ABCMdb

ABCA3 p.Leu982Pro

Predicted by SNAP2:	A: D (59%), C: N (53%), D: D (75%), E: D (59%), F: N (61%), G: D (75%), H: N (53%), I: N (87%), K: D (63%), M: N (87%), N: D (66%), P: D (71%), Q: N (53%), R: D (63%), S: D (63%), T: N (57%), V: N (78%), W: D (71%), Y: N (61%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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[hide] ABCA3 gene mutations in newborns with fatal surfac... N Engl J Med. 2004 Mar 25;350(13):1296-303. 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. 2004 Mar 25;350(13):1296-303., [PMID:15044640]

Abstract [show]
BACKGROUND: Pulmonary surfactant forms a lipid-rich monolayer that coats the airways of the lung and is essential for proper inflation and function of the lung. Surfactant is produced by alveolar type II cells, stored intracellularly in organelles known as lamellar bodies, and secreted by exocytosis. The gene for ATP-binding cassette transporter A3 (ABCA3) is expressed in alveolar type II cells, and the protein is localized to lamellar bodies, suggesting that it has an important role in surfactant metabolism. METHODS: We sequenced each of the coding exons of the ABCA3 gene in blood DNA from 21 racially and ethnically diverse infants with severe neonatal surfactant deficiency for which the etiologic process was unknown. Lung tissue from four patients was examined by high-resolution light and electron microscopy. RESULTS: Nonsense and frameshift mutations, as well as mutations in highly conserved residues and in splice sites of the ABCA3 gene were identified in 16 of the 21 patients (76 percent). In five consanguineous families with mutations, each pair of siblings was homozygous for the same mutation and each mutation was found in only one family. Markedly abnormal lamellar bodies were observed by ultrastructural examination of lung tissue from four patients with different ABCA3 mutations, including nonsense, splice-site, and missense mutations. CONCLUSIONS: Mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. ABCA3 is critical for the proper formation of lamellar bodies and surfactant function and may also be important for lung function in other pulmonary diseases. Since it is closely related to ABCA1 and ABCA4, proteins that transport phospholipids in macrophages and photoreceptor cells, it may have a role in surfactant phospholipid metabolism.

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44 Family History/ Consanguinity Outcome Histologic Findings ABCA3 Mutation 1 White F 1 Yes/Yes Death within 3 mo after birth DIP, PAP W1142X/W1142X 2 White F 1 Yes/Yes Death during neonatal period DIP, PAP W1142X/W1142X 3 Black M 2 Yes/Yes Death during neonatal period NA L101P/L101P 4 Black M 2 Yes/Yes Death during neonatal period NA L101P/L101P 5 White F 3 Yes/No Death during neonatal period NA 4552insT/L1580P 6 White F 3 Yes/No Death during neonatal period NA 4552insT/L1580P 7 White M 4 Yes/No Death within 3 mo after birth PAP G1221S/L982P 8 White M 4 Yes/No Death during neonatal period PAP G1221S/L982P 9 Middle Eastern M 5 Yes/Yes Death during neonatal period DIP, PAP L1553P/L1553P 10 Middle Eastern M 5 Yes/Yes Death during neonatal period NA L1553P/L1553P 11 White M 6 Yes/No Recovery from RDS NA None found 12 White M 6 Yes/No Recovery from RDS NA None found 13 Middle Eastern M 7 No/Yes Unknown NA 1644delC/1644delC 14 Middle Eastern M 8 Yes/No Death during neonatal period DIP, PAP R106X/R106X 15 Asian F 9† Yes/Yes Death during neonatal period NA 4909+1G>A/4909+1G>A 16 White M 10 Yes/Yes Death during neonatal period NA None found 17 White M 11 No/No Recovery from RDS NA None found 18 White F 12 No/No Death during neonatal period NA None found 19 White M 13 Yes/No Chronic lung disease CPI, DIP Q1591P/-‡ 20 Hispanic M 14 No/No Death after lung transplantation PAP N568D/-‡ 21 Asian F 9† Yes/Yes Death during neonatal period PAP 4909+1G>A/4909+1G>A entorganisms,weusedthededucedaminoacidse- quence of ABCA3 (GenBank accession number NP_001080) to search the sequence data base using the BLAST program (http://www.ncbi.nlm.nih. Login to comment
59 Seven missense muta- tionswereidentifiedinconservedaminoacids(Fig. 2), including homozygous substitutions of proline for leucine in codons 101 and 1553 (L101P and L1553P, respectively) and heterozygous substitutions of aspartic acid for asparagine at position 568 (N568D), proline for leucine at position 982 (L982P), serine for glycine at position 1221 (G1221S), proline for leucine at position 1580 (L1580P), and proline for glutamine at position 1591 (Q1591P). Login to comment
77 Electron micrographs of lung tissue from Patient 21 (who was homozygous for the 4909+1G>A mutation) revealed lamellarbodies(Fig.3)thatweresmallerthanthose from control lung tissue, with more densely packed membranes and eccentrically placed, dense inclusion bodies, similar to those previously described in Patients 1 and 2, who were homozygous for the W1142X mutation.17 Similarly abnormal lamellar bodies were observed in lung tissue from Patient 8 (who was heterozygous for the G1221S and L982P mutations) and Patient 9 (who was homozygous for the L1553P mutation). Login to comment
85 The sequences of the puffer fish and the zebra fish are not complete, resulting in some gaps in this information in the case of L982P, G1221S, L1552P, L1553P, and L1580P. Login to comment
87 Human Mouse Rat Puffer fish ETVRRALVIN ETVKREFMIK EAVRREFMIK EDVRGKLELS QDVQQNLVRG L101P NGAGKTT NGAGKTT NGAGKTT NGAGKTT NGAGKTT N568D VARRLL VARRLL VARRLL VARRLL L1553P Q1591P T301C(L101P) C316T(R106X) A1702G(N568D) 1644delC G3426A(W1142X) G3661A(G1221S) T4657C(L1553P) 4552insT A4771C(Q1591P) 4909+1G>A G1221S LSGIAT LSGIAT LSGIAT ATP-binding domains L982P QQLSEHL QQLSENL QQLSEHL T2945C(L982P) ECEALC LAIMVQGQFKC ECEALC ECEALC L1580P T4739C(L1580P) Nonsense MissenseSpliceFrameshift LAIMVQGQFKC LAIMVQGQFKC LAVMVNGQFKC Zebra fish LAVMVNGQFKC tified occur in residues that are highly conserved (Fig.2).Theaminoacidalignmentwasusedtopro- duce a phylogenetic tree of the ABCA3-related proteins showing the relation of the proteins from different organisms (see Supplementary Appendix 2, available with the full text of this article at www. nejm.org).ThefishABCA3proteinsclusterwiththe mammalian ABCA3 proteins and are distinct from other, more distant ABCA-family proteins, such as the mouse Abca14, Abca15, and Abca16 proteins and the sea-urchin ABCA proteins (see Supplementary Appendix 2. Login to comment
106 Nucleotide Affected* Site Affected or Outcome SNP No.† Mutation Exon 5 3, 4 T301C L101P‡ Exon 5 14 C316T R106X‡ Exon 14 20 A1702G N568D Exon 14 13 1644delC Frameshift‡ Exon 21 7, 8 T2945C L982P Exon 23 1, 2 G3426A W1142X‡ Exon 24 7, 8 G3661A G1221S Exon 30 9, 10 T4657C L1553P Exon 30 5, 6 4552insT Frameshift Exon 31 15, 21 4909+1G>A Splice site‡ Exon 31 5, 6 T4739C L1580P Exon 31 19 A4772C Q1591P Polymorphism Exon 5 Multiple Exon 5+50A/G Intron rs46725 Exon 6 20 393C/T A131A Exon 6 18 Exon 6+119G/A Intron rs323059 Exon 7 19 Exon 7-14C/G Intron Exon 8 14 681C/T A227A Exon 10 Multiple Exon 10-105C/A Intron rs323066 Exon 10 Multiple Exon 10-20C/T Intron Exon 10 Multiple 1058C/T F353F Exon 14 Multiple Exon 14+33G/A Intron rs170447 Exon 15 Multiple 1755C/G P585P rs323043 Exon 18 13 Exon 18-17G/A Intron Exon 18 1 2340C/T H780H Exon 21 Multiple Exon 21-20C/G Intron rs313908 Exon 21 Multiple Exon 21+34C/T Intron rs313909 Exon 27 Multiple 4116C/T S1372S rs149532 Exon 32 11 4944C/T V1648V In two patients, a mutation was identified on only one allele. Login to comment
43 Family History/ Consanguinity Outcome Histologic Findings ABCA3 Mutation 1 White F 1 Yes/Yes Death within 3 mo after birth DIP, PAP W1142X/W1142X 2 White F 1 Yes/Yes Death during neonatal period DIP, PAP W1142X/W1142X 3 Black M 2 Yes/Yes Death during neonatal period NA L101P/L101P 4 Black M 2 Yes/Yes Death during neonatal period NA L101P/L101P 5 White F 3 Yes/No Death during neonatal period NA 4552insT/L1580P 6 White F 3 Yes/No Death during neonatal period NA 4552insT/L1580P 7 White M 4 Yes/No Death within 3 mo after birth PAP G1221S/L982P 8 White M 4 Yes/No Death during neonatal period PAP G1221S/L982P 9 Middle Eastern M 5 Yes/Yes Death during neonatal period DIP, PAP L1553P/L1553P 10 Middle Eastern M 5 Yes/Yes Death during neonatal period NA L1553P/L1553P 11 White M 6 Yes/No Recovery from RDS NA None found 12 White M 6 Yes/No Recovery from RDS NA None found 13 Middle Eastern M 7 No/Yes Unknown NA 1644delC/1644delC 14 Middle Eastern M 8 Yes/No Death during neonatal period DIP, PAP R106X/R106X 15 Asian F 9ߤ Yes/Yes Death during neonatal period NA 4909+1G>A/4909+1G>A 16 White M 10 Yes/Yes Death during neonatal period NA None found 17 White M 11 No/No Recovery from RDS NA None found 18 White F 12 No/No Death during neonatal period NA None found 19 White M 13 Yes/No Chronic lung disease CPI, DIP Q1591P/-ߥ 20 Hispanic M 14 No/No Death after lung transplantation PAP N568D/-ߥ 21 Asian F 9ߤ Yes/Yes Death during neonatal period PAP 4909+1G>A/4909+1G>A entorganisms,weusedthededucedaminoacidse- quence of ABCA3 (GenBank accession number NP_001080) to search the sequence data base using the BLAST program (http://www.ncbi.nlm.nih. Login to comment
58 Seven missense muta- tionswereidentifiedinconservedaminoacids(Fig. 2), including homozygous substitutions of proline for leucine in codons 101 and 1553 (L101P and L1553P, respectively) and heterozygous substitutions of aspartic acid for asparagine at position 568 (N568D), proline for leucine at position 982 (L982P), serine for glycine at position 1221 (G1221S), proline for leucine at position 1580 (L1580P), and proline for glutamine at position 1591 (Q1591P). Login to comment
76 Electron micrographs of lung tissue from Patient 21 (who was homozygous for the 4909+1G>A mutation) revealed lamellarbodies(Fig.3)thatweresmallerthanthose from control lung tissue, with more densely packed membranes and eccentrically placed, dense inclusion bodies, similar to those previously described in Patients 1 and 2, who were homozygous for the W1142X mutation.17 Similarly abnormal lamellar bodies were observed in lung tissue from Patient 8 (who was heterozygous for the G1221S and L982P mutations) and Patient 9 (who was homozygous for the L1553P mutation). Login to comment
84 The sequences of the puffer fish and the zebra fish are not complete, resulting in some gaps in this information in the case of L982P, G1221S, L1552P, L1553P, and L1580P. Login to comment
86 Human Mouse Rat Puffer fish ETVRRALVIN ETVKREFMIK EAVRREFMIK EDVRGKLELS QDVQQNLVRG L101P NGAGKTT NGAGKTT NGAGKTT NGAGKTT NGAGKTT N568D VARRLL VARRLL VARRLL VARRLL L1553P Q1591P T301C(L101P) C316T(R106X) A1702G(N568D) 1644delC G3426A(W1142X) G3661A(G1221S) T4657C(L1553P) 4552insT A4771C(Q1591P) 4909+1G>A G1221S LSGIAT LSGIAT LSGIAT ATP-binding domains L982P QQLSEHL QQLSENL QQLSEHL T2945C(L982P) ECEALC LAIMVQGQFKC ECEALC ECEALC L1580P T4739C(L1580P) Nonsense Missense Splice Frameshift LAIMVQGQFKC LAIMVQGQFKC LAVMVNGQFKC Zebra fish LAVMVNGQFKC tified occur in residues that are highly conserved (Fig.2).Theaminoacidalignmentwasusedtopro- duce a phylogenetic tree of the ABCA3-related proteins showing the relation of the proteins from different organisms (see Supplementary Appendix 2, available with the full text of this article at www. nejm.org).ThefishABCA3proteinsclusterwiththe mammalian ABCA3 proteins and are distinct from other, more distant ABCA-family proteins, such as the mouse Abca14, Abca15, and Abca16 proteins and the sea-urchin ABCA proteins (see Supplementary Appendix 2. Login to comment
105 Nucleotide Affected* Site Affected or Outcome SNP No.ߤ Mutation Exon 5 3, 4 T301C L101Pߥ Exon 5 14 C316T R106Xߥ Exon 14 20 A1702G N568D Exon 14 13 1644delC Frameshiftߥ Exon 21 7, 8 T2945C L982P Exon 23 1, 2 G3426A W1142Xߥ Exon 24 7, 8 G3661A G1221S Exon 30 9, 10 T4657C L1553P Exon 30 5, 6 4552insT Frameshift Exon 31 15, 21 4909+1G>A Splice siteߥ Exon 31 5, 6 T4739C L1580P Exon 31 19 A4772C Q1591P Polymorphism Exon 5 Multiple Exon 5+50A/G Intron rs46725 Exon 6 20 393C/T A131A Exon 6 18 Exon 6+119G/A Intron rs323059 Exon 7 19 Exon 7-14C/G Intron Exon 8 14 681C/T A227A Exon 10 Multiple Exon 10-105C/A Intron rs323066 Exon 10 Multiple Exon 10-20C/T Intron Exon 10 Multiple 1058C/T F353F Exon 14 Multiple Exon 14+33G/A Intron rs170447 Exon 15 Multiple 1755C/G P585P rs323043 Exon 18 13 Exon 18-17G/A Intron Exon 18 1 2340C/T H780H Exon 21 Multiple Exon 21-20C/G Intron rs313908 Exon 21 Multiple Exon 21+34C/T Intron rs313909 Exon 27 Multiple 4116C/T S1372S rs149532 Exon 32 11 4944C/T V1648V In two patients, a mutation was identified on only one allele. Login to comment

[hide] Identification and characterization of a novel ABC... Physiol Genomics. 2010 Jan 8;40(2):94-9. Epub 2009 Oct 27. Park SK, Amos L, Rao A, Quasney MW, Matsumura Y, Inagaki N, Dahmer MK
Identification and characterization of a novel ABCA3 mutation.
Physiol Genomics. 2010 Jan 8;40(2):94-9. Epub 2009 Oct 27., [PMID:19861431]

Abstract [show]
Mutations in the gene coding for ATP-binding cassette protein A3 (ABCA3) are recognized as a genetic cause of lung disease of varying severity. Characterization of a number of mutant ABCA3 proteins has demonstrated that the mutations generally affect intracellular localization or the ability of the protein to hydrolyze ATP. A novel heterozygous mutation that results in the substitution of cysteine for arginine at amino acid 295 in ABCA3 was identified in a premature infant with chronic respiratory insufficiency and abnormal lamellar bodies. Sequencing of DNA performed in study participants demonstrated that this was a mutation and not a common variant. Plasmid vectors containing ABCA3 with the identified novel mutation tagged with green fluorescent protein on the carboxy terminus were generated. The effect of the mutation on protein function was characterized by examining the glycosylation state of the mutant protein in transiently transfected HEK293 cells and by examining ATP hydrolysis activity of the mutant protein with a vanadate-induced nucleotide trapping assay in stably transfected HEK293 cells. The ABCA3 protein containing the R295C mutation undergoes normal glycosylation and intracellular localization but has dramatically reduced ATP hydrolysis activity (12% of wild type). The identification of one copy of this novel mutation in a premature infant with chronic respiratory insufficiency suggests that ABCA3 haploinsufficiency together with lung prematurity may result in more severe, or more prolonged, respiratory failure.

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53 Transient transfections of HEK293 cells with wild-type ABCA3-GFP and ABCA3 mutants L101P-GFP, N568D-GFP, and L982P-GFP (14), as well as the new pEGFPN1 construct for R295C-GFP, were performed with FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) as previously described (14). Login to comment
99 Type I mutations include L101P, L982P, L1553P, and Q1591P; type II mutations include E292V, N568D, E690K, T1114, G1221S, and L1580P (13, 14). Login to comment
100 B: alignment of sequences surrounding the R295C mutation in ICL-1 in various members of the ABCA subfamily. Login to comment
105 As reported previously, the N568D variant shows resistance to Endo H (Fig. 3A, lanes 8 and 9) at a level similar to that of the wild-type protein; however, the L101P and L982P variants (Fig. 3A, lanes 4 and 5 and lanes 10 and 11, respectively) show no Endo H resistance, indicating that these mutants have not left the endoplasmic reticulum (14). Login to comment
126 A: 20 ␮g of membrane fraction from HEK293 cells transiently transfected with WT ABCA3-GFP (lanes 2 and 3) or with ABCA3-GFP mutants L101P (lanes 4 and 5), R295C (lanes 6 and 7), N568D (lanes 8 and 9), and L982P (lanes 10 and 11) were treated without (-) or with (ϩ) endoglycosidase H (Endo H) and analyzed by 5% SDS-PAGE followed by immunoblotting with anti-GFP antibody. Lane 1, immunoblotting of untransfected HEK293 cells. Login to comment
127 B: WT ABCA3-GFP (lanes 2 and 3) or ABCA3-GFP mutants L101P (lanes 4 and 5), R295C (lanes 6 and 7), N568D (lanes 8 and 9), and L982P (lanes 10 and 11) were treated without (-) or with (ϩ) peptide N-glycosidase F (PNGase F) and were then analyzed by 5% SDS-PAGE followed by immunoblotting with anti-GFP antibody. Lane 1, immunoblotting of untransfected HEK293 cells. Login to comment
48 Transient transfections of HEK293 cells with wild-type ABCA3-GFP and ABCA3 mutants L101P-GFP, N568D-GFP, and L982P-GFP (14), as well as the new pEGFPN1 construct for R295C-GFP, were performed with FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) as previously described (14). Login to comment
94 Type I mutations include L101P, L982P, L1553P, and Q1591P; type II mutations include E292V, N568D, E690K, T1114, G1221S, and L1580P (13, 14). Login to comment
122 A: 20 òe;g of membrane fraction from HEK293 cells transiently transfected with WT ABCA3-GFP (lanes 2 and 3) or with ABCA3-GFP mutants L101P (lanes 4 and 5), R295C (lanes 6 and 7), N568D (lanes 8 and 9), and L982P (lanes 10 and 11) were treated without (afa;) or with (af9;) endoglycosidase H (Endo H) and analyzed by 5% SDS-PAGE followed by immunoblotting with anti-GFP antibody. Lane 1, immunoblotting of untransfected HEK293 cells. Login to comment
123 B: WT ABCA3-GFP (lanes 2 and 3) or ABCA3-GFP mutants L101P (lanes 4 and 5), R295C (lanes 6 and 7), N568D (lanes 8 and 9), and L982P (lanes 10 and 11) were treated without (afa;) or with (af9;) peptide N-glycosidase F (PNGase F) and were then analyzed by 5% SDS-PAGE followed by immunoblotting with anti-GFP antibody. Lane 1, immunoblotting of untransfected HEK293 cells. Login to comment

[hide] Aberrant catalytic cycle and impaired lipid transp... Am J Physiol Lung Cell Mol Physiol. 2008 Oct;295(4):L698-707. Epub 2008 Aug 1. 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., [PMID:18676873]

Abstract [show]
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-[(32)P]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.

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23 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
230 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
233 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
252 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
18 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
249 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

[hide] Heterozygous ABCA3 mutation associated with non-fa... Eur J Pediatr. 2008 Jun;167(6):691-3. Epub 2007 Jul 6. Yokota T, Matsumura Y, Ban N, Matsubayashi T, Inagaki N
Heterozygous ABCA3 mutation associated with non-fatal evolution of respiratory distress.
Eur J Pediatr. 2008 Jun;167(6):691-3. Epub 2007 Jul 6., [PMID:17618459]

Abstract [show]
A boy without symptoms up to 12 months of age started with persisting cough followed by respiratory failure at 18 months of age, resulting in mechanical ventilation because of alveolar proteinosis. Lung biopsy showed PAS-positive material. PCR was negative for CMV, Pneumocystis jiroveci and adenovirus. BALF showed mature SP-B. Analysis of the ATP-binding cassette transporter A3 (ABCA3; OMIM 601615) gene showed a compound heterozygous mutation from paternal W1148X and maternal T1114A. Alveolar lavage with 720 mg of bovine surfactant allowed weaning from ventilator support. Heterozygous mutation in the ABCA3 gene could be associated with a milder evolution as compared to the homozygous frequently lethal evolution.

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40 All of the patients reported with fatal surfactant deficiency carrying type I/type II compound heterozygous ABCA3 mutation (L982P/G1221S or Ins1518fs/L1580P) died within the neonatal period [4], and ATP-hydrolysis activity was severely impaired in the case of the G1221S and L1580P mutants [2]. Login to comment

[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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3 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
35 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 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
100 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 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
106 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 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
122 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 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
129 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
143 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
216 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
221 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 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
236 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
215 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
220 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
234 *, 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

[hide] Lost after translation: insights from pulmonary su... Am J Physiol Lung Cell Mol Physiol. 2015 Sep 15;309(6):L507-25. doi: 10.1152/ajplung.00139.2015. Epub 2015 Jul 17. Mulugeta S, Nureki S, Beers MF
Lost after translation: insights from pulmonary surfactant for understanding the role of alveolar epithelial dysfunction and cellular quality control in fibrotic lung disease.
Am J Physiol Lung Cell Mol Physiol. 2015 Sep 15;309(6):L507-25. doi: 10.1152/ajplung.00139.2015. Epub 2015 Jul 17., [PMID:26186947]

Abstract [show]
Dating back nearly 35 years ago to the Witschi hypothesis, epithelial cell dysfunction and abnormal wound healing have reemerged as central concepts in the pathophysiology of idiopathic pulmonary fibrosis (IPF) in adults and in interstitial lung disease in children. Alveolar type 2 (AT2) cells represent a metabolically active compartment in the distal air spaces responsible for pulmonary surfactant biosynthesis and function as a progenitor population required for maintenance of alveolar integrity. Rare mutations in surfactant system components have provided new clues to understanding broader questions regarding the role of AT2 cell dysfunction in the pathophysiology of fibrotic lung diseases. Drawing on data generated from a variety of model systems expressing disease-related surfactant component mutations [surfactant proteins A and C (SP-A and SP-C); the lipid transporter ABCA3], this review will examine the concept of epithelial dysfunction in fibrotic lung disease, provide an update on AT2 cell and surfactant biology, summarize cellular responses to mutant surfactant components [including endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and intrinsic apoptosis], and examine quality control pathways (unfolded protein response, the ubiquitin-proteasome system, macroautophagy) that can be utilized to restore AT2 homeostasis. This integrated response and its derangement will be placed in the context of cell stress and quality control signatures found in patients with familial or sporadic IPF as well as non-surfactant-related AT2 cell dysfunction syndromes associated with a fibrotic lung phenotype. Finally, the need for targeted therapeutic strategies for pulmonary fibrosis that address epithelial ER stress, its downstream signaling, and cell quality control are discussed.

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267 Summary of reported phenotypic features for surfactant component mutations Mutation (Domain) Clinical Diagnosis Lung Phenotype (in vivo) Subcellular Localization Trafficking Cellular Responses (in vitro) References SFTPA2 F198S (CRD) G231V (CRD) Familial pulmonary fibrosis Total BAL [SP-A] Normal ER retention Intracellular aggregation Not secreted (af9;) ER stress, cleared by ERAD (af9;) TGFbeta1 elaboration 99, 100, 175 SFTPC Group A1 èc;Exon4 (BRICHOS) L188Q (BRICHOS) G100S (BRICHOS) NSIP (Children) IPF/UIP (Adult) Absence of mature SP-C (humans) Arrested lung development (mice) ER stress (humans; mice) 1Sensitivity to bleomycin (mice) Epithelial cytotoxicity ER retention&#a1; aggresomes Intracellular aggregates ERAD requires Erdj 4/5 MG132 blocks degradation 4-PBA improves aggregates (af9;) ER stress (af9;) Apoptosis (af9;) Incomplete or absent proSP-C processing (af9;) IL-8/TGFbeta1 expression (af9;) Polyubiquitinated isoforms 21, 39, 97, 98, 100, 111, 112, 116, 117, 120, 153, 159, 160, 173, 193 Group A2 L110R (BRICHOS) P115L (BRICHOS) A116D (BRICHOS) Unspecified ILD Unspecified ILD Unspecified chILD Phenotype not reported EEA-1 (af9;); Syntaxin2 (afa;) Intracellular aggregation 2 PC secretion (af9;) Aberrant processing, 2 cell viability 1 HSP response (af9;) Congo red aggregates 160, 193 Group B1 E66K (Linker) I73T (Linker) NSIP/PAP (Child) IPF/UIP (Adult) 1 Phospholipid; 1SP-A, PAS positive staining Biopsy: PM and EE localization Misprocessed SP-C (BAL) Misprocessed SP-B (BAL) Plasma membrane&#a1;EE&#a1;LE/MVB (af9;) Aberrantly processed protein (af9;) Late autophagy block 2 Mitophagy 1 Mysfunctional mitochondria 1, 19, 24, 26, 49, 116, 118, 128, 152 Group B2 èc;91-93 (Non-BRICHOS) NSIP/PAP 2 BAL SP-B 1 BAL SP-A 2 Surfactant surface tension (af9;) Intracellular aggregates (af9;) Congo red staining Plasma membraneߥ EEA1 (af9;) compartmentsߥ Not reported 55, 181 Group C P30L (NH2-terminal) Unspecified ILD Phenotype not reported (af9;) ER retention 1 Bip expression (af9;) Polyubiquitinated isoforms 13, 116, 160 ABCA3 Group I (Trafficking Defective) L101P (1st luminal loop) R280C (1st cytosolic loop) L982P (3rd luminal loop) G1221S (11th TM domain) L1553P (COOH-terminal) Q1591P (COOH-terminal) Surfactant deficiency* RDS* chILDߤ Phenotype not reported Phenotype not reported Phenotype not reported (af9;) ER retention Non-LRO cytosolic vesicles (af9;) ER stress 30, 31, 103, 147, 172, 177 Group II (Functionally Defective) R43L (1st luminal loop) D253H (1st luminal loop) E292V (1st cytosolic loop) N568D (ABC1) E690K (ABC1) T1114M (8thTM domain) T1173R (1st luminal loop) L1580P (COOH-terminal) Surfactant deficiency* RDS* chILD (CPI)ߤ Reduced SP-B and SP-C (afa;) ER retention Lysosomes or LROs (normal) Impaired lipid transport Impaired ATP hydrolysis Impaired ATP binding Abnormal LBs 1 IL8 secretion 20, 25, 103, 104, 147, 148, 177 *Seen with homozygous or compound heterozygous ABCA3 expression; ߤfound with heterozugous ABCA3 expression. Login to comment
304 Although not restricted to any particular domain, functional characterization of a subgroup of ABCA3 mutations (including L101P, L982P, L1553P, Q1591P, G1221S) by transient or stable expression results in their total or partial retention in the ER (30, 103). Login to comment