ABCMdb

PMID: 15897603

Krimbou L, Hajj Hassan H, Blain S, Rashid S, Denis M, Marcil M, Genest J
Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines.
J Lipid Res. 2005 Aug;46(8):1668-77. Epub 2005 May 16., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCA1 p.Gln597Arg Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only -nascent apolipoprotein A-I-containing particles (-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Login to comment 2 ABCA1 p.Gln597Arg Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only ␣-nascent apolipoprotein A-I-containing particles (␣-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Login to comment 26 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg MATERIALS AND METHODS Patient selection For the present study, we selected fibroblasts from three normal control subjects and two patients with TD (TD1, homozygous for Q597R at the ABCA1 gene; and TD2, compound heterozygous carrying the mutations C1477R and the splice site G→C in exon 24), as described previously (13, 14). Login to comment 75 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg However, the incomplete removal of lipid-free apoA-I from the medium may result in an artifactual presence of prebeta1-LpA-I. To ensure the quality of the complete removal of lipid-free apoA-I, the filtration of the medium from either HepG2 cells or macrophages found to form prebeta1-LpA-I was carefully controlled for each experiment by the filtration of the medium from normal fibroblasts, ABCA1 mutant Q597R fibroblasts, or lipid-free 125I-apoA-I incubated without cells. Login to comment 77 ABCA1 p.Gln597Arg In separate experiments (see below), we show that both the centrifugal filter and the dialysis membrane retained native plasma pre1-LpA-I having an apparent molecular mass of 67 kDa. As shown in Fig. 1 (upper panels), incubation of stimulated normal fibroblasts, CaCo-2 cells, or CHO-overexpressing ABCA1 (CHO-ABCA1) with 10 g/ml exogenously added 125I-apoA-I for 6 h at 37C followed by the removal of lipid-free apoA-I as described above, and then separation of samples by 2D-PAGGE, generated only -LpA-I with a particle size ranging from 8 to 20 nm, whereas lipid-free apoA-I incubated with either HUVEC or ABCA1 mutant (Q597R) was unable to form such particles. Login to comment 78 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg As shown in Fig. 1 (upper panels), incubation of stimulated normal fibroblasts, CaCo-2 cells, or CHO-overexpressing ABCA1 (CHO-ABCA1) with 10 ␮g/ml exogenously added 125I-apoA-I for 6 h at 37ЊC followed by the removal of lipid-free apoA-I as described above, and then separation of samples by 2D-PAGGE, generated only ␣-LpA-I with a particle size ranging from 8 to 20 nm, whereas lipid-free apoA-I incubated with either HUVEC or ABCA1 mutant (Q597R) was unable to form such particles. Login to comment 79 ABCA1 p.Cys1477Arg In separate experiments, we found that ABCA1 mutant C1477R also failed to form ␣-LpA-I (data not shown). Login to comment 82 ABCA1 p.Gln597Arg Furthermore, inactivation of ABCA1 with glyburide in fibroblasts, CaCo-2, or CHO-ABCA1 led to the formation of smaller -LpA-I having a diameter of 8 nm; these particles were not formed in the presence of ABCA1 mutant Q597R cells. Login to comment 83 ABCA1 p.Gln597Arg Furthermore, inactivation of ABCA1 with glyburide in fibroblasts, CaCo-2, or CHO-ABCA1 led to the formation of smaller ␣-LpA-I having a diameter of ‫8ف‬ nm; these particles were not formed in the presence of ABCA1 mutant Q597R cells. Login to comment 100 ABCA1 p.Gln597Arg Normal fibroblasts, CaCo-2, human umbilical vein endothelial cells (HUVEC), HepG2, or ABCA1 mutant (Q597R) cells in 100 mm diameter dishes were loaded with 20 g/ml cholesterol for 24 h and then stimulated with 2.5 g/ml 22(R)-hydroxycholesterol and 10 M 9-cis-retinoic acid (22OH/9CRA) for 20 h. THP-1 cells and human monocyte-derived macrophages in 100 mm diameter dishes were loaded with 20 g/ml cholesterol for 24 h and then stimulated with 0.3 mM cAMP for 12 h. CHO-overexpressing ABCA1 cells (CHO-ABCA1) were used as a control. Login to comment 101 ABCA1 p.Gln597Arg Normal fibroblasts, CaCo-2, human umbilical vein endothelial cells (HUVEC), HepG2, or ABCA1 mutant (Q597R) cells in 100 mm diameter dishes were loaded with 20 ␮g/ml cholesterol for 24 h and then stimulated with 2.5 ␮g/ml 22(R)-hydroxycholesterol and 10 ␮M 9-cis-retinoic acid (22OH/9CRA) for 20 h. THP-1 cells and human monocyte-derived macrophages in 100 mm diameter dishes were loaded with 20 ␮g/ml cholesterol for 24 h and then stimulated with 0.3 mM cAMP for 12 h. CHO-overexpressing ABCA1 cells (CHO-ABCA1) were used as a control. Login to comment 105 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg beled with [14C]cholesterol or [32P]orthophosphate as described in Materials and Methods and then incubated with 10 ␮g/ml lipid-free apoA-I for 24 h at 37ЊC. Radiolabeled ABCA1 mutant Q597R cells were used as a control in the present experiment. Login to comment 107 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg At the same time, we have not been able to detect any ␣-LpA-I formation during incubation with ABCA1 mutant Q597R (Fig. 4). Login to comment 128 ABCA1 p.Gln597Arg In contrast, lipid-free apoA-I was unable to form such particles in the presence of HUVEC and ABCA1 mutant Q597R cells (Fig. 1), consistent with previous studies showing that lipid-free apoA-I did not promote cholesterol efflux from endothelial cells in which the expression of ABCA1 protein is very low and seems not to be sensitive to treatment with cholesterol or 22-hydroxycholesterol (24, 28). Login to comment 130 ABCA1 p.Gln597Arg Here, we present evidence that incubation of exogenously added lipid-free apoA-I with various cell lines, including human fibroblasts, CaCo-2, and CHO-ABCA1, generates only -LpA-I. In contrast, lipid-free apoA-I was unable to form such particles in the presence of HUVEC and ABCA1 mutant Q597R cells (Fig. 1), consistent with previous studies showing that lipid-free apoA-I did not promote cholesterol efflux from endothelial cells in which the expression of ABCA1 protein is very low and seems not to be sensitive to treatment with cholesterol or 22-hydroxycholesterol (24, 28). Login to comment 151 ABCA1 p.Gln597Arg This is also strongly supported by our results showing that glyburide did not affect the formation of prebeta1-LpA-I but had a dramatic effect on ␣-LpA-I, providing strong support for the Fig. 4. Characterization of lipidated apoA-I-containing particles generated during lipid-free apoA-I incubation with normal fibroblasts and ABCA1 mutant (Q597R). Login to comment 152 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg Normal fibroblasts or ABCA1 mutant (Q597R) were labeled with either [14C]cholesterol or [32P]orthophosphate as described in Materials and Methods and incubated with 10 ␮g/ml apoA-I for 24 h. Media were recovered and concentrated with a size-exclusion filter with a MWCO of 10,000 (without the removal of lipid-free apoA-I). Login to comment 153 ABCA1 p.Gln597Arg Normal fibroblasts or ABCA1 mutant (Q597R) were labeled with either [14C]cholesterol or [32P]orthophosphate as described in Materials and Methods and incubated with 10 g/ml apoA-I for 24 h. Login to comment