hand, there is no evidence for lipid-free 125 I-apoA-I aggregation, as assessed by the complete removal of lipid-free 125 I-apoA-I incubated with ABCA1 mutant (Q597R) fibroblasts, under treatment or not with phospholipases by filtration with a 50,000 molecular weight cut off filter system (23). Login to comment 217 ABCA1 p.Gln597Arg On the other hand, there is no evidence for lipid-free 125 I-apoA-I aggregation, as assessed by the complete removal of lipid-free 125 I-apoA-I incubated with ABCA1 mutant (Q597R) fibroblasts, under treatment or not with phospholipases by filtration with a 50,000 molecular weight cut off filter system (23). Login to comment 227 ABCA1 p.Gln597Arg This is consistent with the results showing that overexpressing ABCA1 in BHK cells promoted the formation of the HCBS, whereas the inhibition of ABCA1 activity by glyburide or the presence of ABCA1 mutant fibroblasts (Q597R) drastically reduced the binding of apoA-I to the HCBS (Figs. 2, 3). Login to comment 228 ABCA1 p.Gln597Arg This is consistent with the results showing that overexpressing ABCA1 in BHK cells promoted the formation of the HCBS, whereas the inhibition of ABCA1 activity by glyburide or the presence of ABCA1 mutant fibroblasts (Q597R) drastically reduced the binding of apoA-I to the HCBS (Figs. 2, 3). Login to comment 244 ABCA1 p.Gln597Arg A: Confluent stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I for 45 min at 37jC. Login to comment 245 ABCA1 p.Gln597Arg A: Confluent stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I for 45 min at 37jC. Login to comment 265 ABCA1 p.Gln597Arg For example: 1) inhibition of ABCA1 activity by glyburide decreased the binding of apoA-I to both ABCA1 and the HCBS; 2) rLpA-I exhibited reduced affinity for ABCA1 and also for the HCBS (Fig. 4A); and 3) ABCA1 mutants (Q597R and C1447R) that failed to bind apoA-I also failed to mediate the formation of the HCBS. Login to comment 266 ABCA1 p.Gln597Arg For example: 1) inhibition of ABCA1 activity by glyburide decreased the binding of apoA-I to both ABCA1 and the HCBS; 2) rLpA-I exhibited reduced affinity for ABCA1 and also for the HCBS (Fig. 4A); and 3) ABCA1 mutants (Q597R and C1447R) that failed to bind apoA-I also failed to mediate the formation of the HCBS. Login to comment 319 ABCA1 p.Gln597Arg Furthermore, the presence of ABCA1 mutant Q597R abolished the association of apoA-I with both compartments, consistent with the idea that ABCA1 seems to be required for the association of apoA-I with both the PM and ICCs. Login to comment 320 ABCA1 p.Gln597Arg Furthermore, the presence of ABCA1 mutant Q597R abolished the association of apoA-I with both compartments, consistent with the idea that ABCA1 seems to be required for the association of apoA-I with both the PM and ICCs. Login to comment