ABCMdb

PMID: 12084722

Fitzgerald ML, Morris AL, Rhee JS, Andersson LP, Mendez AJ, Freeman MW
Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I.
J Biol Chem. 2002 Sep 6;277(36):33178-87. Epub 2002 Jun 25., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCA1 p.Trp590Ser Four of five mutants had a marked decline in cross-linking to apoA-I, whereas one (W590S) retained full cross-linking activity. Login to comment 7 ABCA1 p.Trp590Ser The behavior of the W590S mutant indicates that although binding of apoA-I by ABCA1 may be necessary, it is not sufficient for stimulation of cholesterol efflux. Login to comment 37 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu DNA Constructs-Five missense mutants of ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) were generated using overlap polymerase chain reaction methods, as described previously (17). Login to comment 39 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu DNA Constructs-Five missense mutants of ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) were generated using overlap polymerase chain reaction methods, as described previously (17). Login to comment 67 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu Missense Mutations in Two Putative Extracellular Loops of ABCA1 Ablate Efflux Activity-Five missense mutations (R587W, W590S, Q597R, C1477R, and S1506L) were introduced into a wild type ABCA1 cDNA using PCR mutagenesis techniques. Login to comment 68 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Three of these mutations (R587W, W590S, and Q597R) fall in a tight cluster within the large N-terminal loop at a point near the putative second transmembrane domain shown in Fig. 3. Login to comment 69 ABCA1 p.Cys1477Arg ABCA1 p.Ser1506Leu The other two mutations (C1477R and S1506L) fall within the central loop of the protein. Login to comment 70 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu Missense Mutations in Two Putative Extracellular Loops of ABCA1 Ablate Efflux Activity-Five missense mutations (R587W, W590S, Q597R, C1477R, and S1506L) were introduced into a wild type ABCA1 cDNA using PCR mutagenesis techniques. Login to comment 71 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Three of these mutations (R587W, W590S, and Q597R) fall in a tight cluster within the large N-terminal loop at a point near the putative second transmembrane domain shown in Fig. 3. Login to comment 72 ABCA1 p.Cys1477Arg ABCA1 p.Ser1506Leu The other two mutations (C1477R and S1506L) fall within the central loop of the protein. Login to comment 112 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu The cells were transfected with either empty vector (mock), wild type ABCA1 (WT), or ABCA1 constructs carrying the indicated point mutations (R587W, W590S, Q597R, C1477R, and S1506L). Login to comment 115 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu Absolute apoA-I and medium efflux values, respectively, are as follows: mock, 1.59 afe; 0.04% versus 1.21 afe; 0.39%; WT, 3.92 afe; 0.13% versus 1.9 afe; 0.08%; R587W, 1.78 afe; 0.11% versus 1.61 afe; 0.24%; W590S, 1.92 afe; 0.24% versus 1.63 afe; 0.08%; Q597R, 1.5 afe; 0.14% versus 1.49 afe; 0.03%; C1477R, 1.67 afe; 0.18% versus 1.52 afe; 0.15%; and S1506L, 1.66 afe; 0.28% versus 1.6 afe; 0.13%. Login to comment 116 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu The cells were transfected with either empty vector (mock), wild type ABCA1 (WT), or ABCA1 constructs carrying the indicated point mutations (R587W, W590S, Q597R, C1477R, and S1506L). Login to comment 119 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu Absolute apoA-I and medium efflux values, respectively, are as follows: mock, 1.59 Ϯ 0.04% versus 1.21 Ϯ 0.39%; WT, 3.92 Ϯ 0.13% versus 1.9 Ϯ 0.08%; R587W, 1.78 Ϯ 0.11% versus 1.61 Ϯ 0.24%; W590S, 1.92 Ϯ 0.24% versus 1.63 Ϯ 0.08%; Q597R, 1.5 Ϯ 0.14% versus 1.49 Ϯ 0.03%; C1477R, 1.67 Ϯ 0.18% versus 1.52 Ϯ 0.15%; and S1506L, 1.66 Ϯ 0.28% versus 1.6 Ϯ 0.13%. Login to comment 190 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Ser1506Leu Three of the mutants (Q597R, C1477R, and S1506L) had dramatic reductions in their cross-linking efficiency to apoA-I, relative to the wild type transporter (90% or greater reduction). Login to comment 191 ABCA1 p.Arg587Trp The R587W mutant showed an intermediate phenotype, with cross-linking efficiency reduced b03;50%. Login to comment 192 ABCA1 p.Trp590Ser Surprisingly, the W590S mutant consistently cross-linked to apoA-I at an efficiency that was equal to or greater than that of wild type ABCA1. Login to comment 194 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu DISCUSSION In this study, we have established that several naturally occurring missense mutations in ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) located in the two largest loop domains of the protein (comprising amino acids b03;44-640 and b03;1371-1649, respectively) are, in fact, loss-of-function mutations. Login to comment 198 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Ser1506Leu Three of the mutants (Q597R, C1477R, and S1506L) had dramatic reductions in their cross-linking efficiency to apoA-I, relative to the wild type transporter (90% or greater reduction). Login to comment 199 ABCA1 p.Arg587Trp The R587W mutant showed an intermediate phenotype, with cross-linking efficiency reduced ϳ50%. Login to comment 200 ABCA1 p.Trp590Ser Surprisingly, the W590S mutant consistently cross-linked to apoA-I at an efficiency that was equal to or greater than that of wild type ABCA1. Login to comment 202 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu DISCUSSION In this study, we have established that several naturally occurring missense mutations in ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) located in the two largest loop domains of the protein (comprising amino acids ϳ44-640 and ϳ1371-1649, respectively) are, in fact, loss-of-function mutations. Login to comment 204 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu Although three of the mutations (Q597R, C1477R, and S1506L) showed no appreciable cross-linking to apoA-I, the R587W mutant had an intermediate activity, and the W590S mutant retained full, if not enhanced, cross-linking to the apoprotein. Login to comment 210 ABCA1 p.Cys1477Arg Only one cysteine mutant was utilized in this study (C1477R), and work is currently underway to determine whether this mutation affects the formation of a disulfide linkage in ABCA1. Login to comment 212 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg ABCA1 p.Arg587Trp ABCA1 p.Ser1506Leu Although three of the mutations (Q597R, C1477R, and S1506L) showed no appreciable cross-linking to apoA-I, the R587W mutant had an intermediate activity, and the W590S mutant retained full, if not enhanced, cross-linking to the apoprotein. Login to comment 254 ABCA1 p.Trp590Ser What is clear is that a direct interaction between apoA-I and ABCA1 is not sufficient for the movement of cholesterol out of the cell, because the W590S mutant retained full cross-linking activity and yet could not efflux cholesterol. Login to comment 263 ABCA1 p.Trp590Ser What is clear is that a direct interaction between apoA-I and ABCA1 is not sufficient for the movement of cholesterol out of the cell, because the W590S mutant retained full cross-linking activity and yet could not efflux cholesterol. Login to comment