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Am J Physiol Lung Cell Mol Physiol 295: L698-L707, 2008. First published August 1, 2008; doi:10.1152/ajplung.90352.2008
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Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

Yoshihiro Matsumura,1 Nobuhiro Ban,1,2 and Nobuya Inagaki1,2

1Department of Physiology, Akita University School of Medicine, Akita; and 2Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University, and Core Research for Evolutional Science and Technology of Japan Science and Technology Agency, Kyoto, Japan

Submitted 18 June 2008 ; accepted in final form 31 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The ATP-binding cassette transporter ABCA3 mediates uptake of choline-phospholipids into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have shown previously that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[32P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.

ATP-binding cassette A3 mutant; pediatric interstitial lung disease; lamellar body; lipid transporter; phosphatidylcholine

THE FAMILY OF ATP-BINDING CASSETTE (ABC) transporters is involved in ATP-dependent transport of various substrates across membranes (14). ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells and is proposed to be a surfactant lipid transporter (20, 36). Exogenous expression of ABCA3 in cultured cells promotes lipid uptake into intracellular vesicles that generate lamellar body-like vesicles (7, 18, 21). ABCA3 deficiency in human and mice leads to decreased phosphatidylcholine and phosphatidylglycerol in surfactant, dysgenesis of lamellar bodies, and respiratory distress (1, 3, 8, 11, 12, 27). Considered together, these results indicate that ABCA3 is an essential lipid transporter in surfactant metabolism.

In addition, ABCA3 mutations cause lung disease of differing severity. We previously found that ABCA3 mutations in fatal surfactant deficiency can result in abnormal intracellular localization (type I) or impaired ATP hydrolysis of ABCA3 protein (type II) (17). For example, patients with type I homozygous ABCA3 mutations such as W1142X/W1142X or type I/type II compound heterozygous ABCA3 mutations such as L982P/G1221S die of surfactant deficiency within the neonatal period (27). On the other hand, patients with the common missense mutation E292V and a second, specific mutation such as E690K or T1114M develop pediatric interstitial lung disease (pILD), the phenotype of which is milder than that of fatal surfactant deficiency, suggesting that the E292V ABCA3 mutation is responsible for the development of pILD (4). However, the mechanism underlying the phenotypic heterogeneity of lung disease associated with ABCA3 mutation remains unknown.

In this study, we characterized E292V, E690K, and T1114M mutant ABCA3 proteins identified in pILD. Analysis of these ABCA3 mutants demonstrates the importance of a functional catalytic cycle in the lipid transport function of ABCA3 and suggests various therapeutic targets in lung disease due to ABCA3 mutation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
DNA construction. The plasmid pEGFPN1-ABCA3 (17), which encodes ABCA3 protein fused with enhanced green fluorescent protein (GFP) at the COOH terminus (ABCA3-GFP), and its mutants containing pILD mutations (E292V, E690K, and T1114M) and other site-directed mutations (E292D, E292K, T1114S, E690D, and E690R) were generated as described previously and used for transient transfection experiment. Plasmid pCAGIpuro-ABCA3-GFP (17), which encodes ABCA3-GFP and its mutants described above, driven by a CAG promoter containing an internal ribosomal entry site and puromycin N-acetyltransferase gene cassette (22), was generated as described previously and used for stable transfection experiments.

Confocal microscopy. Human embryonic kidney (HEK-293) cells were grown at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum and penicillin/streptomycin. HEK-293 cells (3 x 105) were seeded into 35-mm dishes with a poly-L-lysine-coated cover glass. After 24 h, HEK-293 cells were transfected with wild-type or mutant pEGFP plasmid (1 µg) using FuGENE transfection reagent (Roche Applied Science). The transfected cells were cultured for 48 h, fixed with 4% paraformaldehyde, and viewed with a Zeiss confocal microscope LSM510-META.

Glycosylation of ABCA3-GFP and mutant proteins. HEK-293 cells (3 x 106) were seeded into 100-mm dishes 24 h before transfection. Forty-eight hours after transfection with pEGFP vectors (6.25 µg) using FuGENE reagent, the cells were homogenized in 50 mM Tris·HCl buffer (pH 7.5) containing Complete protease inhibitor mixture (Roche Applied Science), and the total membrane fraction (100,000-g pellet) was obtained as described previously (17). Ten micrograms of total membrane fraction were treated with 1 unit of peptide N-glycosidase F (PNGase F) or 5 milliunits of endoglycosidase H (Endo H) for 30 min at 37°C. The deglycosylated proteins were separated by SDS-PAGE and analyzed by immunoblot analysis using anti-GFP monoclonal antibody (Santa Cruz Biotechnology).

Establishment of HEK-293 cells stably expressing ABCA3-GFP and mutant proteins. HEK-293 cells (3 x 105) were seeded into 35-mm dishes and, after 24 h, transfected with linearized pCAGIpuro plasmids (1 µg) with PvuI using FuGENE reagent. Forty-eight hours after transfection, the cells were trypsinized, seeded into 100-mm dishes, and selected by 2.5 µg/ml puromycin for 7 days. Resistant colonies were combined and used for lipid analysis, whereas single colonies were isolated and used for nucleotide trapping and photoaffinity labeling experiments. The expression of ABCA3-GFP, LAMP3, and GRP78 was examined by immunoblot analysis using anti-GFP, anti-LAMP3 (Chemicon), and anti-GRP78 (Santa Cruz Biotechnology) antibodies, respectively.

Sucrose gradient fractionation. Confluent cells (five 100-mm dishes) were harvested and disrupted in 20 mM Tris·HCl buffer (pH 7.3) containing 1 M sucrose and Complete protease inhibitor mixture by N2 cavitation, followed by centrifugation at 1,000 g for 10 min to obtain postnuclear supernatant (PNS). A sucrose gradient consisting of 1 ml each of 0.8, 0.7, 0.6, 0.5, 0.4, and 0.3 M sucrose and 0.5 ml of 0.2 M sucrose was layered successively above 3.5 ml of PNS (4 mg of protein). The gradient was spun in a P40ST rotor (Hitachi) at 125 g for 15 min and then at 80,000 g for 3 h. After centrifugation, 1 ml of each fraction was collected from the top.

Lipids analysis. Total lipids in PNS and sucrose gradient fractions were extracted using the method of Bligh and Dyer (2). The amount of total cholesterol and choline-phospholipids was measured using an enzymatic assay kit (Kyowa Medex and Wako, respectively). Data normalized by the protein content are represented as means ± SD, and statistical analysis was performed using the Bonferroni/Dunn procedure for post hoc testing.

Vanadate-induced nucleotide trapping of ABCA3-GFP and mutant proteins. A 20,000-g membrane fraction was obtained from HEK-293 cells stably expressing wild-type ABCA3-GFP or mutants as described previously (17). A 20,000-g membrane fraction (18–24 µg of protein) was incubated with 10 µM 8-azido-[{alpha}-32P]ATP, 2 mM ouabain, 0.1 mM EGTA, 3 mM MgCl2, and 40 mM Tris·HCl (pH 7.5) in a total volume of 12 µl for 10 min at 37°C in the presence or absence of 0.4 mM orthovanadate. In some experiments, 8-azido-[{alpha}-32P]ATP was replaced with 8-azido-[{gamma}-32P]ATP. The reaction was stopped by adding 400 µl of ice-cold TEM buffer [40 mM Tris·HCl buffer (pH 7.5) containing 0.1 mM EGTA and 1 mM MgCl2]. The supernatant containing unbound nucleotides was removed from the membrane pellet after centrifugation (20,000 g, 10 min, 2°C), and the procedure was repeated once. In the release experiment, following the initial removal of unbound nucleotides, the pellets were suspended in 100 µl of TEM buffer and incubated for 0–15 min at 37°C to release trapped nucleotides. After incubation, 300 µl of TEM buffer were added and supernatant containing released nucleotides was removed from the membrane pellet after centrifugation (20,000 g, 10 min, 2°C). The pellets were resuspended in 10 µl of TEM buffer and irradiated for 5 min (254 nm, 8.2 mW/cm2) on ice. The samples were then electrophoresed on a 5% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The radioactivities of photoaffinity-labeled protein (total 220-kDa noncleaved form plus 180-kDa cleaved form) were quantified using FLA-5000 (Fujifilm). Radioactivities in the absence of orthovanadate were subtracted from radioactivities in the presence of orthovanadate and presented as means ± SD after normalization to the level of ABCA3-GFP protein (total 220-kDa noncleaved form plus 180-kDa cleaved form). Statistical analysis was performed as described above.

Photoaffinity labeling of ABCA3-GFP and mutant proteins with 8-azido-[{gamma}-32P]ATP or 8-azido-[{alpha}-32P]ADP. A 20,000-g membrane fraction was incubated with 40 µM 8-azido-[{gamma}-32P]ATP or 8-azido-[{alpha}-32P]ADP, 2 mM ouabain, 0.1 mM EGTA, 3 mM MgCl2, and 40 mM Tris·HCl (pH 7.5) in a total volume of 12 µl for 10 min at 0°C. After irradiation for 5 min (254 nm, 8.2 mW/cm2) on ice, 400 µl of TEM buffer were added and supernatant containing unbound nucleotides was removed from the membrane pellet after centrifugation (20,000 g, 10 min, 2°C). The pellets were solubilized in RIPA buffer [50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate] containing protease inhibitor mixture for 30 min at 4°C. After centrifugation (20,000 g, 20 min, 2°C), proteins were immunoprecipitated from the supernatant with the anti-human ABCA3 antibody (38). Samples were electrophoresed on a 5% SDS-polyacrylamide gel and transferred to a PVDF membrane. The radioactivities of photoaffinity-labeled protein (total 220-kDa noncleaved form plus 180-kDa cleaved form) were quantified using FLA-5000. To confirm the expression level of ABCA3-GFP proteins, the membrane was further analyzed by immunoblotting using anti-GFP antibody. Data normalized to the level of ABCA3-GFP proteins (total 220-kDa noncleaved form plus 180-kDa cleaved form) are presented as means ± SD. Statistical analysis was performed as described above.

Homology modeling of nucleotide binding domain 1 of ABCA3. The secondary structures of nucleotide binding domains (NBDs) of ABCA3 and other ABC transporters were predicted using the PSIPRED program (19). Amino acids sequences were aligned using the ClustalW program (15) and manually corrected based on the predicted secondary structures. The amino acids residues 545–766 in NBD-1 of ABCA3 share 29.7%, 25.7%, and 23.4% sequence identities with Escherichia coli maltose transporter MalK, Staphylococcus aureus Sav1866, and E. coli hemolysin transporter HlyB, respectively (6, 9, 39). The structure of NBD-1 of ABCA3 was modeled based on the ATP-bound closed form of E. coli MalK (Protein Data Bank entry 1Q12; Ref. 6) using SWISS-MODEL (26). Amino acid substitution was performed on Swiss-PDB Viewer, considering the best rotamer conformation.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Subcellular localization and glycosylation of ABCA3-GFP and pILD mutant proteins. We previously found that GFP-tagged wild-type ABCA3 protein (ABCA3-GFP) expressed in cultured cells is localized mainly at the limiting membrane of LAMP3-positive vesicles, whereas type I mutant protein (e.g., L101P) in fatal surfactant deficiency remains localized in the endoplasmic reticulum, accompanied by impaired processing of oligosaccharide (17). The E292V, E690K, and T1114M mutations identified in pILD (4) are located at intracellular loop 1 (ICL-1), adjacent to the Walker B motif in NBD-1 and the 8th putative transmembrane segment (TM-8), respectively (Fig. 1A). When E292V, E690K, and T1114M mutant ABCA3-GFP proteins were transiently expressed in HEK-293 cells, most of the GFP fluorescence was located at intracellular vesicles, as with the wild-type protein (Fig. 1B). ABCA3 is expressed as 190- and 150-kDa forms, with the latter suggested to be produced by proteolytic cleavage at extracellular domain 1 (ECD1) within lysosomal vesicles (21, 36). In native lung tissue, the cleaved form is predominant, whereas both forms are detected when overexpressed in cultured cells. Immunoblot analysis of total membrane fraction from transiently transfected HEK-293 cells using anti-GFP antibody showed two bands at ~220 kDa (noncleaved form) and 180 kDa (cleaved form) in wild-type and three mutant ABCA3-GFP proteins (Fig. 1C). In E690K mutant protein, the amount of the 180-kDa cleaved form was increased compared with that of wild-type protein. PNGase F digestion of total membrane fraction showed that the 220-kDa forms of all three mutants are N-glycosylated, as is wild-type ABCA3-GFP protein (Fig. 1D). In the E292V, E690K, and T1114M mutant proteins, 50–60% of the 220-kDa protein remained as Endo H-insensitive complex-type protein (Fig. 1E), indicating that intracellular trafficking and processing of oligosaccharide of these mutant proteins are largely preserved and that these mutations are not type I mutations.


Figure 1
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  		Fig. 1. Structural model of ATP-binding cassette transporter ABCA3 protein and intracellular localization and glycosylation of wild-type ABCA3-green fluorescent protein (GFP) and pediatric interstitial lung disease (pILD) mutant proteins. A: locations of pILD mutations characterized in this study are indicated. ABCA3 is constituted by 2 transmembrane domains (TMD), each of which contains 6 putative transmembrane segments (TM), 2 extracellular domains (ECD), and 2 nucleotide binding domains (NBD). There are 3 asparagine residues (Asp53, Asp124, and Asp140) with a consensus N-glycosylation motif (NXS/T) in ECD1 but not in ECD2. B: intracellular localization of GFP, wild-type ABCA3-GFP protein (WT), and its mutants (E292V, E690K, and T1114M) transiently expressed in human embryonic kidney HEK-293 cells were determined by confocal microscopy. Scale bar, 2 µm. C: total membrane fractions from HEK-293 cells transiently transfected with GFP, WT ABCA3-GFP, or mutants were subjected to SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and analyzed using anti-GFP monoclonal antibody. The positions of noncleaved (220 kDa) and cleaved (180 kDa) ABCA3-GFP proteins are indicated. D: total membrane fraction with (+) or without (–) treatment of peptide N-glycosidase F (PNGase F) was subjected to SDS-PAGE and analyzed by immunoblotting. ABCA3-GFP proteins modified with oligosaccharide (220 kDa) were deglycosylated by PNGase F, producing 210-kDa proteins. E: total membrane fraction with or without treatment of endoglycosidase H (Endo H) was subjected to SDS-PAGE and analyzed by immunoblotting. Band I shows Endo H-insensitive 220-kDa ABCA3-GFP proteins containing complex-type sugar chains, whereas band II shows Endo H-sensitive 210-kDa proteins modified with high-mannose-type sugar chains.

 
Lipid transport function of ABCA3-GFP and pILD mutant proteins. The lipid transport function of ABCA3 protein has been investigated using non-lung HEK-293 cells or lung adenocarcinoma A549 cells transfected with ABCA3 (7, 18). In this study, we established HEK-293 cells stably expressing pILD mutant ABCA3-GFP proteins that can be used to analyze both lipid transport function and nucleotide trapping of ABCA3 proteins. ABCA3 mediates uptake of choline-phospholipids into LAMP3-positive vesicles to convert lysosomal organelles into lamellar body-like organelles (7, 18). The lipid transport function of ABCA3 protein can therefore be evaluated by comparing endogenous content of choline-phospholipid-rich vesicles and the level of LAMP3, a marker of lysosomal organelles such as multivesicular bodies and lamellar bodies (34, 35). Expression of wild-type ABCA3-GFP in HEK-293 cells was found to increase the level of LAMP3, as well as the choline-phospholipid level and low-density and choline-phospholipid-rich vesicle contents (Fig. 2, A, B, and D), as we previously found in A549 cells (18). Immunoblot analysis using anti-GFP antibody revealed that three mutant ABCA3-GFP proteins were expressed at a level similar to that in wild-type protein (Fig. 2A). The level of LAMP3 in E292V transfectant was comparable to that in wild-type transfectant, whereas those in E690K and T1114M transfectants were lower than in wild-type transfectant (Fig. 2A and Supplemental Fig. 1A). (Supplemental data for this article is available online at the American Journal of Physiology-Lung Cellular and Molecular Physiology website.) The smeared appearance of the immunoblot signal reflects high glycosylation states of LAMP3 protein (16). On the other hand, the levels of GRP78, a marker of endoplasmic reticulum, were similar in the five cell lines. These results indicate that expression of pILD mutant ABCA3-GFP proteins affects lysosomal organelles in differing degrees.


Figure 2
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  		Fig. 2. Lipids transport function of wild-type ABCA3-GFP and pILD mutant proteins. A: Immunoblot analysis of the level of ABCA3-GFP, LAMP3, and GRP78 in HEK-293 cells stably expressing WT ABCA3-GFP, E292V, E690K, T1114M, or untransfected HEK-293 cells. B and C: the contents of endogenous choline-phospholipids (B) and total cholesterol (C) in postnuclear supernatant (PNS) of each cell type. Data are means ± SD (n = 6–9). *P < 0.05; **P < 0.01 vs. HEK-293 cells. N.S., not significant. D: sucrose gradient fractionation of intracellular compartments from each cell type. PNS (4 mg of protein) was fractionated in a sucrose gradient. Fractions 1 and 10 are the lowest and highest density fractions, respectively. The contents of endogenous choline-phospholipids are shown. For comparison, the lipid content of HEK-293 cells stably expressing wild-type ABCA3 (solid line) and untransfected HEK-293 cells (broken line) are shown. Experiments were performed 4 times, and representative data are shown.

 
To investigate the effect of ABCA3 expression on the endogenous lipid level, we analyzed the contents of choline-phospholipids and total cholesterol in these cell lines. The levels of choline-phospholipids were significantly increased 1.38- and 1.13-fold in wild-type and E292V transfectants, respectively, compared with the level in HEK-293 cells, whereas levels in E690K and T1114M transfectants were similar to that in HEK-293 cells (Fig. 2B). On the other hand, the levels of total cholesterol were similar in the five cell lines (Fig. 2C).

We then investigated sucrose gradient fractionation to identify low-density and choline-phospholipid-rich vesicle formation in mutant transfectants. In this gradient system, choline-phospholipid-rich vesicles generated by ABCA3-mediated lipid transport migrate to the lower fractions in a manner similar to lamellar bodies from native lung tissue (18). In wild-type transfectant, endogenous choline-phospholipid content in the lower fractions (fractions 2–4) was higher than in HEK-293 cells (Fig. 2D). In addition, in wild-type transfectant, LAMP3 was detected mainly in fractions 2–4 and 10, whereas LAMP3 was detected mainly in fraction 10 and partly in fractions 4–6 in HEK-293 cells (Supplemental Fig. 1B). Thus the increased choline-phospholipid content in the lower fractions and the shift of LAMP3 distribution into lower fractions reflect ABCA3-mediated uptake of choline-phospholipids into LAMP3-positive vesicles to convert lysosomal organelles into lower density and lipid-rich vesicles (7, 18). In E292V transfectant, choline-phospholipid content in fractions 2–4 was higher than that in HEK-293 cells and lower than that in wild-type transfectant. In E690K transfectant, the distribution of choline-phospholipid content was similar to that in HEK-293 cells. In T1114M transfectant, choline-phospholipid content in fractions 2–4 was somewhat higher than that in HEK-293 cells, but the difference was not statistically significant (n = 4, Supplemental Fig. 1C). Considered together, these results suggest that although the lipid transport function of the E292V mutant protein is moderate, those of the E690K and T1114M mutant proteins are severely impaired.

Abnormalities of ATP binding and/or hydrolysis in pILD mutant proteins. To clarify the mechanism of impaired lipid transport in the pILD mutant proteins, we compared ATP hydrolysis of these mutants by using a vanadate-induced nucleotide trapping technique (28, 32). ABCA3 protein efficiently traps Mg-ADP in the presence of orthovanadate, an analog of phosphate, and forms a stable inhibitory intermediate during the ATP hydrolysis cycle (17, 21). This intermediate can be specifically photoaffinity labeled in the membrane after ATP hydrolysis when 8-azido-[{alpha}-32P]ATP is used as an ATP analog, allowing assessment of ATP hydrolysis with production of a stable intermediate based on the intensity of photoaffinity labeling.

As previously reported (17), among 20,000-g membrane fractions of cells expressing wild-type ABCA3-GFP, 220-kDa (noncleaved form) and 180-kDa (cleaved form) proteins were slightly photoaffinity labeled with 8-azido-[{alpha}-32P]ATP in the absence of orthovanadate (Fig. 3A, lane 3), and photoaffinity labeling was further induced in the presence of orthovanadate (Fig. 3A, lane 4). In vanadate-induced nucleotide trapping by the E292V and T1114M mutant proteins, ATP hydrolysis with production of a photoaffinity-labeled intermediate was decreased to 40 and 52% of that of wild-type protein, respectively (Fig. 3A, lanes 5, 6, 9, and 10, and B). On the other hand, in the E690K mutant protein, it was increased to 200% of that of wild-type protein (Fig. 3A, lanes 7 and 8, and B).


Figure 3
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  		Fig. 3. Vanadate-induced nucleotide trapping and photoaffinity labeling of ABCA3-GFP and pILD mutant proteins using 8-azido-[32P]ATP. A: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4), E292V (lanes 5 and 6), E690K (lanes 7 and 8), T1114M (lanes 9 and 10), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 µM 8-azido-[{alpha}-32P]ATP in the absence (–) or presence (+) of 0.4 mM orthovanadate (Vi) and 3 mM MgCl2 for 10 min at 37°C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by autoradiography (top) and immunoblotting (IB) using anti-GFP antibody (bottom). B: radioactivity of photoaffinity-labeled protein (total 220-kDa noncleaved form plus 180-kDa cleaved form) was quantified by FLA-5000. Radioactivity in the absence of orthovanadate was subtracted from that in the presence of orthovanadate and is expressed after normalization to the level of ABCA3-GFP protein (total 220-kDa noncleaved form plus 180-kDa cleaved form). Data are means ± SD (n = 4). *P < 0.05; **P < 0.01 vs. WT. C: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP, E292V, E690K, T1114M, or untransfected HEK-293 cells was incubated with 40 µM 8-azido-[{gamma}-32P] and 3 mM MgCl2 for 10 min at 0°C. Protein was photoaffinity labeled with UV irradiation, immunoprecipitated with anti-human ABCA3 antibody, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by FLA-5000 (top) and IB using anti-GFP antibody (bottom). D: radioactivity of photoaffinity-labeled protein (total 220-kDa noncleaved form plus 180-kDa cleaved form) was quantified by FLA-5000 and is expressed after normalization to the level of ABCA3-GFP protein (total 220-kDa noncleaved form plus 180-kDa cleaved form). Data are means ± SD (n = 3). *P < 0.01 vs. WT.

 
To investigate the altered ATP hydrolysis with production of a photoaffinity-labeled intermediate in the mutant proteins, we examined ATP binding of ABCA3-GFP proteins by photoaffinity labeling with 8-azido-[{gamma}-32P]ATP at 0°C. In this nonhydrolytic condition, ABCA3-GFP proteins are labeled only with nucleotides before hydrolysis, allowing determination of ATP binding based on the intensity of photoaffinity labeling. Among the 20,000-g membrane fractions of cells expressing wild-type ABCA3-GFP, 220- and 180-kDa proteins were photoaffinity labeled with 8-azido-[{gamma}-32P]ATP, whereas membrane fractions of untransfected cells were not photoaffinity labeled (Fig. 3C). ATP binding of the E292V and T1114M mutant proteins determined by photoaffinity labeling with 8-azido-[{gamma}-32P]ATP was similar to that of wild-type ABCA3-GFP protein (Fig. 3, C and D). However, in E690K mutant protein, photoaffinity labeling of the 220-kDa protein was similar to that of wild type protein regardless of decreased noncleaved form protein, and photoaffinity labeling of the 180-kDa protein was dramatically enhanced even when the increased levels of the cleaved form of the protein were taken into consideration. When normalized to the level of ABCA3-GFP proteins (total 220-kDa noncleaved form plus 180-kDa cleaved form), photoaffinity labeling of the E690K mutant protein was increased to 250% of that of wild-type protein. These results show that the catalytic cycles of these mutant proteins differ from that of wild-type protein and that these mutations in pILD are type II mutations.

Mutational analysis of Glu292 in ICL-1 and Thr1114 in putative TM-8. To investigate the mechanism of loss of ATP hydrolysis with production of a photoaffinity-labeled intermediate in E292V and T1114M mutant proteins, we performed mutational analyses of Glu292 residue in ICL-1 and Thr1114 in putative TM-8. The Glu or Asp residue in ICL-1 is conserved in members of the ABCA subfamily (Fig. 4A), suggesting the importance of negatively charged amino acids in ICL-1 for ATP hydrolysis. To clarify this, we substituted Glu292 with Asp and Lys, which are negatively and positively charged, respectively. In vanadate-induced nucleotide trapping by E292D mutant protein, production of a photoaffinity-labeled intermediate during ATP hydrolysis was decreased to 37% of that of wild-type protein, as also was the case in E292V mutant protein (Fig. 4B, lanes 5–8, and C). In E292K mutant protein, it was decreased to 4% of that of wild-type protein (Fig. 4B, lanes 9 and 10, and C). These results indicate that not only a negative charge but also an appropriate side chain length of Glu292 at ICL-1 is important for production of a photoaffinity-labeled intermediate during ATP hydrolysis of ABCA3 protein.


Figure 4
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  		Fig. 4. Alignment of amino acid sequence of intracellular loop 1 (ICL-1) and vanadate-induced nucleotide trapping in site-directed mutant proteins of Glu292. A: alignment of amino acid sequence of ICL-1 of some members of the human ABCA subfamily is shown. The amino acid residues that are conserved in 6 transporters and in 4 or 5 transporters are indicated by asterisks and dots, respectively. B: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4), E292V (lanes 5 and 6), E292D (lanes 7 and 8), E292K (lanes 9 and 10), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 µM 8-azido-[{alpha}-32P]ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37°C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by autoradiography (top) and IB using anti-GFP antibody (bottom). C: radioactivity of photoaffinity-labeled protein (total 220-kDa noncleaved form plus 180-kDa cleaved form) was quantified by FLA-5000. Radioactivity in the absence of orthovanadate was subtracted from that in the presence of orthovanadate and is expressed after normalization to the level of ABCA3-GFP protein (total 220-kDa noncleaved form plus 180-kDa cleaved form). Data are means ± SD (n = 3). *P < 0.01 vs. WT.

 
Recently, we identified a novel compound heterozygous mutation (maternal T1114A and paternal W1148X) from a Japanese boy with respiratory distress from age 18 mo (37). The T1114A mutation decreased vanadate-induced nucleotide trapping by ABCA3 protein similarly to the T1114M mutation. Because ABCA1, ABCA2, ABCA4, ABCA7, and ABCA12 have conserved Ser residues rather than a Thr residue in putative TM-8 (Fig. 5A), loss of the hydroxyl group-containing amino acid might well act on putative TM-8 to hamper communication between transmembrane domains (TMDs) and NBDs, resulting in loss of ATP hydrolysis activity. To clarify this, we substituted Thr1114 with Ser and examined vanadate-induced nucleotide trapping. In vanadate-induced nucleotide trapping by T1114S mutant protein, production of a photoaffinity-labeled intermediate during ATP hydrolysis was found to be similar to that of wild-type protein, whereas that of T1114M and T1114A mutant protein was 56 and 47% of wild-type protein, respectively (Fig. 5B, lanes 5–10, and C). These results indicate that loss of the hydroxyl group of the 1114th amino acid is responsible for the impaired ATP hydrolysis with production of a photoaffinity-labeled intermediate in both the T1114M and T1114A mutant proteins.


Figure 5
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  		Fig. 5. Alignment of amino acid sequence of the 8th putative transmembrane segment (TM-8) and vanadate-induced nucleotide trapping in site-directed mutant proteins of Thr1114. A: alignment of amino acid sequence of putative TM-8 of some members of the human ABCA subfamily is shown. The amino acids residues that are conserved in 6 transporters and in 4 or 5 transporters are indicated by asterisks and dots, respectively. B: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4), T1114M (lanes 5 and 6), T1114A (lanes 7 and 8), T1114S (lanes 9 and 10), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 µM 8-azido-[{alpha}-32P]ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37°C. Proteins were photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by autoradiography (top) and IB using anti-GFP antibody (bottom). C: radioactivity of photoaffinity-labeled protein (total 220-kDa noncleaved form plus 180-kDa cleaved form) was quantified by FLA-5000. Radioactivity in the absence of orthovanadate was subtracted from that in the presence of orthovanadate and is expressed after normalization to the level of ABCA3-GFP protein (total 220-kDa noncleaved form plus 180-kDa cleaved form). Data are means ± SD (n = 3). *P < 0.01 vs. WT.

 
Interaction of E690K mutant ABCA3-GFP protein with nucleotides. To investigate the mechanism of enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in E690K mutant protein, we performed the trapping experiments using 8-azido-[{gamma}-32P]ATP. In this procedure, a posthydrolyzed trapped nucleotide should not be detected by vanadate-induced trapping because of hydrolytic loss of [{gamma}-32P]PO4. Indeed, in the presence of orthovanadate, photoaffinity labeling of wild-type and E690K mutant proteins with 8-azido-[{gamma}-32P]ATP was barely detectable (Fig. 6A). These data combined with vanadate-induced trapping using 8-azido-[{alpha}-32P]ATP indicate that E690K mutant protein hydrolyzes 8-azido-[{alpha}-32P]ATP and releases {gamma}-phosphate and that the nucleotide trapped by E690K mutant protein is mostly in the ADP form.


Figure 6
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  		Fig. 6. Interaction of E690K mutant ABCA3-GFP protein with nucleotides. A: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4) E690K (lanes 5 and 6), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 µM 8-azido-[{gamma}-32P] ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37°C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by autoradiography (top) and IB using anti-GFP antibody (bottom). B: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP, E690K, or untransfected HEK-293 cells was incubated with 40 µM 8-azido-[{alpha}-32P]ADP and 3 mM MgCl2 for 10 min at 0°C. Protein was photoaffinity labeled with UV irradiation, immunoprecipitated with anti-human ABCA3 antibody, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by FLA-5000 (top) and IB using anti-GFP antibody (bottom). C: radioactivity of photoaffinity-labeled protein (total 220-kDa noncleaved form plus 180-kDa cleaved form) was quantified by FLA-5000 and is expressed after normalization to the level of ABCA3-GFP protein (total 220-kDa noncleaved form plus 180-kDa cleaved form). Data are means ± SD (n = 3). D: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP or E690K was first incubated for 10 min at 37°C with 10 µM 8-azido-[{alpha}-32P]ATP in the presence of orthovanadate, and unbound nucleotides were removed by washing. A membrane fraction was reincubated at 37°C for 0–15 min in a buffer containing MgCl2, before cross-linking. After removal of released nucleotides and cross-linking, protein was electrophoresed on SDS-PAGE and transferred to a PVDF membrane. Membrane was analyzed by autoradiography (top) and IB using anti-GFP antibody (bottom). E: radioactivity of photoaffinity-labeled protein (total 220-kDa noncleaved form plus 180-kDa cleaved form) was quantified by FLA-5000 and is expressed as a percentage of the 0-min value after normalization to the level of ABCA3-GFP protein (total 220-kDa noncleaved form plus 180-kDa cleaved form). Data are means ± SD (n = 4). *P < 0.05; **P < 0.01 vs. WT.

 
In E690K mutant protein, ATP binding determined by photoaffinity labeling with 8-azido-[{gamma}-32P]ATP was dramatically increased compared with that of wild-type protein (see Fig. 3, C and D). To determine whether the E690K mutation alters ADP binding, we performed labeling experiments using 8-azido-[{alpha}-32P]ADP at 0°C. In this condition, photoaffinity labeling of E690K mutant protein was comparable to that of wild-type ABCA3-GFP protein (Fig. 6, B and C), suggesting that {gamma}-phosphate of 8-azido-[{gamma}-32P]ATP contributes to enhanced photoaffinity labeling in E690K mutant protein.

Since some mutations in the Glu residues following the Walker B motif have been reported to interfere with the ATP-hydrolysis cycle including the ADP release step (5, 24, 25, 33), we examined release of trapped nucleotides from wild-type and E690K mutant ABCA3-GFP proteins. A 20,000-g membrane fraction was first incubated for 10 min at 37°C with 8-azido-[{alpha}-32P]ATP in the presence of orthovanadate, and unbound nucleotides were removed by washing. The membrane fraction was reincubated at 37°C for 0–15 min in buffer containing MgCl2 before cross-linking. In wild-type ABCA3-GFP protein, trapped nucleotides were time-dependently reduced by reincubation, and photoaffinity labeling at 5 min was 32% of that at 0 min (Fig. 6, D and E). In contrast, in E690K mutant protein, trapped nucleotides were more slowly released than in wild-type protein, and photoaffinity labeling at 5 min was 73% of that at 0 min, suggesting that E690K mutant protein forms a more stable inhibitory intermediate after hydrolysis of 8-azido-[{alpha}-32P]ATP than wild-type protein. Thus these data further suggest abnormal interaction with nucleotides in E690K mutant protein.

Mutational analysis of Glu690 adjacent to walker B motif in NBD-1. To further clarify the enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in E690K mutant, we performed mutational analyses of the Glu690 residue adjacent to the Walker B motif in NBD-1. Because Glu and Lys are negatively and positively charged amino acids, respectively, alteration of charge at the 690th amino acid residue could well be responsible for the abnormal interaction with nucleotides in the E690K mutant. Accordingly, Glu690 was substituted with Asp and Arg, which are negatively and positively charged, respectively. Substitution with Asp and Arg caused a dramatic decrease in ATP hydrolysis with production of a photoaffinity-labeled intermediate to 11 and 12% of that of wild-type protein, respectively (Fig. 7, A and B). These results show that both negative charge and side chain length of Glu690 are important for ATP hydrolysis of wild-type ABCA3 protein and that not only positive charge but also side chain length of Lys690 contribute to enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in the E690K mutant protein.


Figure 7
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  		Fig. 7. Vanadate-induced nucleotide trapping in site-directed mutant proteins of Glu690. A: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4), E690K (lanes 5 and 6), E690D (lanes 7 and 8), E690R (lanes 9 and 10), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 µM 8-azido-[{alpha}-32P]ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37°C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by autoradiography (top) and IB using anti-GFP antibody (bottom). B: radioactivity of photoaffinity-labeled protein (total 220-kDa noncleaved form plus 180-kDa cleaved form) was quantified by FLA-5000. Radioactivity in the absence of orthovanadate was subtracted from that in the presence of orthovanadate and is expressed after normalization to the level of ABCA3-GFP protein (total 220-kDa noncleaved form plus 180-kDa cleaved form). Data are means ± SD (n = 3). *P < 0.01 vs. WT.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, to clarify the phenotypic heterogeneity of lung disease associated with ABCA3 mutation, we characterized the ABCA3 mutant proteins identified in pILD patients, a more mild disease than fatal surfactant deficiency. Although E292V, E690K, and T1114M mutant proteins were found to traffic to intracellular vesicles, the lipid transport function of E292V mutant protein was partially impaired, and those of E690K and T1114M mutant protein were severely impaired, accompanied by an aberrant catalytic cycle. We recently found that fatal surfactant deficiency due to ABCA3 mutation comprises defects of abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP hydrolysis of ABCA3 protein (type II) (17). Accordingly, E292V, E690K, and T1114M are type II mutations.

Patients with fatal surfactant deficiency carrying a type I homozygous ABCA3 mutation (W1142X/W1142X, L101P/L101P, or L1553P/L1553P) or a type I/type II compound heterozygous mutation (L982P/G1221S or Ins1518/L1580P) die within the neonatal period (Table 1) (27). On the other hand, patients carrying a type II/type II ABCA3 mutation (E292V/T1114M or E292V/E690K) exhibit pILD (4), suggesting that the type II/type II ABCA3 mutation produces a milder phenotype. Although an exception has been identified in a Japanese patient with a type I/type II ABCA3 mutation (W1148X/T1114A) (37), the moderately preserved lipid transport function of the E292V mutant protein may underlie the generally milder phenotype of pILD patients. Further analysis using transgenic mice is required to understand the impacts of these mutations on surfactant metabolism in vivo.


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  		Table 1. Genotype-phenotype correlation for ABCA3 mutation

 
The E292V mutant protein exhibits moderately preserved lipid transport function and vanadate-induced nucleotide trapping, and mutational analysis of Glu292 in ICL-1 indicates the significance of both negative charge and side chain length of Glu292 for ATP hydrolysis with production of a photoaffinity-labeled intermediate in ABCA3 protein. In addition, the recently solved crystal structure of the bacterial ABC transporter Sav1866 suggests that ICLs interact with NBDs and TMDs to transmit conformational changes generated by ATP binding and hydrolysis from NBDs to TMDs (9). Thus the E292V mutation might impede the transmission of conformational changes, resulting in moderate impairment of lipid transport in ABCA3 protein.

The T1114M mutant protein exhibits impaired lipid transport function accompanied by moderately preserved vanadate-induced nucleotide trapping. In addition, mutational analysis of Thr1114 in putative TM-8 shows the importance of the hydroxyl group of the 1114th amino acid residue for ATP hydrolysis with production of a photoaffinity-labeled intermediate in ABCA3 protein. In membrane proteins, the hydroxyl group of Ser and that of Thr are known to form a hydrogen bond with backbone nitrogen to contribute to tight helix packing (10), suggesting that loss of the hydroxyl group from the 1114th amino acid in putative TM-8 might well hamper conformational change of TMDs during ATP-hydrolysis that results in impaired lipid transport of the ABCA3 protein.

The glutamate residue following the Walker B motif in ABC transporters has been suggested to be a catalytic carboxylate that facilitates nucleophilic attack on ATP via a water molecule (13, 23). However, it has been reported that some mutations in the Glu residues interfere with ADP release (5, 24, 25, 33) and tight dimerization of NBDs (29, 30, 31). In E690K mutant ABCA3 protein, lipid transport function is severely impaired, accompanied by abnormal interaction with nucleotides: enhanced vanadate-induced nucleotide trapping at 37°C, enhanced photoaffinity labeling with 8-azido-[{gamma}-32P]ATP at 0°C, and delayed release of 8-azido-[{alpha}-32P]ADP after vanadate-induced nucleotide trapping. Furthermore, mutational analysis of Glu690 indicates that side chain length of Lys690 contributes to enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in the E690K mutant protein. To clarify the origin of the abnormal interaction with nucleotides in E690K mutant protein, we modeled the structure of NBD-1 of ABCA3 based on the crystal structure of E. coli MalK using SWISS-MODEL (Supplemental Fig. 2). In the wild-type ABCA3 model, the distance from side chain oxygen of Glu690 to {gamma}-phosphate oxygen of ATP is ~7.1 Å, similar to the distance from side chain oxygen of Glu159 in MalK. On the other hand, in the model of E690K mutant, the distance from side chain nitrogen of Lys690 to {gamma}-phosphate oxygen of ATP is ~3.6 Å. One possible interpretation of these biochemical results and modeling is that ionic interaction of Lys690 and {gamma}-phosphate of ATP in the E690K mutant protein may tighten the binding of ATP in NBD-1, resulting in delayed ADP release after ATP hydrolysis, probably in NBD-2. Another possible interpretation is that the interaction of Lys690 with {gamma}-phosphate of ATP may alter the directionality of the adenine moiety of ATP, increasing the efficiency of photoaffinity labeling with 8-azido-ATP. Analysis using purified ABCA3 protein would further clarify the aberrant catalytic cycle and impaired lipid transport in E690K mutant protein.

The cleaved form of ABCA3 protein is predominantly expressed in native lung tissue (11, 18, 36) and shows increased vanadate-induced trapping compared with the noncleaved form of the protein in HEK-293 cells. Furthermore, cleavage of ABCA3 protein at ECD1 is suggested to be important for fully active transport function (unpublished observation). Since the cleaved form was rarely detected in ABCA3 mutant protein that remained localized to endoplasmic reticulum (17), the cleavage of ABCA3 protein may occur in Golgi or post-Golgi compartments. Interestingly, in E690K mutant protein, the amount of 180-kDa cleaved form was increased compared with that of wild-type protein. The increased level of 180-kDa cleaved form E690K mutant protein might be due to the abnormal nucleotide-bound conformation of E690K mutant protein being preferentially cleaved by enzymes within intracellular vesicles, compared with that in wild-type protein. Further studies to determine cleavage sites and the enzymes involved in the cleavage of ABCA3 protein are needed.

In summary, the moderately preserved lipid transport function of E292V mutant protein may be responsible for the milder phenotype in pILD caused by ABCA3 mutation compared with that in fatal surfactant deficiency. The molecular mechanisms of the impaired lipid transport function of ABCA3 could be key to developing treatment strategies to restore ABCA3 function in patients with ABCA3 mutation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Scientific Research Grants and Grant-in-Aid for Creative Scientific Research 15GS0301 from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Core Research for Evolutional Science and Technology of Japan Science and Technology Agency; the 21st Century Center of Excellence Program; and the Manpei Suzuki Diabetes Foundation.


   ACKNOWLEDGMENTS
 
We thank Drs. Jun-ichi Miyazaki (Osaka University) and Hitoshi Niwa (RIKEN, Kobe) for providing the pCAGIpuro plasmid.

Present address of Y. Matsumura: Dept. of Biochemistry and Molecular Biology, Oregon Health and Science Univ., Portland, OR 97239.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Inagaki, Dept. of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto Univ., 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan (e-mail: inagaki{at}metab.kuhp.kyoto-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ban N, Matsumura Y, Sakai H, Takanezawa Y, Sasaki M, Arai H, Inagaki N. ABCA3 as a lipid transporter in pulmonary surfactant biogenesis. J Biol Chem 282: 9628–9634, 2007.[Abstract/Free Full Text]
  2. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917, 1959.[Medline]
  3. Brasch F, Schimanski S, Muhlfeld C, Barlage S, Langmann T, Aslanidis C, Boettcher A, Dada A, Schroten H, Mildenberger E, Prueter E, Ballmann M, Ochs M, Johnen G, Griese M, Schmitz G. Alteration of the pulmonary surfactant system in full-term infants with hereditary ABCA3 deficiency. Am J Respir Crit Care Med 174: 571–580, 2006.[Abstract/Free Full Text]
  4. Bullard JE, Wert SE, Whitsett JA, Dean M, Nogee LM. ABCA3 mutations associated with pediatric interstitial lung disease. Am J Respir Crit Care Med 172: 1026–1031, 2005.[Abstract/Free Full Text]
  5. Carrier I, Julien M, Gros P. Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein. Biochemistry 42: 12875–12885, 2003.[CrossRef][Web of Science][Medline]
  6. Chen J, Lu G, Lin J, Davidson AL, Quiocho FA. A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. Mol Cell 12: 651–661, 2003.[CrossRef][Web of Science][Medline]
  7. Cheong N, Madesh M, Gonzales LW, Zhao M, Yu K, Ballard PL, Shuman H. Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome. J Biol Chem 281: 9791–9800, 2006.[Abstract/Free Full Text]
  8. Cheong N, Zhang H, Muniswamy M, Zhao M, Yu K, Dodia C, Fisher AB, Savani RC, Shuman H. ABCA3 is critical for lamellar body biogenesis in vivo. J Biol Chem 282: 23811–23817, 2007.[Abstract/Free Full Text]
  9. Dawson RJ, Locher KP. Structure of a bacterial multidrug ABC transporter. Nature 443: 180–185, 2006.[CrossRef][Web of Science][Medline]
  10. Eilers M, Shekar SC, Shieh T, Smith SO, Fleming PJ. Internal packing of helical membrane proteins. Proc Natl Acad Sci USA 97: 5796–5801, 2000.[Abstract/Free Full Text]
  11. Fitzgerald ML, Xavier R, Haley KJ, Welti R, Goss JL, Brown CE, Zhuang DZ, Bell SA, Lu N, McKee M, Seed B, Freeman MW. ABCA3 inactivation in mice causes respiratory failure, loss of pulmonary surfactant, and depletion of lung phosphatidylglycerol. J Lipid Res 48: 621–632, 2007.[Abstract/Free Full Text]
  12. Garmany TH, Moxley MA, White FV, Dean M, Hull WM, Whitsett JA, Nogee LM, Hamvas A. Surfactant composition and function in patients with ABCA3 mutations. Pediatr Res 59: 801–805, 2006.[CrossRef][Web of Science][Medline]
  13. Geourjon C, Orelle C, Steinfels E, Blanchet C, Deleage G, Di Pietro A, Jault JM. A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies. Trends Biochem Sci 26: 539–544, 2001.[CrossRef][Web of Science][Medline]
  14. Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8: 67–113, 1992.[CrossRef][Web of Science][Medline]
  15. Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680, 1994.[Abstract/Free Full Text]
  16. Kobayashi T, Beuchat MH, Chevallier J, Makino A, Mayran N, Escola JM, Lebrand C, Cosson P, Kobayashi T, Gruenberg J. Separation and characterization of late endosomal membrane domains. J Biol Chem 277: 32157–32164, 2002.[Abstract/Free Full Text]
  17. Matsumura Y, Ban N, Ueda K, Inagaki N. Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency. J Biol Chem 281: 34503–34514, 2006.[Abstract/Free Full Text]
  18. Matsumura Y, Sakai H, Sasaki M, Ban N, Inagaki N. ABCA3-mediated choline-phospholipids uptake into intracellular vesicles in A549 cells. FEBS Lett 581: 3139–3144, 2007.[CrossRef][Web of Science][Medline]
  19. McGuffin LJ, Bryson K, Jones DT. The PSIPRED protein structure prediction server. Bioinformatics 16: 404–405, 2000.[Abstract/Free Full Text]
  20. Mulugeta S, Gray JM, Notarfrancesco KL, Gonzales LW, Koval M, Feinstein SI, Ballard PL, Fisher AB, Shuman H. Identification of LBM180, a lamellar body limiting membrane protein of alveolar type II cells, as the ABC transporter protein ABCA3. J Biol Chem 277: 22147–22155, 2002.[Abstract/Free Full Text]
  21. Nagata K, Yamamoto A, Ban N, Tanaka AR, Matsuo M, Kioka N, Inagaki N, Ueda K. Human ABCA3, a product of a responsible gene for abca3 for fatal surfactant deficiency in newborns, exhibits unique ATP hydrolysis activity and generates intracellular multilamellar vesicles. Biochem Biophys Res Commun 324: 262–268, 2004.[CrossRef][Web of Science][Medline]
  22. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193–199, 1991.[CrossRef][Web of Science][Medline]
  23. Orelle C, Dalmas O, Gros P, Di Pietro A, Jault JM. The conserved glutamate residue adjacent to the Walker-B motif is the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter BmrA. J Biol Chem 278: 47002–47008, 2003.[Abstract/Free Full Text]
  24. Payen LF, Gao M, Westlake CJ, Cole SP, Deeley RG. Role of carboxylate residues adjacent to the conserved core Walker B motifs in the catalytic cycle of multidrug resistance protein 1 (ABCC1). J Biol Chem 278: 38537–38547, 2003.[Abstract/Free Full Text]
  25. Sauna ZE, Muller M, Peng XH, Ambudkar SV. Importance of the conserved Walker B glutamate residues, 556 and 1201, for the completion of the catalytic cycle of ATP hydrolysis by human P-glycoprotein (ABCB1). Biochemistry 41: 13989–14000, 2002.[CrossRef][Web of Science][Medline]
  26. Schwede T, Kopp J, Guex N, Peitsch MC. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31: 3381–3385, 2003.[Abstract/Free Full Text]
  27. Shulenin S, Nogee LM, Annilo T, Wert SE, Whitsett JA, Dean M. ABCA3 gene mutations in newborns with fatal surfactant deficiency. N Engl J Med 350: 1296–1303, 2004.[Abstract/Free Full Text]
  28. Taguchi Y, Yoshida A, Takada Y, Komano T, Ueda K. Anti-cancer drugs and glutathione stimulate vanadate-induced trapping of nucleotide in multidrug resistance-associated protein (MRP). FEBS Lett 401: 11–14, 1997.[CrossRef][Web of Science][Medline]
  29. Tombline G, Bartholomew LA, Urbatsch IL, Senior AE. Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly. J Biol Chem 279: 31212–31220, 2004.[Abstract/Free Full Text]
  30. Tombline G, Bartholomew LA, Tyndall GA, Gimi K, Urbatsch IL, Senior AE. Properties of P-glycoprotein with mutations in the "catalytic carboxylate" glutamate residues. J Biol Chem 279: 46518–46526, 2004.[Abstract/Free Full Text]
  31. Tombline G, Muharemagic A, White LB, Senior AE. Involvement of the "occluded nucleotide conformation" of P-glycoprotein in the catalytic pathway. Biochemistry 44: 12879–12886, 2005.[CrossRef][Web of Science][Medline]
  32. Urbatsch IL, Sankaran B, Weber J, Senior AE. P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site. J Biol Chem 270: 19383–19390, 1995.[Abstract/Free Full Text]
  33. Urbatsch IL, Julien M, Carrier I, Rousseau ME, Cayrol R, Gros P. Mutational analysis of conserved carboxylate residues in the nucleotide binding sites of P-glycoprotein. Biochemistry 39: 14138–14149, 2000.[CrossRef][Web of Science][Medline]
  34. Voorhout WF, Veenendaal T, Haagsman HP, Weaver TE, Whitsett JA, van Golde LM, Geuze HJ. Intracellular processing of pulmonary surfactant protein B in an endosomal/lysosomal compartment. Am J Physiol Lung Cell Mol Physiol 263: L479–L486, 1992.[Abstract/Free Full Text]
  35. Wang WJ, Russo SJ, Mulugeta S, Beers MF. Biosynthesis of surfactant protein C (SP-C). J Biol Chem 277: 19929–19937, 2002.[Abstract/Free Full Text]
  36. Yamano G, Funahashi H, Kawanami O, Zhao LX, Ban N, Uchida Y, Morohoshi T, Ogawa J, Shioda S, Inagaki N. ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells. FEBS Lett 508: 221–225, 2001.[CrossRef][Web of Science][Medline]
  37. Yokota T, Matsumura Y, Ban N, Matsubayashi T, Inagaki N. Heterozygous ABCA3 mutation associated with non-fatal evolution of respiratory distress. Eur J Pediatr 167: 691–693, 2008.[CrossRef][Web of Science][Medline]
  38. Yoshida I, Ban N, Inagaki N. Expression of ABCA3, a causative gene for fatal surfactant deficiency, is up-regulated by glucocorticoids in lung alveolar type II cells. Biochem Biophys Res Commun 323: 547–555, 2004.[CrossRef][Web of Science][Medline]
  39. Zaitseva J, Jenewein S, Jumpertz T, Holland IB, Schmitt L. H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB. EMBO J 24: 1901–1910, 2005.[CrossRef][Web of Science][Medline]



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