ABCMdb

PMID: 24097981

Quazi F, Molday RS
Differential phospholipid substrates and directional transport by ATP-binding cassette proteins ABCA1, ABCA7, and ABCA4 and disease-causing mutants.
J Biol Chem. 2013 Nov 29;288(48):34414-26. doi: 10.1074/jbc.M113.508812. Epub 2013 Oct 4., [PubMed]

Sentences

No. Mutations Sentence Comment

64 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr929Ile ABCA1 p.Asn935Ser ABCA1 p.Thr1512Met ABCA1 p.Ser100Cys ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp Mutations introduced by overlap extension PCR using Pfu AD DNA polymerase in ABCA1 included S100C, W590S, F593L, N935S, T929I, C1477R, T1512M, R2081W, and P2150L. Login to comment 65 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr929Ile ABCA1 p.Asn935Ser ABCA1 p.Thr1512Met ABCA1 p.Ser100Cys ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp ABCA4 p.Asn965Ser ABCA4 p.Phe608Leu ABCA4 p.Thr959Ile ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Pro2180Leu ABCA4 p.Trp605Ser ABCA4 p.Arg2107Pro ABCA1 p.Ser100Pro Mutations introduced by overlap extension PCR using Pfu AD DNA polymerase in ABCA1 included S100C, W590S, F593L, N935S, T929I, C1477R, T1512M, R2081W, and P2150L. Login to comment 66 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA4 p.Asn965Ser ABCA4 p.Lys969Met ABCA4 p.Phe608Leu ABCA4 p.Thr959Ile ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Pro2180Leu ABCA4 p.Trp605Ser ABCA4 p.Arg2107Pro ABCA1 p.Ser100Pro ABCA7 p.Lys1833Met ABCA7 p.Lys847Met Corresponding ABCA4 mutations determined by amino acid alignment with ABCA1 included S100P, W605S, F608L, T959I, N965S, C1502R, T1537M, R2107P, and P2180L. Login to comment 67 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr929Ile ABCA1 p.Asn935Ser ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 p.Thr1512Met ABCA1 p.Ser100Cys ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp ABCA4 p.Lys969Met ABCA7 p.Lys1833Met ABCA7 p.Lys847Met ABCA1-MM was constructed to harbor the Walker A-motif lysine-to-methionine mutations K939M/K1952M by the nested PCR method; ABCA4-MM had the corresponding K969M/ K1969M Walker A mutations (37), and ABCA7-MM had the Lipid Transport Activity of ABCA Transporters NOVEMBER 29, 2013ߦVOLUME 288ߦNUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 34415 at SEMMELWEIS UNIV OF MEDICINE on December 3, K847M/K1833M Walker A mutations. Login to comment 68 ABCA4 p.Asn965Ser ABCA4 p.Phe608Leu ABCA4 p.Thr959Ile ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Pro2180Leu ABCA4 p.Trp605Ser ABCA4 p.Arg2107Pro ABCA1 p.Ser100Pro Corresponding ABCA4 mutations determined by amino acid alignment with ABCA1 included S100P, W605S, F608L, T959I, N965S, C1502R, T1537M, R2107P, and P2180L. Login to comment 69 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA4 p.Lys969Met ABCA7 p.Lys1833Met ABCA7 p.Lys847Met ABCA1-MM was constructed to harbor the Walker A-motif lysine-to-methionine mutations K939M/K1952M by the nested PCR method; ABCA4-MM had the corresponding K969M/ K1969M Walker A mutations (37), and ABCA7-MM had the Lipid Transport Activity of ABCA Transporters NOVEMBER 29, 2013ߦVOLUME 288ߦNUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 34415 at SEMMELWEIS UNIV OF MEDICINE on December 3, K847M/K1833M Walker A mutations. Login to comment 206 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr929Ile ABCA1 p.Asn935Ser ABCA1 p.Thr1512Met ABCA1 p.Ser100Cys ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp Expression and Purification of Disease-causing ABCA1 and ABCA4 Mutants-As part of this study, we have generated a number of disease-causing mutations in ABCA1 and ABCA4 to determine their effect on the expression and functional properties of these transporters. We focused our studies on nine missense mutations in ABCA1 known to cause Tangier disease, including three (S100C, W590S, and F593L) in ECD1, two (T929I and N935S) in the NBD1, two (C1477R and T1512M) in ECD2, one (R2081W) in NBD2, and one (P2150L) in the C-terminal segment as shown in Fig. 6A (blue). Login to comment 208 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr929Ile ABCA1 p.Asn935Ser ABCA1 p.Thr1512Met ABCA1 p.Ser100Cys ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp Expression and Purification of Disease-causing ABCA1 and ABCA4 Mutants-As part of this study, we have generated a number of disease-causing mutations in ABCA1 and ABCA4 to determine their effect on the expression and functional properties of these transporters. We focused our studies on nine missense mutations in ABCA1 known to cause Tangier disease, including three (S100C, W590S, and F593L) in ECD1, two (T929I and N935S) in the NBD1, two (C1477R and T1512M) in ECD2, one (R2081W) in NBD2, and one (P2150L) in the C-terminal segment as shown in Fig. 6A (blue). Login to comment 211 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr929Ile ABCA1 p.Asn935Ser ABCA1 p.Thr1512Met ABCA1 p.Ser100Cys ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp Expression and Purification of Disease-causing ABCA1 and ABCA4 Mutants-As part of this study, we have generated a number of disease-causing mutations in ABCA1 and ABCA4 to determine their effect on the expression and functional properties of these transporters. We focused our studies on nine missense mutations in ABCA1 known to cause Tangier disease, including three (S100C, W590S, and F593L) in ECD1, two (T929I and N935S) in the NBD1, two (C1477R and T1512M) in ECD2, one (R2081W) in NBD2, and one (P2150L) in the C-terminal segment as shown in Fig. 6A (blue). Login to comment 218 ABCA4 p.Ser100Pro ABCA4 p.Asn965Ser ABCA4 p.Phe608Leu ABCA4 p.Thr959Ile ABCA4 p.Thr1537Met ABCA4 p.Arg2107Pro Error bars show S.E. Lipid Transport Activity of ABCA Transporters 34420 gardt disease (S100P, F608L, N965S, T959I, T1537M, and R2107P) (Fig. 6A, red). Login to comment 220 ABCA4 p.Ser100Pro ABCA4 p.Asn965Ser ABCA4 p.Phe608Leu ABCA4 p.Thr959Ile ABCA4 p.Thr1537Met ABCA4 p.Pro2180Leu ABCA4 p.Trp605Ser ABCA4 p.Arg2107Pro Error bars show S.E. Lipid Transport Activity of ABCA Transporters 34420 gardt disease (S100P, F608L, N965S, T959I, T1537M, and R2107P) (Fig. 6A, red). Login to comment 221 ABCA1 p.Cys1477Arg ABCA4 p.Cys1502* ABCA4 p.Cys1502Arg Finally, the null mutant C1502X associated with Stargardt disease was modified to C1502R to reflect the primary sequence change of the corresponding C1477R mutant in ABCA1 linked to Tangier disease. Login to comment 222 ABCA4 p.Pro2180Leu ABCA4 p.Trp605Ser These two residues were replaced with serine and leucine, respectively, (W605S and P2180L) corresponding to the disease-causing substitutions in ABCA1. Login to comment 223 ABCA1 p.Cys1477Arg ABCA4 p.Ser100Pro ABCA4 p.Asn965Ser ABCA4 p.Phe608Leu ABCA4 p.Thr959Ile ABCA4 p.Cys1502* ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Arg2107Pro Finally, the null mutant C1502X associated with Stargardt disease was modified to C1502R to reflect the primary sequence change of the corresponding C1477R mutant in ABCA1 linked to Tangier disease. Login to comment 224 ABCA1 p.Ser100Cys ABCA1 p.Arg2081Trp ABCA4 p.Ser100Pro ABCA4 p.Arg2107Pro The levels of expression of the ABCA1 and ABCA4 mutants were generally lower than the corresponding WT proteins with the ABCA1 mutants S100C and R2081W and the corresponding ABCA4 mutants S100P and R2107P expressing at levels less than 25% of WT and the remaining mutants expressing in the range of 35-90% of the WT proteins. Login to comment 225 ABCA4 p.Pro2180Leu ABCA4 p.Trp605Ser These two residues were replaced with serine and leucine, respectively, (W605S and P2180L) corresponding to the disease-causing substitutions in ABCA1. Login to comment 226 ABCA1 p.Cys1477Arg ABCA1 p.Ser100Cys ABCA1 p.Arg2081Trp ABCA4 p.Ser100Pro ABCA4 p.Cys1502* ABCA4 p.Cys1502Arg ABCA4 p.Arg2107Pro The levels of expression of the ABCA1 and ABCA4 mutants were generally lower than the corresponding WT proteins with the ABCA1 mutants S100C and R2081W and the corresponding ABCA4 mutants S100P and R2107P expressing at levels less than 25% of WT and the remaining mutants expressing in the range of 35-90% of the WT proteins. Login to comment 227 ABCA1 p.Trp590Ser ABCA1 p.Thr929Ile ABCA1 p.Asn935Ser ABCA1 p.Ser100Cys ABCA1 p.Phe593Leu ABCA1 p.Arg2081Trp Variants in the ECD1 (S100C, W590S, and F593L), NBD1 (T929I and N935S), and NBD2 (R2081W) of ABCA1 showed significantly reduced ATPase activities in the range of 20-35% of WT activity (Fig. 7A). Login to comment 228 ABCA1 p.Thr1512Met ABCA1 p.Pro2150Leu By contrast, the T1512M mutation in ECD2 and P2150L in the C-terminal segment exhibited ATPase activity similar to the WT protein. Login to comment 229 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr929Ile ABCA1 p.Asn935Ser ABCA1 p.Ser100Cys ABCA1 p.Ser100Cys ABCA1 p.Phe593Leu ABCA1 p.Arg2081Trp ABCA1 p.Arg2081Trp ABCA4 p.Ser100Pro ABCA4 p.Arg2107Pro Variants in the ECD1 (S100C, W590S, and F593L), NBD1 (T929I and N935S), and NBD2 (R2081W) of ABCA1 showed significantly reduced ATPase activities in the range of 20-35% of WT activity (Fig. 7A). Login to comment 230 ABCA1 p.Thr1512Met ABCA1 p.Thr1512Met ABCA1 p.Pro2150Leu ABCA4 p.Thr1537Met By contrast, the T1512M mutation in ECD2 and P2150L in the C-terminal segment exhibited ATPase activity similar to the WT protein. Login to comment 231 ABCA1 p.Cys1477Arg The C1477R mutant in ECD2 showed an intermediate reduction in activity. Login to comment 232 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr929Ile ABCA1 p.Asn935Ser ABCA1 p.Thr1512Met ABCA1 p.Ser100Cys ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp ABCA4 p.Thr1537Met ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Pro2180Leu ABCA4 variants showed a similar ATPase activity profile as the ABCA1 mutants with the exception of the T1537M mutation of ABCA4, which was significantly lower than the corresponding T1512M mutant in ABCA1. Login to comment 233 ABCA1 p.Thr1512Met ABCA1 p.Pro2150Leu By contrast, the T1512M mutation in ECD2 and P2150L in the C-terminal segment exhibited ATPase activity similar to the WT protein. Login to comment 234 ABCA1 p.Cys1477Arg ABCA1 p.Cys1477Arg ABCA1 p.Thr1512Met ABCA1 p.Pro2150Leu ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Pro2180Leu As shown in Fig. 8, A and B, the Fl-PC flippase activity of the ABCA1 mutants and the Fl-PE flippase activity of ABCA4 mutants have a similar profile with the ABCA1 variants C1477R, T1512M, and P2150L and corresponding ABCA4 variants C1502R, T1537M, and P2180L showing transport activities ranging from 60 to 80% of the WT protein and the other mutants showing reduced activity in the range of 20-40% of the WT protein. Login to comment 235 ABCA1 p.Thr1512Met ABCA4 p.Thr1537Met ABCA4 variants showed a similar ATPase activity profile as the ABCA1 mutants with the exception of the T1537M mutation of ABCA4, which was significantly lower than the corresponding T1512M mutant in ABCA1. Login to comment 237 ABCA1 p.Cys1477Arg ABCA1 p.Thr929Ile ABCA1 p.Thr1512Met ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp ABCA4 p.Thr959Ile ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Pro2180Leu ABCA4 p.Arg2107Pro As shown in Fig. 8, A and B, the Fl-PC flippase activity of the ABCA1 mutants and the Fl-PE flippase activity of ABCA4 mutants have a similar profile with the ABCA1 variants C1477R, T1512M, and P2150L and corresponding ABCA4 variants C1502R, T1537M, and P2180L showing transport activities ranging from 60 to 80% of the WT protein and the other mutants showing reduced activity in the range of 20-40% of the WT protein. Login to comment 239 ABCA1 p.Thr929Ile ABCA1 p.Arg2081Trp ABCA4 p.Thr959Ile ABCA4 p.Arg2107Pro In contrast, some of the mutants, including ABCA1 mutants T929I and R2081W and related ABCA4 mutants T959I and R2107P, showed partial or complete co-localization with calnexin in a reticular pattern characteristic of the ER. Login to comment 242 ABCA1 p.Thr929Ile ABCA1 p.Arg2081Trp ABCA4 p.Thr959Ile ABCA4 p.Arg2107Pro In contrast, some of the mutants, including ABCA1 mutants T929I and R2081W and related ABCA4 mutants T959I and R2107P, showed partial or complete co-localization with calnexin in a reticular pattern characteristic of the ER. Login to comment 249 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Pro2150Leu Some disease alleles such as W590S, C1477R, and P2150L (ABCA1) have conserved residues in ABCA4, but mutations in these positions in ABCA4 have yet to be linked to Stargardt disease. Login to comment 251 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Pro2150Leu Some disease alleles such as W590S, C1477R, and P2150L (ABCA1) have conserved residues in ABCA4, but mutations in these positions in ABCA4 have yet to be linked to Stargardt disease. Login to comment 254 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Pro2150Leu Some disease alleles such as W590S, C1477R, and P2150L (ABCA1) have conserved residues in ABCA4, but mutations in these positions in ABCA4 have yet to be linked to Stargardt disease. Login to comment 293 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr1512Met ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA4 p.Ser100Pro ABCA4 p.Phe608Leu ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Pro2180Leu ABCA4 p.Trp605Ser ABCA4 p.Ser100Cys The ABCA1/ABCA4 mutants in the ECD1 (S100C/S100P, W590S/ W605S, and F593L/F608L) displayed the lowest activities (2030% WT), whereas those in the ECD2 (C1477R/C1502R and T1512M/T1537M) and the P2150L/P2180L mutants in the C terminus showed the highest activities (60-100% WT). Login to comment 295 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr1512Met ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA4 p.Ser100Pro ABCA4 p.Phe608Leu ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Pro2180Leu ABCA4 p.Trp605Ser ABCA4 p.Ser100Cys The ABCA1/ABCA4 mutants in the ECD1 (S100C/S100P, W590S/ W605S, and F593L/F608L) displayed the lowest activities (20-30% WT), whereas those in the ECD2 (C1477R/C1502R and T1512M/T1537M) and the P2150L/P2180L mutants in the C terminus showed the highest activities (60-100% WT). Login to comment 298 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg ABCA1 p.Thr1512Met ABCA1 p.Phe593Leu ABCA1 p.Pro2150Leu ABCA4 p.Ser100Pro ABCA4 p.Phe608Leu ABCA4 p.Thr1537Met ABCA4 p.Cys1502Arg ABCA4 p.Pro2180Leu ABCA4 p.Trp605Ser ABCA4 p.Ser100Cys The ABCA1/ABCA4 mutants in the ECD1 (S100C/S100P, W590S/ W605S, and F593L/F608L) displayed the lowest activities (20-30% WT), whereas those in the ECD2 (C1477R/C1502R and T1512M/T1537M) and the P2150L/P2180L mutants in the C terminus showed the highest activities (60-100% WT). Login to comment 300 ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp For example, the P2150L showed only mild loss in phospholipid transport and efflux, whereas R2081W showed a more pro- FIGURE 9. Login to comment 302 ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp For example, the P2150L showed only mild loss in phospholipid transport and efflux, whereas R2081W showed a more pro- FIGURE 9. Login to comment 305 ABCA1 p.Pro2150Leu ABCA1 p.Arg2081Trp For example, the P2150L showed only mild loss in phospholipid transport and efflux, whereas R2081W showed a more pro- FIGURE 9. Login to comment 309 ABCA1 p.Cys1477Arg For example, the C1477R mutant shows intermediate (b03;60% WT) phospholipid transport activity, whereas it shows minimum (b0d;20% WT) phospholipid efflux activity. Login to comment 311 ABCA1 p.Cys1477Arg For example, the C1477R mutant shows intermediate (b03;60% WT) phospholipid transport activity, whereas it shows minimum (b0d;20% WT) phospholipid efflux activity. Login to comment 314 ABCA1 p.Cys1477Arg For example, the C1477R mutant shows intermediate (b03;60% WT) phospholipid transport activity, whereas it shows minimum (b0d;20% WT) phospholipid efflux activity. Login to comment