ABCMdb

PMID: 18676873

Matsumura Y, Ban N, Inagaki N
Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease.
Am J Physiol Lung Cell Mol Physiol. 2008 Oct;295(4):L698-707. Epub 2008 Aug 1., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 8 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 12 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 13 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 18 ABCA3 p.Gly1221Ser ABCA3 p.Trp1142* ABCA3 p.Trp1142* ABCA3 p.Leu982Pro 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). Login to comment 19 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 21 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met In this study, we characterized E292V, E690K, and T1114M mutant ABCA3 proteins identified in pILD. Login to comment 23 ABCA3 p.Gly1221Ser ABCA3 p.Trp1142* ABCA3 p.Trp1142* ABCA3 p.Leu982Pro 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). Login to comment 24 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ser ABCA3 p.Glu690Arg ABCA3 p.Glu292Lys ABCA3 p.Glu292Asp ABCA3 p.Glu690Asp 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). Login to comment 26 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met In this study, we characterized E292V, E690K, and T1114M mutant ABCA3 proteins identified in pILD. Login to comment 29 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ser ABCA3 p.Glu690Arg ABCA3 p.Glu292Lys ABCA3 p.Glu292Asp ABCA3 p.Glu690Asp 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. Login to comment 85 ABCA3 p.Leu101Pro 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). Login to comment 86 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 87 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 89 ABCA3 p.Leu101Pro 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). Login to comment 90 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 91 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 93 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 95 ABCA3 p.Glu690Lys In E690K mutant protein, the amount of the 180-kDa cleaved form was increased compared with that of wild-type protein. Login to comment 97 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 103 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 107 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 113 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 117 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 119 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 123 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 126 ABCA3 p.Glu292Val 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. Login to comment 127 ABCA3 p.Glu690Lys In E690K transfectant, the distribution of choline-phospholipid content was similar to that in HEK293 cells. Login to comment 128 ABCA3 p.Thr1114Met In T1114M transfectant, choline-phospholipid content in fractions 2-4 was somewhat higher than that in HEK293 cells, but the difference was not statistically significant (n afd; 4, Supplemental Fig. 1C). Login to comment 129 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 130 ABCA3 p.Glu292Val 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. Login to comment 131 ABCA3 p.Glu690Lys In E690K transfectant, the distribution of choline-phospholipid content was similar to that in HEK-293 cells. Login to comment 132 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met ABCA3 p.Thr1114Met 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 ϭ 4, Supplemental Fig. 1C). Login to comment 133 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 136 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 147 ABCA3 p.Glu292Val ABCA3 p.Thr1114Met 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). Login to comment 148 ABCA3 p.Glu690Lys 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). Login to comment 151 ABCA3 p.Glu292Val ABCA3 p.Thr1114Met 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). Login to comment 152 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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). Login to comment 153 ABCA3 p.Glu690Lys 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. Login to comment 154 ABCA3 p.Glu690Lys 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. Login to comment 156 ABCA3 p.Glu292Val ABCA3 p.Thr1114Met ATP binding of the E292V and T1114M mutant proteins determined by photoaffinity labeling with 8-azido-[␥-32 P]ATP was similar to that of wild-type ABCA3-GFP protein (Fig. 3, C and D). Login to comment 157 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 158 ABCA3 p.Glu690Lys 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. Login to comment 159 ABCA3 p.Glu292Asp To clarify this, we substituted Glu292 with Asp and Lys, which are negatively and positively charged, respectively. Login to comment 160 ABCA3 p.Glu292Asp In vanadate-induced nucleotide trapping by E292D mutant protein, production of a photoaffinity-labeled Fig. 3. Login to comment 161 ABCA3 p.Glu292Val ABCA3 p.Thr1114Met 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. Login to comment 162 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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 òe;M 8-azido-[ॷ-32 P]ATP in the absence (afa;) or presence (af9;) of 0.4 mM orthovanadate (Vi) and 3 mM MgCl2 for 10 min at 37&#b0;C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Login to comment 163 ABCA3 p.Glu292Asp To clarify this, we substituted Glu292 with Asp and Lys, which are negatively and positively charged, respectively. Login to comment 164 ABCA3 p.Glu292Asp In vanadate-induced nucleotide trapping by E292D mutant protein, production of a photoaffinity-labeled Fig. 3. Login to comment 166 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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 ␮M 8-azido-[␣-32 P]ATP in the absence (-) or presence (ϩ) 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. Login to comment 167 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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 òe;M 8-azido-[ॹ-32 P] and 3 mM MgCl2 for 10 min at 0&#b0;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). Login to comment 171 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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 ␮M 8-azido-[␥-32 P] 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). Login to comment 172 ABCA3 p.Glu292Lys In E292K mutant protein, it was decreased to 4% of that of wild-type protein (Fig. 4B, lanes 9 and 10, and C). Login to comment 174 ABCA3 p.Thr1114Ala ABCA3 p.Trp1148* 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). Login to comment 175 ABCA3 p.Glu292Val ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ala L702 CHARACTERIZATION OF ABCA3 MUTANTS ASSOCIATED WITH pILD AJP-Lung Cell Mol Physiol • VOL 295 022; OCTOBER 2008 • www.ajplung.org 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). Login to comment 176 ABCA3 p.Glu292Lys In E292K mutant protein, it was decreased to 4% of that of wild-type protein (Fig. 4B, lanes 9 and 10, and C). Login to comment 177 ABCA3 p.Thr1114Ser To clarify this, we substituted Thr1114 with Ser and examined vanadate-induced nucleotide trapping. Login to comment 178 ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ser ABCA3 p.Thr1114Ala ABCA3 p.Thr1114Ala ABCA3 p.Trp1148* 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). Login to comment 179 ABCA3 p.Thr1114Met ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ala ABCA3 p.Thr1114Ala The T1114A mutation decreased vanadate-induced nucleotide trapping by ABCA3 protein similarly to the T1114M mutation. Login to comment 180 ABCA3 p.Glu690Lys Interaction of E690K mutant ABCA3-GFP protein with nucleotides. Login to comment 181 ABCA3 p.Glu690Lys ABCA3 p.Thr1114Ser To clarify this, we substituted Thr1114 with Ser and examined vanadate-induced nucleotide trapping. Login to comment 182 ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ser ABCA3 p.Thr1114Ala 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). Login to comment 183 ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ala 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. Login to comment 184 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Glu292Lys ABCA3 p.Glu292Asp Interaction of E690K mutant ABCA3-GFP protein with nucleotides. Login to comment 185 ABCA3 p.Glu690Lys To investigate the mechanism of enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in E690K mutant protein, we performed the trap- Fig. 4. Login to comment 188 ABCA3 p.Glu292Val ABCA3 p.Glu292Lys ABCA3 p.Glu292Asp 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 ␮M 8-azido-[␣-32 P]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). Login to comment 191 ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ser ABCA3 p.Thr1114Ala 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 òe;M 8-azido-[ॷ-32 P]ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37&#b0;C. Login to comment 195 ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ser ABCA3 p.Thr1114Ala 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 ␮M 8-azido-[␣-32 P]ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37°C. Login to comment 198 ABCA3 p.Glu690Lys Indeed, in the presence of orthovanadate, photoaffinity labeling of wild-type and E690K mutant proteins with 8-azido-[ॹ-32 P]ATP was barely detectable (Fig. 6A). Login to comment 199 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys These data combined with vanadate-induced trapping using 8-azido- [ॷ-32 P]ATP indicate that E690K mutant protein hydrolyzes 8-azido-[ॷ-32 P]ATP and releases ॹ-phosphate and that the nucleotide trapped by E690K mutant protein is mostly in the ADP form. Login to comment 200 ABCA3 p.Glu690Lys In E690K mutant protein, ATP binding determined by photoaffinity labeling with 8-azido-[ॹ-32 P]ATP was dramatically increased compared with that of wild-type protein (see Fig. 3, C and D). Login to comment 201 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys In this procedure, a posthydrolyzed trapped nucleotide should not be detected by vanadate-induced trapping because of hydrolytic loss of [␥-32 P]PO4. Indeed, in the presence of orthovanadate, photoaffinity labeling of wild-type and E690K mutant proteins with 8-azido-[␥-32 P]ATP was barely detectable (Fig. 6A). Login to comment 202 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys These data combined with vanadate-induced trapping using 8-azido- [␣-32 P]ATP indicate that E690K mutant protein hydrolyzes 8-azido-[␣-32 P]ATP and releases ␥-phosphate and that the nucleotide trapped by E690K mutant protein is mostly in the ADP form. Login to comment 203 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys In E690K mutant protein, ATP binding determined by photoaffinity labeling with 8-azido-[␥-32 P]ATP was dramatically increased compared with that of wild-type protein (see Fig. 3, C and D). Login to comment 204 ABCA3 p.Glu690Lys To determine whether the E690K mutation alters ADP binding, we performed labeling experiments using 8-azido-[␣-32 P]ADP at 0°C. Login to comment 205 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys 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 ␥-phosphate of 8-azido-[␥-32 P]ATP contributes to enhanced photoaffinity labeling in E690K mutant protein. Login to comment 206 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys 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-[␣-32 P]ATP in the presence of orthovanadate, and unbound nucleotides were removed by washing. Login to comment 207 ABCA3 p.Glu690Lys Thus these data further suggest abnormal interaction with nucleotides in E690K mutant protein. Login to comment 209 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys 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-[␣-32 P]ATP than wild-type protein. Login to comment 210 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys Thus these data further suggest abnormal interaction with nucleotides in E690K mutant protein. Login to comment 211 ABCA3 p.Glu690Asp Accordingly, Glu690 was substituted with Asp and Arg, which are negatively and positively charged, respectively. Login to comment 212 ABCA3 p.Glu690Lys 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. Login to comment 213 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys 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. Login to comment 214 ABCA3 p.Glu690Lys ABCA3 p.Glu690Asp Accordingly, Glu690 was substituted with Asp and Arg, which are negatively and positively charged, respectively. Login to comment 215 ABCA3 p.Glu690Lys 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 òe;M 8-azido-[ॷ-32 P]ADP and 3 mM MgCl2 for 10 min at 0&#b0;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). Login to comment 216 ABCA3 p.Glu690Lys Interaction of E690K mutant ABCA3-GFP protein with nucleotides. Login to comment 217 ABCA3 p.Glu690Lys 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 ␮M 8-azido-[␥-32 P] 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). Login to comment 218 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys 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 ␮M 8-azido-[␣-32 P]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). Login to comment 221 ABCA3 p.Glu690Lys 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 ␮M 8-azido-[␣-32 P]ATP in the presence of orthovanadate, and unbound nucleotides were removed by washing. Login to comment 225 ABCA3 p.Glu690Lys 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. Login to comment 227 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met ABCA3 p.Thr1114Met 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. Login to comment 228 ABCA3 p.Glu690Lys 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. Login to comment 229 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met Accordingly, E292V, E690K, and T1114M are type II mutations. Login to comment 230 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Trp1142* ABCA3 p.Trp1142* ABCA3 p.Leu982Pro ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met ABCA3 p.Thr1114Met 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. Login to comment 231 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 232 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ala ABCA3 p.Trp1148* Accordingly, E292V, E690K, and T1114M are type II mutations. Login to comment 233 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Trp1142* ABCA3 p.Trp1142* ABCA3 p.Leu982Pro 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). Login to comment 234 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met 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. Login to comment 235 ABCA3 p.Glu292Val ABCA3 p.Thr1114Ala ABCA3 p.Trp1148* 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. Login to comment 236 ABCA3 p.Glu292Val Thus the E292V mutation might impede the transmission of conformational changes, resulting in moderate impairment of lipid transport in ABCA3 protein. Login to comment 237 ABCA3 p.Glu292Val ABCA3 p.Thr1114Met 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. Login to comment 239 ABCA3 p.Glu292Val Thus the E292V mutation might impede the transmission of conformational changes, resulting in moderate impairment of lipid transport in ABCA3 protein. Login to comment 240 ABCA3 p.Thr1114Met The T1114M mutant protein exhibits impaired lipid transport function accompanied by moderately preserved vanadate-induced nucleotide trapping. Login to comment 242 ABCA3 p.Glu690Lys In E690K mutant Fig. 7. Login to comment 244 ABCA3 p.Glu690Lys ABCA3 p.Glu690Arg ABCA3 p.Glu690Asp 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 òe;M 8-azido-[ॷ-32 P]ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37&#b0;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). Login to comment 245 ABCA3 p.Glu690Lys In E690K mutant Fig. 7. Login to comment 247 ABCA3 p.Glu690Lys ABCA3 p.Glu690Arg ABCA3 p.Glu690Asp 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 ␮M 8-azido-[␣-32 P]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). Login to comment 249 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Trp1142* ABCA3 p.Trp1142* ABCA3 p.Leu982Pro ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ala ABCA3 p.Trp1148* Genotype-phenotype correlation for ABCA3 mutation ABCA3 Mutation Age of Symptoms Phenotype Ref. W1142X W1142X Neonate FSD 27 L101P L101P Neonate FSD 27 L1553P L1553P Neonate FSD 27 Ins1518 L1580P Neonate FSD 27 L982P G1221S Neonate FSD 27 E292V T1114M Neonate pILD 4 E292V E690K 5 or 7 yr pILD 4 W1148X T1114A 12 mo pILD 37 Type I and type II ATP binding cassette A3 (ABCA3) mutations are shown in italics and roman, respectively. Login to comment 252 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Trp1142* ABCA3 p.Trp1142* ABCA3 p.Leu982Pro ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys ABCA3 p.Thr1114Met ABCA3 p.Thr1114Ala ABCA3 p.Trp1148* Genotype-phenotype correlation for ABCA3 mutation ABCA3 Mutation Age of Symptoms Phenotype Ref. W1142X W1142X Neonate FSD 27 L101P L101P Neonate FSD 27 L1553P L1553P Neonate FSD 27 Ins1518 L1580P Neonate FSD 27 L982P G1221S Neonate FSD 27 E292V T1114M Neonate pILD 4 E292V E690K 5 or 7 yr pILD 4 W1148X T1114A 12 mo pILD 37 Type I and type II ATP binding cassette A3 (ABCA3) mutations are shown in italics and roman, respectively. Login to comment 253 ABCA3 p.Glu690Lys 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). Login to comment 255 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys 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. Login to comment 256 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys 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). Login to comment 258 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys On the other hand, in the model of E690K mutant, the distance from side chain nitrogen of Lys690 to ␥-phosphate oxygen of ATP is ϳ3.6 Å. Login to comment 259 ABCA3 p.Glu690Lys One possible interpretation of these biochemical results and modeling is that ionic interaction of Lys690 and ␥-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. Login to comment 261 ABCA3 p.Glu690Lys Analysis using purified ABCA3 protein would further clarify the aberrant catalytic cycle and impaired lipid transport in E690K mutant protein. Login to comment 262 ABCA3 p.Glu690Lys Interestingly, in E690K mutant protein, the amount of 180-kDa cleaved form was increased compared with that of wild-type protein. Login to comment 263 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys 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. Login to comment 265 ABCA3 p.Glu292Val ABCA3 p.Glu690Lys Interestingly, in E690K mutant protein, the amount of 180-kDa cleaved form was increased compared with that of wild-type protein. Login to comment 266 ABCA3 p.Glu690Lys ABCA3 p.Glu690Lys 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. Login to comment 268 ABCA3 p.Glu292Val 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. Login to comment