ABCMdb

PMID: 16959783

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. 2006 Nov 10;281(45):34503-14. Epub 2006 Sep 7., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Login to comment 4 ABCA3 p.Asn568Asp ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro ABCA3 p.Leu1580Pro Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. Login to comment 5 ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Login to comment 35 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Gln1591Pro ABCA3 p.Trp1142* ABCA3 p.Leu982Pro Partial cDNA fragments containing various fatal surfactant deficiency mutations (L101P, N568D, L982P, G1221S, L1553P, L1580P, Q1591P, W1142X, and Ins1518fs (abbreviation of Ins1518fs/ter1519 in this study), see Fig. 1A), were generated with PCR methods and replaced with the corresponding fragment of pEGFPN1-ABCA3. Login to comment 98 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro To examine the effect of the mutations found in fatal surfactant deficiency patients on subcellular localization of ABCA3, wild-type and mutant ABCA3-GFP (seven missense mutations L101P, N568D, L982P, G1221S, L1553P, L1580P, and Q1591P, and one nonsense mutation, Ins1518fs) were transiently expressed in HEK293 cells (Fig. 1A). Login to comment 99 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro The N568D, G1221S, and L1580P mutant proteins were mainly localized to the LAMP3-positive intracellular vesicle membrane (Fig. 2B, panels a-c, d-f, and g-i), similar to wild-type ABCA3 protein (Fig. 2A, panels e-h). Login to comment 100 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro In contrast, the L101P, L982P, L1553P, Q1591P, and Ins1518fs mutant proteins were barely detectable at the LAMP3-positive intracellular vesicle membrane (Fig. 2B panels j-l for L101P and data not shown for others). Login to comment 102 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Gln1591Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro ABCA3 p.Leu982Pro In another cell line, mouse lung epithelial MLE12 cells, transiently expressed GFP-tagged wild-type and N568D, G1221S, and L1580P mutant proteins were mainly localized at the intracellular vesicle membrane, whereas the L101P, L982P, L1553P, Q1591P, and Ins1518fs mutant proteins were mainly localized to the ER (data not shown), confirming defective intracellular sorting of L101P, L982P, L1553P, Q1591P, and Ins1518fs ABCA3 mutant proteins. Login to comment 105 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro In the N568D, G1221S, and L1580P mutant proteins, which were mainly localized to the intracellular vesicle membrane, both the 220-kDa noncleaved form and the 180-kDa cleaved form were detected, similar to wild-type protein (Fig. 3A). Login to comment 106 ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro In contrast, in the L101P, L982P, L1553P, and Q1591P mutant proteins, which were mainly localized at the ER, the amount of the 180-kDa cleaved form was considerably decreased, compared with that of wild-type protein, to an undetectable level (Fig. 3A). Login to comment 107 ABCA3 p.Leu1553Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro In the L982P, L1553P, and Q1591P mutant proteins, although the amount of 220-kDa noncleaved-form protein appears increased compared with that of wild-type protein in Fig. 3A, the total amount of ABCA3-GFP (220-kDa noncleaved form plus 180-kDa cleaved form) did not differ significantly among seven missense mutant proteins and the wild-type protein (n ϭ 3, data not shown). Login to comment 121 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro A merged image of panels i-k is shown in panel l. B, HEK293 cells transiently expressing mutant ABCA3-GFP proteins (panels a, d, g, and j) were processed for immunofluorescence labeling of LAMP3 (panels b, e, h, and k): N568D (panels a-c), G1221S (panels d-f), L1580P (panels g-i), and L101P (panels j-l). Login to comment 122 ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro Merged images are shown in panels c, f, i, and l. C, HEK293 cells transiently co-expressing mutant ABCA3-GFP proteins (panels a, d, g, j, and m) and DsRed2-ER (panels b, e, h, k, and n) are shown: L101P (panels a-c), L982P (panels d-f), L1553P (panels g-i), Q1591P (panels j-l), and Ins1518fs (panels m-o). Login to comment 124 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Leu1580Pro ABCA3 p.Gln1591Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro ABCA3 p.Leu982Pro The scale bar represents 5 ␮m. kDa wild-type ABCA3-GFP and the seven missense mutant (L101P, N568D, L982P, G1221S, L1553P, L1580P, and Q1591P) proteins to produce a 210-kDa deglycosylated protein (Fig. 3B). Login to comment 128 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro In the N568D, G1221S, and L1580P mutant proteins, about 30-40% of the 220-kDa protein remained as Endo H-insensitive complex-type protein (Fig. 3, C and D, band I), indicating that processing of oligosaccharide from high mannose type to complex type is largely preserved in these mutants. Login to comment 129 ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro However, in the L101P, L982P, L1553P, Q1591P, and Ins1518fs mutant proteins, the levels of complex-type protein (band I) were dramatically decreased compared with that of wild-type protein (Fig. 3, C and D). Login to comment 131 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Asp These results indicate that the N568D, G1221S, and L1580P mutant proteins are mainly localized at the intracellular vesicle membrane accompanied by processing of oligosaccharide from high mannose type to complex type, whereas the four missense mutant (L101P, L982D, L1553P, and Q1591P) and one nonsense mutant (Ins1518fs) proteins remain localized at the ER, with impaired processing of oligosaccharide. Login to comment 132 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro ATP Hydrolysis of ABCA3-GFP and Mutants-To investigate the mechanism of loss of function of the N568D, G1221S, and L1580P mutant proteins that are trafficked to intracellular vesicles accompanied by processing of sugar chains as is wild-type ABCA3 protein, we examined ATP hydrolysis of wild-type ABCA3-GFP and the mutant proteins. Login to comment 143 ABCA3 p.Asn568Asp ABCA3 p.Leu1553Pro ABCA3 p.Leu982Pro Band I shows Endo H-insensitive 220-kDa ABCA3-GFP proteins (wild-type, N568D, L982P, and L1553P) containing complex-type sugar chains. Login to comment 148 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro *, p Ͻ 0.05; **, p Ͻ 0.005 versus wild type. N.S., not significant. Characterization and Classification of ABCA3 Mutants 34508 expressing wild-type or mutant (N568D, G1221S, and L1580P) ABCA3-GFP fusion proteins were established. Login to comment 152 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro In the N568D, G1221S, and L1580P mutant proteins, vanadate-induced nucleotide trapping was significantly decreased to 12, 33, and 9% of that of the wild-type protein, respectively (Fig. 4, A, lanes 5-10, and C). Login to comment 154 ABCA3 p.Leu101Pro To examine ATP hydrolysis of the mutant retained to the ER, cells stably expressing L101P mutant protein were established. Login to comment 155 ABCA3 p.Leu101Pro L101P mutant protein was mainly localized to the ER, consistent with the result in transiently expressing cells (data not shown). Login to comment 156 ABCA3 p.Leu101Pro The vanadate-induced nucleotide trapping also was significantly decreased compared with that of wild-type protein (Fig. 4B, lanes 3 and 4), indicating that ATP hydrolysis activity as well as intracellular trafficking is impaired in the L101P mutant protein. Login to comment 157 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro ATP Binding of ABCA3-GFP and Mutants-To clarify the mechanism of loss of ATP hydrolysis activity of the N568D, G1221S, and L1580P mutant proteins, we examined ATP binding of wild-type ABCA3-GFP and the mutant proteins. Login to comment 160 ABCA3 p.Gly1221Ser The level of photoaffinity labeling of the G1221S mutant protein FIGURE 4. Login to comment 162 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro A, 20,000 ϫ g membrane fraction prepared from HEK293 cells stably expressing the wild-type (Wt) ABCA3-GFP (lanes 3 and 4), N568D (lanes 5 and 6), G1221S (lanes 7 and 8), L1580P (lanes 9 and 10), or untransfected HEK293 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. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE (5%), and transferred to a PVDF membrane. Login to comment 164 ABCA3 p.Leu101Pro B, 20,000 ϫ g membrane fraction prepared from HEK293 cells stably expressing the wild-type (Wt) ABCA3-GFP (lanes 1 and 2) or L101P (lanes 3 and 4) was similarly analyzed. Login to comment 170 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser A, 20,000 ϫ g membrane fraction prepared from HEK293 cells stably expressing wild-type (Wt) ABCA3-GFP, N568D, G1221S, L101P, or untransfected HEK293 cells was incubated with 20 ␮M 8-azido-[␥-32 P]ATP and 3 mM MgCl2 for 10 min at 0 °C. Proteins were photoaffinity-labeled with UV irradiation, immunoprecipitated using anti-human ABCA3 antibody, electrophoresed on SDS-PAGE (5%), and transferred to a PVDF membrane. Login to comment 175 ABCA3 p.Asn568Asp ABCA3 p.Leu1580Pro However, the levels of photoaffinity labeling of N568D and L1580P mutant proteins were moderately decreased to 60 and 54% of that of wild-type protein, respectively. Login to comment 176 ABCA3 p.Leu101Pro The level of photoaffinity labeling of L101P protein remaining localized at the ER was considerably decreased to 25% that of wild-type protein. Login to comment 177 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro These results suggest that decreased ATP binding contributes to impaired ATP hydrolysis in the N568D, L1580P, and L101P mutants but not in the G1221S mutant. Login to comment 178 ABCA3 p.Gly1221Ser ATP Hydrolysis of Site-directed Mutants of Gly-1221 in 11th Transmembrane Segment-Since transmembrane domains and NBDs are suggested to communicate during the ATP hydrolysis cycle (31), local environmental changes in the 11th transmembrane segment (TM-11) resulting from the G1221S mutation might allosterically impair ATP hydrolysis activity of the ABCA3 protein. Login to comment 179 ABCA3 p.Gly1221Ala ABCA3 p.Gly1221Thr ABCA3 p.Gly1221Val To address this, the effects of introducing hydroxyl groups or alteration of side-chain size on ATP hydrolysis activity were investigated by generating three site-directed mutants (G1221A, G1221V, and G1221T), which were stably expressed in HEK293 cells. Login to comment 181 ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Thr In the G1221T mutant protein, which had hydroxyl-containing amino acids, vanadate-induced nucleotide trapping was decreased to 36% of that of wild-type protein, as also in G1221S mutant protein (Fig. 6, A, lanes 9-12, and B). Login to comment 182 ABCA3 p.Gly1221Ala ABCA3 p.Gly1221Val In the G1221A and G1221V mutant proteins, which have a hydrophobic side chain, vanadate-induced nucleotide trapping was decreased to 15 and 18% of that of wild-type protein, respectively (Fig. 6, A, lanes 5-8, and B). Login to comment 184 ABCA3 p.Leu1580Pro ATP Hydrolysis of Site-directed Mutants of Leu-1580 in NBD-2-Because both leucine and proline are hydrophobic amino acids, alteration of side-chain size could be responsible for the impaired ATP hydrolysis in the L1580P mutant. Login to comment 198 ABCA3 p.Leu1580Pro Substitution of Leu-1580 with Pro, considering the best rotamer conformation, extended the distance from ␥-carbon of Pro-1580 to beta-carbon of Trp-1554 to 5.4 Å (Fig. 8D). Login to comment 199 ABCA3 p.Leu1580Val In the L1580V mutant protein, FIGURE 6. Login to comment 201 ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ala ABCA3 p.Gly1221Thr ABCA3 p.Gly1221Val A, 20,000 ϫ g membrane fraction prepared from HEK293 cells stably expressing wild-type (Wt) ABCA3-GFP (lanes 3 and 4), G1221A (lanes 5 and 6), G1221V (lanes 7 and 8), G1221T (lanes 9 and 10), G1221S (lanes 11 and 12), or untransfected HEK293 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. Proteins werephotoaffinity-labeledwithUVirradiationafterremovalofunboundATP, electrophoresed on SDS-PAGE (5%), and transferred to a PVDF membrane. Login to comment 207 ABCA3 p.Leu1580Ala ABCA3 p.Leu1580Phe In the L1580A and L1580F mutant proteins, which had dramatically impaired ATP hydrolysis, the distance from beta-carbon of Ala-1580 and ॼ-carbon of Phe-1580 was extended to 6.3 &#c5; and shortened to 2.2 &#c5;, respectively, compared with that of wild-type protein (Fig. 8, E and G). Login to comment 208 ABCA3 p.Leu1580Ala ABCA3 p.Leu1580Phe In the L1580A and L1580F mutant proteins, which had dramatically impaired ATP hydrolysis, the distance from beta-carbon of Ala-1580 and ␨-carbon of Phe-1580 was extended to 6.3 Å and shortened to 2.2 Å, respectively, compared with that of wild-type protein (Fig. 8, E and G). Login to comment 209 ABCA3 p.Leu1580Pro Thus, impaired ATP hydrolysis in the L1580P mutant protein may result in part from the alteration of side-chain size. Login to comment 210 ABCA3 p.Leu1580Pro Thus, impaired ATP hydrolysis in the L1580P mutant protein may result in part from the alteration of side-chain size. Login to comment 215 ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro Investigating the intracellular localization and N-glycosylation of these ABCA3 mutant proteins in HEK293 cells, we found the missense L101P, L982P, L1553P, Q1591P, and nonsense Ins1518fs mutant proteins to be predominantly localized at the ER, with impaired processing of oligosaccharide. Login to comment 216 ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Gln1591Pro ABCA3 p.Trp1142* ABCA3 p.Leu982Pro Investigating the intracellular localization and N-glycosylation of these ABCA3 mutant proteins in HEK293 cells, we found the missense L101P, L982P, L1553P, Q1591P, and nonsense Ins1518fs mutant proteins to be predominantly localized at the ER, with impaired processing of oligosaccharide. Login to comment 217 ABCA3 p.Trp1142* W1142X mutant ABCA3 protein, another nonsense mutant reported in fatal surfactant deficiency (12), also was predominantly localized at the ER with impaired processing of oligosaccharide (data not shown). Login to comment 218 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp For example, R587W and Q597R mutations of ABCA1, which are found in Tangier disease patients with high density lipoprotein deficiency, appear to be impaired in intracellular trafficking and localized predominantly to the ER (36). Login to comment 219 ABCA1 p.Gln597Arg ABCA1 p.Arg587Trp For example, R587W and Q597R mutations of ABCA1, which are found in Tangier disease patients with high density lipoprotein deficiency, appear to be impaired in intracellular trafficking and localized predominantly to the ER (36). Login to comment 220 ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro Interestingly, a single amino acid is substituted with a proline residue in the four mutant ABCA3 proteins (L101P, L982P, L1553P, and Q1591P) that are retained at the ER. Login to comment 221 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Leu1553Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro ABCA3 p.Leu982Pro Interestingly, a single amino acid is substituted with a proline residue in the four mutant ABCA3 proteins (L101P, L982P, L1553P, and Q1591P) that are retained at the ER. Login to comment 222 ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Trp1142* ABCA3 p.Leu982Pro As three of these mutations (L101P, L982P, and L1553P) are located in the predicted ␣-helical structure of the ABCA3 protein (32) and the proline residue is known to be helix breaker (39), its introduction into the ABCA3 protein might well disrupt the ␣-helical structure and hamper proper folding and intracellular translocation. Login to comment 223 ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Leu1553Pro ABCA3 p.Trp1142* ABCA3 p.Trp1142* ABCA3 p.Trp1142* The large C-terminal deletion of the ABCA3 protein (Ins1518fs and W1142X) also might hamper this process. Login to comment 224 ABCA3 p.Leu101Pro ABCA3 p.Leu1553Pro ABCA3 p.Leu1553Pro ABCA3 p.Trp1142* ABCA3 p.Trp1142* Indeed, patients with homozygous mutations of L101P, L1553P, and W1142X have been reported to die of surfactant deficiency during the neonatal period, and electron microscopic study of lung tissue from patients with homozygous L1553P and W1142X mutations revealed smaller lamellar bodies than those in normal lung tissue (12). Login to comment 225 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro In contrast, the N568D, G1221S, and L1580P mutant proteins were localized to intracellular vesicle membrane accompanied by processing of oligosaccharide from high mannose type to complex type as found in wild-type ABCA3 protein. Login to comment 226 ABCA3 p.Asn568Asp ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro ABCA3 p.Leu1580Pro In contrast, the N568D, G1221S, and L1580P mutant proteins were localized to intracellular vesicle membrane accompanied by processing of oligosaccharide from high mannose type to complex type as found in wild-type ABCA3 protein. Login to comment 227 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro However, vanadate-induced nucleotide trapping analysis revealed ATP hydrolysis activity to be significantly decreased in N568D, G1221S, and L1580P mutant ABCA3 proteins compared with wild type. Login to comment 230 ABCA3 p.Leu1580Pro ABCA3 p.Leu1580Val ABCA3 p.Leu1580Ala ABCA3 p.Leu1580Phe A, 20,000 afb; g membrane fraction prepared from HEK293 cells stably expressing wild-type (Wt) ABCA3-GFP (lanes 3 and 4), L1580A (lanes 5 and 6), L1580V (lanes 7 and 8), L1580F (lanes 9 and 10), L1580P (lanes 11 and 12), or untransfected HEK293 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. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE (5%), and transferred to a PVDF membrane. Login to comment 231 ABCA3 p.Leu1580Pro ABCA3 p.Leu1580Val ABCA3 p.Leu1580Ala ABCA3 p.Leu1580Phe A, 20,000 ϫ g membrane fraction prepared from HEK293 cells stably expressing wild-type (Wt) ABCA3-GFP (lanes 3 and 4), L1580A (lanes 5 and 6), L1580V (lanes 7 and 8), L1580F (lanes 9 and 10), L1580P (lanes 11 and 12), or untransfected HEK293 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. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE (5%), and transferred to a PVDF membrane. Login to comment 234 ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro ABCA3 p.Leu982Pro ABCA3 p.Leu982Pro *, p b0d; 0.01 versus wild type. Characterization and Classification of ABCA3 Mutants NOVEMBER 10, 2006ߦVOLUME 281ߦNUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY homozygous type II ABCA3 mutations have not been reported, patients with type I/type II compound heterozygous ABCA3 mutations (L982P/G1221S and Ins1518fs/L1580P) died of surfactant deficiency during the neonatal period, and the lamellar bodies of lung tissue from a patient with L982P/G1221S were reported to be smaller than those from normal lung tissue (12). Login to comment 236 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro ABCA3 p.Leu1580Pro ABCA3 p.Leu982Pro ABCA3 p.Leu982Pro homozygous type II ABCA3 mutations have not been reported, patients with type I/type II compound heterozygous ABCA3 mutations (L982P/G1221S and Ins1518fs/L1580P) died of surfactant deficiency during the neonatal period, and the lamellar bodies of lung tissue from a patient with L982P/G1221S were reported to be smaller than those from normal lung tissue (12). Login to comment 238 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro Type II mutations lie in various locations as follows: N568D in the Walker A motif of NBD-1, G1221S in TM-11, and L1580P in NBD-2 (Fig. 1A), and all of these amino acids are conserved in the ABCA subfamily (Fig. 1, B-D). Login to comment 247 ABCA3 p.Asn568Asp Characterization and Classification of ABCA3 Mutants 34512 from the crystal structure of other ABC transporters (40), both ATP binding and ATP hydrolysis activities should be impaired in the N568D mutant protein. Login to comment 249 ABCA3 p.Asn568Asp Characterization and Classification of ABCA3 Mutants 34512 from the crystal structure of other ABC transporters (40), both ATP binding and ATP hydrolysis activities should be impaired in the N568D mutant protein. Login to comment 251 ABCC1 p.Glu1204Leu For example, the E1204L mutation in TM-16 of MRP1(multidrug resistance associated protein 1) affects vanadate-induced nucleotide trapping as well as transport activity of the protein (41). Login to comment 253 ABCA3 p.Leu1580Pro Mutational analysis of Leu-1580 suggested that impaired ATP hydrolysis in the L1580P mutant protein is in part due to the change in side-chain size. Login to comment 254 ABCA3 p.Leu1580Pro However, because Pro is known as a helix breaker (39), disruption of helix 7 by the introduction of the Pro residue may also contribute to the impaired ATP hydrolysis in the case of the L1580P mutant protein. Login to comment 255 ABCA3 p.Leu1580Pro Mutational analysis of Leu-1580 suggested that impaired ATP hydrolysis in the L1580P mutant protein is in part due to the change in side-chain size. Login to comment 256 ABCA3 p.Leu1580Pro However, because Pro is known as a helix breaker (39), disruption of helix 7 by the introduction of the Pro residue may also contribute to the impaired ATP hydrolysis in the case of the L1580P mutant protein. Login to comment 257 ABCA1 p.Ala665Thr ABCA4 p.Glu2131Lys ABCA4 p.Arg2106Cys Although further confirmation of this interaction might be provided by mutational analysis of Trp-1554, many disease-related mutations at helix 6 and helix 7 of NBDs such as R2106C and E2131K in ABCA4 (44-47), F587I and L610S in ABCC7/CFTR (48-50), and A665T in ABCB3/TAP2 (51) (Fig. 8A) support the importance of these helices for the function of the ABC transporter. Login to comment 259 ABCC7 p.Leu610Ser ABCC7 p.Phe587Ile ABCA1 p.Ala665Thr ABCA4 p.Glu2131Lys ABCA4 p.Arg2106Cys Although further confirmation of this interaction might be provided by mutational analysis of Trp-1554, many disease-related mutations at helix 6 and helix 7 of NBDs such as R2106C and E2131K in ABCA4 (44-47), F587I and L610S in ABCC7/CFTR (48-50), and A665T in ABCB3/TAP2 (51) (Fig. 8A) support the importance of these helices for the function of the ABC transporter. Login to comment 264 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro We examined ATP binding and ATP hydrolysis of the type I mutant ABCA3 protein by using cells stably expressing L101P mutant protein, and we found that both ATP binding and ATP hydrolysis activities as well as intracellular trafficking were impaired in the L101P mutant protein. Login to comment 266 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro We examined ATP binding and ATP hydrolysis of the type I mutant ABCA3 protein by using cells stably expressing L101P mutant protein, and we found that both ATP binding and ATP hydrolysis activities as well as intracellular trafficking were impaired in the L101P mutant protein. Login to comment 268 ABCA3 p.Glu292Val Very recently, it has been reported that the E292V mutation of the ABCA3 gene is responsible in the genetic etiology of pediatric interstitial lung disease related to abnormal surfactant function (24), the phenotype of which is milder than that of fatal surfactant deficiency. Login to comment 269 ABCA3 p.Glu292Val It is possible that the E292V mutation causes less severe disruption of intracellular trafficking or ATP hydrolysis activity. Login to comment 270 ABCA3 p.Glu292Val Very recently, it has been reported that the E292V mutation of the ABCA3 gene is responsible in the genetic etiology of pediatric interstitial lung disease related to abnormal surfactant function (24), the phenotype of which is milder than that of fatal surfactant deficiency. Login to comment 271 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Glu292Val ABCA3 p.Gly1221Ser It is possible that the E292V mutation causes less severe disruption of intracellular trafficking or ATP hydrolysis activity. Login to comment 272 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser In this study, the difference in degree of defect between N568D and G1221S mutant proteins is slight, if any. Login to comment 273 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser Very recently, Cheong et al. (19) reported that L101P, G1221S, and N568D mutant proteins have the most severe, moderate, and the least severe trafficking and processing defects. Login to comment 274 ABCA3 p.Asn568Asp ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser In this study, the difference in degree of defect between N568D and G1221S mutant proteins is slight, if any. Login to comment 276 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser With regard to the function of the ABCA3 mutant proteins, their findings indicating impaired co-localization of fluorescence-labeled phosphatidylcholine with ABCA3-positive vesicles in G1221S and N568D may well correlate with our findings indicating impaired ATP hydrolysis activities of these mutants. Login to comment