ABCMdb

PMID: 14660648

Denis M, Haidar B, Marcil M, Bouvier M, Krimbou L, Genest J Jr
Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1).
J Biol Chem. 2004 Feb 27;279(9):7384-94. Epub 2003 Dec 4., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited ␣-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated ␣-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. Login to comment 37 ABCA1 p.Gln597Arg EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from 3 normal control subjects and 1 patient with TD (homozygous for Q597R at the ABCA1 gene). Login to comment 38 ABCA1 p.Gln597Arg EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from 3 normal control subjects and 1 patient with TD (homozygous for Q597R at the ABCA1 gene). Login to comment 61 ABCA1 p.Gln597Arg ABCA1 mutant (Q597R) was used as a negative control. Login to comment 62 ABCA1 p.Gln597Arg ABCA1 mutant (Q597R) was used as a negative control. Login to comment 124 ABCA1 p.Gln597Arg In addition, ABCA1 mutant (Q597R) that has been shown previously to not cross-link to apoA-I (13) was used as a negative control for the present experiment and showed no binding or cross-linking to ABCA1 (Fig. 3, A and B). Login to comment 126 ABCA1 p.Gln597Arg In addition, ABCA1 mutant (Q597R) that has been shown previously to not cross-link to apoA-I (13) was used as a negative control for the present experiment and showed no binding or cross-linking to ABCA1 (Fig. 3, A and B). Login to comment 127 ABCA1 p.Gln597Arg Also, to rule out the possibility that treatment with phospholipases might induce membrane aggregation or affect ABCA1 protein structure, which may trap apoA-I and result in nonspecific cross-linking, we examined the effect of phospholipases treatment on the cross-linking of 125 I-apoA-I to ABCA1 mutant (Q597R). Login to comment 128 ABCA1 p.Gln597Arg As shown in Fig. 3B (upper panel), 125 I-apoA-I did not cross-link to Q597R mutant whether treated with PC-PLC, SM-ase, or left intact. Login to comment 129 ABCA1 p.Gln597Arg Also, to rule out the possibility that treatment with phospholipases might induce membrane aggregation or affect ABCA1 protein structure, which may trap apoA-I and result in nonspecific cross-linking, we examined the effect of phospholipases treatment on the cross-linking of 125 I-apoA-I to ABCA1 mutant (Q597R). Login to comment 130 ABCA1 p.Gln597Arg As shown in Fig. 3B (upper panel), 125 I-apoA-I did not cross-link to Q597R mutant whether treated with PC-PLC, SM-ase, or left intact. Login to comment 137 ABCA1 p.Gln597Arg In addition, to ensure that cholesterol efflux was ABCA1-dependent in our cell culture system, apoA-I-mediated cholesterol efflux from ABCA1 mutant (Q597R) cells was also monitored. Login to comment 138 ABCA1 p.Gln597Arg In order to investigate the nature of apoA-I-containing particles generated by ABCA1 activity, stimulated cells from either normal or from TD (Q597R) subjects in 100 mm diameter dishes were incubated with 10 òe;g/ml of 125 I-apoA-I in 8 ml of DMEM for 24 h at 37 &#b0;C. Login to comment 139 ABCA1 p.Gln597Arg In addition, to ensure that cholesterol efflux was ABCA1-dependent in our cell culture system, apoA-I-mediated cholesterol efflux from ABCA1 mutant (Q597R) cells was also monitored. Login to comment 140 ABCA1 p.Gln597Arg In order to investigate the nature of apoA-I-containing particles generated by ABCA1 activity, stimulated cells from either normal or from TD (Q597R) subjects in 100 mm diameter dishes were incubated with 10 ␮g/ml of 125 I-apoA-I in 8 ml of DMEM for 24 h at 37 °C. Login to comment 141 ABCA1 p.Gln597Arg In contrast, lipid-free apoA-I incubated with stimulated mutant Q597R cells was unable to form such particles (panel C), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (panel A). Login to comment 143 ABCA1 p.Gln597Arg In contrast, lipid-free apoA-I incubated with stimulated mutant Q597R cells was unable to form such particles (panel C), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (panel A). Login to comment 194 ABCA1 p.Gln597Arg ABCA1 mutant (Q597R) was used as a negative control. Login to comment 195 ABCA1 p.Gln597Arg B, upper panel, intact stimulated normal or Q597R cells in 100-mm diameter dishes were incubated or not with PC-PLC or SM-ase as described above and then incubated with 10 òe;g/ml of 125 I-apoA-I for 1 h at 37 &#b0;C in the presence or absence of a 20-fold excess of unlabeled apoA-I (200 òe;g/ml). Login to comment 196 ABCA1 p.Gln597Arg ABCA1 mutant (Q597R) was used as a negative control. Login to comment 197 ABCA1 p.Gln597Arg B, upper panel, intact stimulated normal or Q597R cells in 100-mm diameter dishes were incubated or not with PC-PLC or SM-ase as described above and then incubated with 10 ␮g/ml of 125 I-apoA-I for 1 h at 37 °C in the presence or absence of a 20-fold excess of unlabeled apoA-I (200 ␮g/ml). Login to comment 230 ABCA1 p.Gln597Arg B, stimulated normal or Q597R cells were radiolabeled with [3 H]cholesterol and incubated with 10 òe;g/ml of apoA-I or 1 mg/ml of BSA at 37 &#b0;C for the indicated time points. Login to comment 233 ABCA1 p.Gln597Arg B, stimulated normal or Q597R cells were radiolabeled with [3 H]cholesterol and incubated with 10 ␮g/ml of apoA-I or 1 mg/ml of BSA at 37 °C for the indicated time points. Login to comment 239 ABCA1 p.Gln597Arg 125 I-apoA-I was incubated in DMEM/BSA for 24 h at 37 &#b0;C without cells (A) or with both stimulated normal and Q597R cells (B and C, respectively) for 24 h at 37 &#b0;C. Login to comment 242 ABCA1 p.Gln597Arg 125 I- apoA-I was incubated in DMEM/BSA for 24 h at 37 °C without cells (A) or with both stimulated normal and Q597R cells (B and C, respectively) for 24 h at 37 °C. Login to comment 249 ABCA1 p.Gln597Arg Results shown are representative of two different independent experiments. Lipidation of ApoA-I by ABCA1 to form larger particles during its incubation with Q597R mutant cells. Login to comment 253 ABCA1 p.Gln597Arg to form larger particles during its incubation with Q597R mutant cells. Login to comment