ABCMdb

PMID: 12509412

Tanaka AR, Abe-Dohmae S, Ohnishi T, Aoki R, Morinaga G, Okuhira K, Ikeda Y, Kano F, Matsuo M, Kioka N, Amachi T, Murata M, Yokoyama S, Ueda K
Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.
J Biol Chem. 2003 Mar 7;278(10):8815-9. Epub 2002 Dec 31., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. Login to comment 5 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Login to comment 6 ABCA1 p.Trp590Ser Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Login to comment 7 ABCA1 p.Trp590Ser Photoaffinity labeling of W590S with 8-azido-[␣- 32 P]ATP was stimulated by adding ortho-vanadate in the presence of Mn2؉ as much as in the presence of wild-type ABCA1. Login to comment 8 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis. Login to comment 20 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Three TD mutants (R587W, W590S, Q597R), clustered in ECD1, were examined in the present report. Login to comment 24 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp On the other hand, the two mutants R587W and Q597R were only partially or scarcely localized to the PM, whereas W590S * This work was supported by Grant-in-aid for Scientific Research 10217205 on Priority Areas "ABC Proteins" from the Ministry of Education, Science, Sports, and Culture of Japan and by the Nakajima Foundation. Login to comment 35 ABCA1 p.Trp590Ser Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Login to comment 44 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp DNA Construction-DNA fragments (XhoI-BclI) containing each missense TD mutation (R587W, W590S, or Q597R) were generated using the polymerase chain reaction method with R587W (XhoI) primer (5Ј-GTCCTCGAGCTGACCCCTTTGAGGACATGTGGTACGTC-3Ј), W590S (XhoI) primer (5Ј-GTCCTCGAGCTGACCCCTTTGAGGACAT- GCGGTACGTCTCGGGGGGCTTC-3Ј), or Q597 (XhoI) primer (5Ј-GT- CCTCGAGCTGACCCCTTTGAGGACATGCGGTACGTCTGGGGGGG- CTTCGCCTACTTGCGGGATGTGGTG-3Ј), where the mutated nucleotide is underlined, and BclI primer (5Ј-CGATGCCCTTGATGATCACA- GCCACTGAG-3Ј). Login to comment 47 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp In brief, 10 ␮g of membrane proteins from HEK293 cells stably expressing the wild-type, R587W, W590S, or Q597R ABCA1-GFP were treated with 500 units of Endo H or 0.3 units of PNGaseF for 1 h at 37 °C. Login to comment 53 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Vanadate-induced Nucleotide Trapping in ABCA1 with 8-Azido-[␣- 32 P]ATP-A membrane fraction (20-30 ␮g) was prepared from HEK293 cells stably expressing the wild-type or W590S ABCA1-GFP. Login to comment 64 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Effects of ECD1 Mutations on Subcellular Localization of ABCA1-GFP-Many mutations in patients with TD and FHA have been identified in ECD1 of ABCA1, and three mutations (R587W, W590S, Q597R) cluster in the vicinity between amino acids 587 and 597 (20)(Fig. 2A). Login to comment 67 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Confocal microscopic examination revealed that R587W and Q597R appeared to be localized mainly in the ER and not to the PM (Fig. 2B). Login to comment 68 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Confocal microscopic examination revealed that R587W and Q597R appeared to be localized mainly in the ER and not to the PM (Fig. 2B). Login to comment 69 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser In contrast, W590S was localized to the PM as much as the wild-type ABCA1-GFP was, although more was found with intracellular vesicles than with the wild type (Fig. 2B). Login to comment 70 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Immunostaining with the antibody against ECD1 confirmed the proper orientation of W590S (Fig. 2C). Login to comment 71 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Glycosylation of ABCA1-GFP-Glycosylation of the wild-type ABCA1-GFP and its mutants R587W, W590S, and Q597R was examined by the treatment with PNGaseF and Endo H (Fig. 3A). Login to comment 73 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 with TD mutations, R587W and Q597R ABCA1-GFP, was sensitive to Endo H to produce the deglycosylated form of ABCA1-GFP, whereas the wild-type ABCA1-GFP was little digested by Endo H. Login to comment 74 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp ABCA1 with TD mutations, R587W and Q597R ABCA1-GFP, was sensitive to Endo H to produce the deglycosylated form of ABCA1-GFP, whereas the wild-type ABCA1-GFP was little digested by Endo H. Login to comment 75 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp These results indicated that R587W and Q597R ABCA1-GFP did not contain complex oligosaccharides and supported the confocal microscopy observation, which suggested the localization of these two TD mutants in the ER or the cis-Golgi complex. Login to comment 76 ABCA1 p.Trp590Ser On the other hand, W590S ABCA1-GFP was resistant to Endo H, indicating that it does not contain high mannose oligosaccharides but contains complex oligosaccharides and reached the trans-Golgi complex. Login to comment 83 ABCA1 p.Arg587Trp The R587W mutation resulted in the apoA-I-mediated release of cholesterol and choline-phospholipids to 24 and 23% of the wild-type ABCA1-GFP, respectively. Login to comment 84 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp The R587W mutation resulted in the apoA-I-mediated release of cholesterol and choline-phospholipids to 24 and 23% of the wild-type ABCA1-GFP, respectively. Login to comment 85 ABCA1 p.Gln597Arg The Q597R mutant almost completely lost this activity. Login to comment 86 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The apoA-I-mediated release of cholesterol and choline-phospholipids from HEK293 expressing W590S ABCA1-GFP were 7.4 and 13%, respectively, of those expressing the wild-type ABCA1-GFP, although W590S ABCA1-GFP was localized to the PM as efficiently as the wild type. Login to comment 87 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The apoA-I-mediated release of cholesterol and choline-phospholipids from HEK293 expressing W590S ABCA1-GFP were 7.4 and 13%, respectively, of those expressing the wild-type ABCA1-GFP, although W590S ABCA1-GFP was localized to the PM as efficiently as the wild type. Login to comment 88 ABCA1 p.Trp590Ser The results were similar to those observed with the stable transformants shown in Fig. 4, A and B. Interaction of ABCA1-GFP with 8-Azido-[ॷ-32 P]ATP-To elucidate the mechanism for the loss of function of ABCA1 in the W590S mutant, we examined the interaction of ABCA1-GFP with ATP. Login to comment 89 ABCA1 p.Trp590Ser The results were similar to those observed with the stable transformants shown in Fig. 4, A and B. Interaction of ABCA1-GFP with 8-Azido-[␣-32 P]ATP-To elucidate the mechanism for the loss of function of ABCA1 in the W590S mutant, we examined the interaction of ABCA1-GFP with ATP. Login to comment 101 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp A, a putative secondary structure of ABCA1 and localization of Tangier Disease mutations R587W, W590S, and Q597R in ECD1. Login to comment 102 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp A, a putative secondary structure of ABCA1 and localization of Tangier Disease mutations R587W, W590S, and Q597R in ECD1. Login to comment 103 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp B, GFP fluorescence of HEK293 cells stably expressing the wild-type (WT) ABCA1-GFP and three TD mutants R587W, W590S, and Q597R ABCA1-GFP. Login to comment 104 ABCA1 p.Trp590Ser C, immunofluorescent observation of W590S ABCA1-GFP with anti-ECD1 antibody and anti-rat IgG-Alexa594. Login to comment 106 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp The wild-type (WT), R587W, W590S, and Q597R ABCA1-GFP were treated with none (afa;), Endo H (H), or PNGaseF (F) and separated with 7% SDS-PAGE. Login to comment 107 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp The wild-type (WT), R587W, W590S, and Q597R ABCA1-GFP were treated with none (-), Endo H (H), or PNGaseF (F) and separated with 7% SDS-PAGE. Login to comment 110 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp Cholesterol (A) and choline-phospholipid (B) content in the medium in a 6-well plate containing HEK293 cells stably expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP were measured after a 24-h incubation in the presence (black bars) or absence (white bars) of 10 òe;g/ml apoA-I. The relative amount of cholesterol (C) and choline-phospholipid (D) in the medium in a 6-well plate containing HEK293 cells transiently expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP was measured after a 24-h incubation in the presence of 10 òe;g/ml apoA-I. The expression levels of mutants were normalized with the GFP fluorescence of cells. Login to comment 111 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp Cholesterol (A) and choline-phospholipid (B) content in the medium in a 6-well plate containing HEK293 cells stably expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP were measured after a 24-h incubation in the presence (black bars) or absence (white bars) of 10 ␮g/ml apoA-I. The relative amount of cholesterol (C) and choline-phospholipid (D) in the medium in a 6-well plate containing HEK293 cells transiently expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP was measured after a 24-h incubation in the presence of 10 ␮g/ml apoA-I. The expression levels of mutants were normalized with the GFP fluorescence of cells. Login to comment 112 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser To determine whether the W590S mutation affects the ATP hydrolysis cycle of ABCA1, vanadate-induced nucleotide trapping in W590S ABCA1-GFP was examined in the presence of Mn2af9; (Fig. 5C). Login to comment 113 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser To determine whether the W590S mutation affects the ATP hydrolysis cycle of ABCA1, vanadate-induced nucleotide trapping in W590S ABCA1-GFP was examined in the presence of Mn2ϩ (Fig. 5C). Login to comment 114 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Membrane proteins from HEK293 cells expressing a similar amount of wild-type ABCA1 or W590S ABCA1-GFP (Fig. 5B) were incubated with 8-azido-[␣-32 P]ATP in the absence or presence of ortho-vanadate. Login to comment 115 ABCA1 p.Trp590Ser The photoaffinity labeling of W590S ABCA1-GFP was stimulated by adding ortho-vanadate in the presence of Mn2ϩ as much as in the presence of the wild type. Login to comment 118 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1-GFP with a R587W or Q597R mutation appeared to be impaired with intracellular trafficking and predominantly localized in the ER. Login to comment 119 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1-GFP with a R587W or Q597R mutation appeared to be impaired with intracellular trafficking and predominantly localized in the ER. Login to comment 120 ABCA1 p.Trp590Ser On the other hand, W590S ABCA1-GFP was mainly localized to the PM as much as the wild-type ABCA1 was. Login to comment 121 ABCA1 p.Arg587Trp R587W and Q597 ABCA1-GFP contained high mannose oligosaccharides, indicating that they do not reach the trans-Golgi complex. Login to comment 122 ABCA1 p.Trp590Ser ABCA1 p.Arg587Trp R587W and Q597 ABCA1-GFP contained high mannose oligosaccharides, indicating that they do not reach the trans-Golgi complex. Login to comment 123 ABCA1 p.Trp590Ser In contrast, W590S ABCA1-GFP contained complex-type oligosaccharides as the wild-type does. Login to comment 124 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp R587W and Q597R ABCA1-GFP appeared to be retained in the ER. Login to comment 125 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp R587W and Q597R ABCA1-GFP appeared to be retained in the ER. Login to comment 126 ABCA1 p.Trp590Ser This region (R587 to Q597) in ECD1 would be critical for proper folding of ABCA1 and would probably affect the intracellular translocation process, whereas the W590S mutation does not. Login to comment 127 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp This region (R587 to Q597) in ECD1 would be critical for proper folding of ABCA1 and would probably affect the intracellular translocation process, whereas the W590S mutation does not. Login to comment 128 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp Fitzgerald et al. (17) reported that R587W or Q597R mutation did not affect the PM localization but disrupted the direct interaction with ApoA-I. This supports a major conformational alteration of ECD1 by these mutations. Login to comment 129 ABCA1 p.Trp590Ser W590S ABCA1-GFP was localized to the PM as much as the wild-type ABCA1-GFP when expressed in HEK293. Login to comment 130 ABCA1 p.Trp590Ser W590S ABCA1-GFP was localized to the PM as much as the wild-type ABCA1-GFP when expressed in HEK293. Login to comment 131 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser To elucidate the reason for this functional impairment in the W590S mutant, nucleotide interaction was examined with the wild-type and W590S ABCA1-GFP by using 8-azido-[ॷ-32 P]ATP. Login to comment 132 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser To elucidate the reason for this functional impairment in the W590S mutant, nucleotide interaction was examined with the wild-type and W590S ABCA1-GFP by using 8-azido-[␣-32 P]ATP. Login to comment 138 ABCA1 p.Trp590Ser B, immunoblots of membranes prepared from HEK293 cells stably expressing the wild-type (4 òe;g, lane 1) or W590S (6 òe;g, lane 2) ABCA1-GFP or from untransfected HEK293 cells (6 òe;g, lane 3). Login to comment 139 ABCA1 p.Trp590Ser B, immunoblots of membranes prepared from HEK293 cells stably expressing the wild-type (4 ␮g, lane 1) or W590S (6 ␮g, lane 2) ABCA1-GFP or from untransfected HEK293 cells (6 ␮g, lane 3). Login to comment 140 ABCA1 p.Trp590Ser C, membranes prepared from HEK293 cells stably expressing the wild type (WT) (20 òe;g, lanes 1 and 2) or W590S (30 òe;g, lanes 3 and 4) were incubated with 15 òe;M 8-azido-[ॷ-32 P]ATP in the absence or presence of 1 mM ortho-vanadate (Vi) and 3 mM MnCl2 for 15 min at 37 &#b0;C. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ligands and analyzed as described under "Experimental Procedures." Login to comment 141 ABCA1 p.Trp590Ser C, membranes prepared from HEK293 cells stably expressing the wild type (WT) (20 ␮g, lanes 1 and 2) or W590S (30 ␮g, lanes 3 and 4) were incubated with 15 ␮M 8-azido-[␣-32 P]ATP in the absence or presence of 1 mM ortho-vanadate (Vi) and 3 mM MnCl2 for 15 min at 37 °C. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ligands and analyzed as described under "Experimental Procedures." Login to comment 145 ABCA1 p.Trp590Ser W590S ABCA1-GFP showed vanadate-induced nucleotide trapping in the presence of Mn2af9; . Login to comment 146 ABCA1 p.Trp590Ser W590S ABCA1-GFP showed vanadate-induced nucleotide trapping in the presence of Mn2ϩ . Login to comment 147 ABCA1 p.Trp590Ser It has been reported that apoA-I does not properly interact with ATP hydrolysis mutants of ABCA1 (10) and that apoA-I can be interacted with ABCA1-W590S as with the wild-type ABCA1 (17, 35). Login to comment 148 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser It has been reported that apoA-I does not properly interact with ATP hydrolysis mutants of ABCA1 (10) and that apoA-I can be interacted with ABCA1-W590S as with the wild-type ABCA1 (17, 35). Login to comment 149 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser These results suggest that W590S ABCA1-GFP possesses, at least, minimum ATPase activity, which supports apoA-I binding. Login to comment 150 ABCA1 p.Trp590Ser W590S mutation may impair a step after the interaction with apoA-I, such as proper loading of phospholipid and/or cholesterol or proper release of apoA-I after phospholipid/cholesterol loading. Login to comment 153 ABCA1 p.Trp590Ser ABCA1 p.Arg587Trp Interestingly, clinical manifestations of these mutations are apparently different (20): R587W is associated with coronary heart disease, whereas W590S is associated with splenomegaly. Login to comment 154 ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp ABCA1 p.Arg587Trp Interestingly, clinical manifestations of these mutations are apparently different (20): R587W is associated with coronary heart disease, whereas W590S is associated with splenomegaly. Login to comment 155 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp In this study, we demonstrated that the defect of HDL assembly in R587W and Q597R is due to the impaired localization of ABCA1 to the PM. Login to comment 156 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Subcellular trafficking and vanadate-induced nucleotide trapping in the presence of Mn2ϩ were not impaired in ABCA1-GFP containing the W590S mutation so that W590S seems to have a different type of functional defect. Login to comment