ABCA3 p.Gly1221Val
| Predicted by SNAP2: | A: D (63%), C: D (71%), D: D (66%), E: D (66%), F: D (85%), H: D (66%), I: D (80%), K: D (66%), L: D (85%), M: D (80%), N: N (53%), P: D (71%), Q: D (63%), R: D (71%), S: N (53%), T: D (59%), V: D (75%), W: D (91%), Y: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Characterization and classification of ATP-binding... J Biol Chem. 2006 Nov 10;281(45):34503-14. Epub 2006 Sep 7. 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., [PMID:16959783]
Abstract [show]
The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. 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. 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. 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. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency.
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No. Sentence Comment
179 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.
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ABCA3 p.Gly1221Val 16959783:179:189
status: NEW182 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).
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ABCA3 p.Gly1221Val 16959783:182:18
status: NEW201 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.
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ABCA3 p.Gly1221Val 16959783:201:149
status: NEW