ABCMdb

ABCA3 p.Leu101Pro

ClinVar:	c.302T>C , p.Leu101Pro D , Pathogenic
Predicted by SNAP2:	A: D (53%), C: N (57%), D: D (75%), E: D (59%), F: N (61%), G: D (71%), H: N (53%), I: N (87%), K: D (59%), M: N (93%), N: D (63%), P: D (66%), Q: N (53%), R: D (59%), S: D (59%), T: N (61%), 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: N,

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[hide] Ubiquitin-mediated proteasomal degradation of ABC ... J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12. Nakagawa H, Toyoda Y, Wakabayashi-Nakao K, Tamaki H, Osumi M, Ishikawa T
Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts.
J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12., [PMID:21567408]

Abstract [show]
The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.

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155 Effect of Mutations and Nonsynonymous SNPs on Protein Trafficking, Maturation, or ERAD of ABC Transporters Protein AA Mutation/SNP Effect on Protein Reference ABCA1 W590S Mutation Functional defect 115 R587W Mutation Impaired glycol processing 115 Q597R Mutation Impaired glycol processing, ERAD 115,116 Y1532C Mutation Altered protein trafficking 117 R1925Q Mutation Altered protein trafficking 118 ABCA3 R43L Mutation Altered protein trafficking 119 L101P Mutation Altered protein trafficking 119 R280C Mutation Altered protein trafficking 119 ABCA4 L541P Mutation Mislocalization 120 R602W Mutation Mislocalization 120 A1038V Mutation Mislocalization 120 C1490Y Mutation Mislocalization 120 ABCB1a G268V Mutation ERAD 121 G341C Mutation ERAD 121 I1196S Mutation Reduced glycosylation 122 ABCB4 I541F Mutation Accumulation in ER 123 ABCB11a E135K Mutation Reduced level of mature protein 124 L198P Mutation Reduced level of mature protein 124 E297G Mutation Reduced level of mature protein 124 L413W Mutation Reduced level of mature protein 124 R432T Mutation Reduced level of mature protein 124 D482G Mutation Immature protein in ER 124,125 N490D Mutation Reduced level of mature protein 124 A570T Mutation Reduced level of mature protein 124 T655I Mutation Reduced level of mature protein 124 Y818F SNP Moderate reduction of protein 124 G982R Mutation Retention in ER 125 R1153C Mutation ERAD 125 R1286Q Mutation Retention in ER 125 ABCC2a R768W Mutation Impaired protein trafficking 126 I1173F Mutation Impaired protein maturation 127 R1392 Mutation Impaired protein maturation 128 M1393 Mutation Impaired protein maturation 129 ABCC4a E757K SNP Altered protein trafficking 23 ABCC7 F508 Mutation Misfolding, ERAD 36-39,130 G85E Mutation Impaired protein maturation 130-132 G91R Mutation Impaired protein maturation 130-132 N1303K Mutation Impaired protein maturation 130-132 ABCC8 WT Wild type Ubiquitin-proteasome degradation 133 A116P Mutation Ubiquitin-proteasome degradation 133 V187D Mutation Ubiquitin-proteasome degradation 133 F1388 Mutation Impaired protein trafficking 134 L1544P Mutation Impaired protein trafficking 135,136 ABCC11a G180R SNP Ubiquitin-proteasome degradation 50 27 Mutation Ubiquitin-proteasome degradation 50 ABCG2a V12M SNP Altered protein localization 96 Q141K SNP Ubiquitin-proteasome degradation 102 F208S SNP Ubiquitin-proteasome degradation 78,99 S441N SNP Ubiquitin-proteasome degradation 78,99 Mutations of ABCA1, ABCA3, ABCA4, ABCB4, ABCB11, ABCC2, ABCC7 (CFTR), and ABCC8 are associated with Tangier disease, fatal surfactant deficiency, Stargardt disease, progressive familial intrahepatic cholestasis type 3 (PFIC-3), progressive familial intrahepatic cholestasis type 2 (PFIC-2), Dubin-Johnson syndrome, cystic fibrosis, and familial hyperinsulinism, respectively. Login to comment

[hide] A novel conserved targeting motif found in ABCA tr... J Lipid Res. 2011 Aug;52(8):1471-82. Epub 2011 May 17. Beers MF, Hawkins A, Shuman H, Zhao M, Newitt JL, Maguire JA, Ding W, Mulugeta S
A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments.
J Lipid Res. 2011 Aug;52(8):1471-82. Epub 2011 May 17., [PMID:21586796]

Abstract [show]
The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.

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167 Most prominent of these was L101P, which, when transfected into similar cell lines, produced profound ER retention. Login to comment
193 However, like L101P, xLxxKN mutations would naturally affect functional outcome if the transporters were held from reaching their destined compartments. Login to comment
166 Most prominent of these was L101P, which, when transfected into similar cell lines, produced profound ER retention. Login to comment
192 However, like L101P, xLxxKN mutations would naturally affect functional outcome if the transporters were held from reaching their destined compartments. Login to comment

[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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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
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
97 In the case of the L101P and L1553P mutations, each of which affected one pair of siblings from two different families, the two pairs of siblings were both homozygous for the variant and homozygous for all other polymorphisms that we found in the gene - findings that are consistent with the occurrence of a recessive mutation in these consanguineous families. 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
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
96 In the case of the L101P and L1553P mutations, each of which affected one pair of siblings from two different families, the two pairs of siblings were both homozygous for the variant and homozygous for all other polymorphisms that we found in the gene - findings that are consistent with the occurrence of a recessive mutation in these consanguineous families. Login to comment

[hide] Heterozygosity for E292V in ABCA3, lung function a... Respir Res. 2012 Aug 6;13(1):67. Baekvad-Hansen M, Nordestgaard BG, Dahl M
Heterozygosity for E292V in ABCA3, lung function and COPD in 64,000 individuals.
Respir Res. 2012 Aug 6;13(1):67., [PMID:22866751]

Abstract [show]
ABSTRACT: BACKGROUND: Mutations in ATP-binding-cassette-member A3 (ABCA3) are related to severe chronic lung disease in neonates and children, but frequency of chronic lung disease due to ABCA3 mutations in the general population is unknown. We tested the hypothesis that individuals heterozygous for ABCA3 mutations have reduced lung function and increased risk of COPD in the general population. METHODS: We screened 760 individuals with extreme pulmonary phenotypes and identified three novel (H86Y, A320T, A1086D) and four previously described mutations (E292V, P766S, S1262G, R1474W) in the ABCA3 gene. We genotyped the entire Copenhagen City Heart study (n=10,604) to assess the clinical importance of these mutations. To validate our findings we genotyped an additional 54,395 individuals from the Copenhagen General Population Study. RESULTS: In the Copenhagen City Heart Study individuals heterozygous for E292V had 5% reduced FEV1 % predicted compared with noncarriers (t-test: p=0.008), and an increased odds ratio for COPD of 1.9 (95% CI: 1.1-3.1). In contrast, the A1086D mutation was associated with increased FEV1 % predicted (p=0.03). None of the other ABCA3 mutations associated with lung function or COPD risk in the Copenhagen City Heart Study. In the larger Copenhagen General Population Study, and in the two studies combined, E292V heterozygotes did not have reduced lung function or increased risk of COPD (p=0.11-0.98), while this was the case for the positive controls, surfactant protein-B 121ins2 heterozygotes and alpha1-antitrypsin ZZ homozygotes. CONCLUSION: Our results indicate that partially reduced ABCA3 activity due to E292V is not a major risk factor for reduced lung function and COPD in the general population. This is an important finding as 1.3% in the Danish population has partially reduced ABCA3 function due to E292V.

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146 This is an important finding as 1.3 % in the Danish general population has partially reduced ABCA3 function due to E292V, and since this variant has been linked previously with severe chronic lung disease in heterozygous and compound heterozygous E292V carriers [8-10]. Login to comment
153 Another mutation in this loop, L101P, has been shown to affect ABCA3 protein folding leading to retention of ABCA3 in the endoplasmatic reticulum and subsequent ER stress and apoptosis [27,28], however individuals heterozygous for the H86Y mutation appeared asymptomatic in this study. Login to comment

[hide] Some ABCA3 mutations elevate ER stress and initiat... Respir Res. 2011 Jan 7;12:4. Weichert N, Kaltenborn E, Hector A, Woischnik M, Schams A, Holzinger A, Kern S, Griese M
Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells.
Respir Res. 2011 Jan 7;12:4., [PMID:21214890]

Abstract [show]
BACKGROUND: ABCA3 transporter (ATP-binding cassette transporter of the A subfamily) is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII). It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD) of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER). METHODS: Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level. RESULTS: We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. CONCLUSION: Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD.

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4 Methods: Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Login to comment
8 Results: We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. Login to comment
35 Fibrosis is one of the hallmarks documented in ABCA3-associated ILD [12,16,17] and knowing that ABCA3 mutations can cause ER retention of the mutated transporter [6,20], we investigated the influence of three ABCA3 mutations, R43L, R280C and L101P, found in children with surfactant deficiency and chronic ILD [10,14,19], on ER stress and apoptosis induction in lung epithelial A549 cells. Login to comment
39 Three hABCA3 point mutations R43L, R280C and L101P were introduced in the WT ABCA3 in both vector types by PCR-based site-directed mutagenesis (QuickChange Site-Directed Mutagenesis, Stratagene, La Jolla, CA). Login to comment
40 Mutagenesis primers were as follows: R43L-For 5`-CAT CTG GCT CCTCTT GAA GAT TC-3`, R43L-Rev 5`-GAA TCT TCA AGAGGA GCC AGA TG-3`, L101P-For 5`-CAG TGC GCA GGG CAC CTG TGA TCA AC-3`, L101P-Rev 5`- GTT GAT CAC AGG TGC CCT GCG CAC TG-3`, R280C-For 5`-CAT TGC CTG TGC TGT CGT G-3`, R280C-Rev 5`-CAC GAC AGC ACAGGC AAT G-3`. Login to comment
82 Results General characterization of R43L, R280C and L101P ABCA3 mutations A) Localization and trafficking Three clinically relevant ABCA3 mutations identified in patients with neonatal surfactant deficiency (R43L and L101P) and chronic ILD (R280C) were chosen for the study. Login to comment
83 While cell biology of R43L and R280C mutations has not been studied yet, L101P mutation was previously described as a trafficking/folding defect resulting in the ER accumulation of L101P protein [6,20] and was deliberately chosen for this study as a cause for the ABCA3 ER retention. Login to comment
84 Initially we investigated intracellular localization of the WT and R43L, R280C and L101P transporters. Login to comment
92 L101P mutation did not show any vesicular signal at all (Figure 1A) but presented with a cytoplasmic fluorescence mainly colocalizing with the ER protein calnexin (Figure 1B). Login to comment
93 This suggests correct localization of the WT and R43L transporters in the LAMP3-positive (LAMP3+ ) vesicles and almost full retention of the L101P mutant in the ER, possibly as a result of protein misfolding. Login to comment
97 The processing of ER retained L101P protein was different, showing complete lack of the 180 kDa band, in line with published data (Figure 2A) [6,20]. Login to comment
99 Processing of oligosaccharides and protein progress down the ER-Golgi maturation pathway Figure 1 Intracellular localization of the WT and mutant R43L, R280C and L101P ABCA3 proteins. Login to comment
101 YFP fluorescence of ABCA3-YFP fusions (green) was used to detect ABCA3 WT and R43L, R280C and L101P proteins. Login to comment
103 WT and R43L localized in LAMP3+ vesicles, R280C partially in LAMP3+ vesicles and partially in the ER and L101P completely in the ER. Login to comment
106 Immunoblotting with anti-GFP antibody revealed absence of complex sugars in L101P protein that was susceptible to both enzymes, PNGaseF and EndoH, resulting in both cases in a single shifted deglycosylated 210 kDa band and no 220 kDa band. Login to comment
109 This confirms the localization studies showing retention of the L101P mutant in the ER and ability of WT, R43L and R280C to progress further from the ER to the Golgi. Login to comment
110 C) Functional assay Trafficking/folding defect and ER accumulation of L101P protein exclude the ABCA3 function in the case of this mutant. Login to comment
113 Liposomes containing NBD-labeled major surfactant phospholipid phosphatidyl-choline (C12-NBD-PC) and NBD-labeled minor surfactant phospholipid phosphatidylethanol-amine (C12-NBD-PE) were incubated with A549 cells expressing WT, R43L, R280C and L101P HA-tagged proteins. Login to comment
114 L101P mutant was used to monitor the situation with nonfunctional ABCA3 protein. Login to comment
116 Interestingly, uptake of fluorescent liposomes into A549 cells was prominent in all cells, including those expressing L101P mutants (Figure 3) and therefore Figure 2 Processing of the WT and mutant R43L, R280C and L101P ABCA3. Login to comment
117 (A) Immunodetection with anti-GFP antibody showed two ABCA3 protein bands (180 kDa and 220 kDa) in whole cell lysates of A549 cells expressing WT and R43L and R280C mutations, and only one protein band in the cells expressing L101P mutation. Login to comment
118 (B) Deglycosylation assay with PNGaseF and EndoH on the membrane fractions from A549 cells transfected with pEYFP-N1/ ABCA3 plasmids and subsequent ABCA3-YFP immunodetection with anti-GFP antibody showed presence of high-mannose and complex oligosaccharides in WT, R43L and R280C proteins, as well as only high-mannose and no complex oligosaccharides in the L101P mutant resulting from the L101P ER retention. Login to comment
122 HA-tag was used to detect ABCA3 WT and R43L, R280C and L101P by immunofluorescence (red). Login to comment
125 Uptake through the plasma membrane into the cytoplasm of L101P transfected cells with no functional exogenous ABCA3 was observed as well as numerous cytoplasmic NBD-positive liposome-like vesicles in all A549 cells independent of the ABCA3 mutation shown in (A) and (B). Login to comment
127 WT ABCA3 (green) induced biogenesis of LAMP3+ vesicles (red) increasing their number and size in A549 cells, while R43L, R280C and L101P proteins showed no such effect (the same was observed in A549 pEYFP-N1/ABCA3 transfected cells - not shown). Login to comment
137 Expression of R43L, R280C and L101P mutations had a negative effect on vesicle formation and induced a lower number of smaller compact LAMP3+ vesicles, with the most drastic effect in L101P mutant (Figure 3D). Login to comment
140 L101P and R280C mutation upregulate ER stress marker BiP BiP/Grp78 is an essential ER chaperone of the Hsp70 family, which assists the translocation of a nascent protein chain into the ER and its subsequent folding. Login to comment
142 Immunoblotting of whole cell lysates from A549 cells expressing ABCA3-WT and three mutants revealed significant upregulation of BiP chaperone in the case of L101P mutation and R280C mutation in comparison to WT, caused by complete (L101P) or partial ER retention (R280C) of these two mutated ABCA3 transporters (Figure 4A, B). Login to comment
145 L101P and R280C mutations increase susceptibility of A549 cells to ER stress Upon ER accumulation of misfolded proteins BiP dissociates from the luminal domain of IRE1, allows IRE1 dimerization and synthesis of active XBP1 protein, a UPR transcription factor which regulates expression of ER stress proteins including BiP. Login to comment
147 To confirm previous observation on Figure 4 Increase in the ER stress chaperone BiP in A549 cells expressing R280C and L101P mutations. Login to comment
148 (A) A549 cells with ER retained L101P mutant or partially ER retained R280C protein showed upregulation of the immunodetected ER chaperone BiP in comparison to WT and A549 cells. Login to comment
155 In A549 cells with either WT or one of the three mutations, increase of XBP1 splicing was measured only in the case of L101P mutation (Figure 5D). Login to comment
156 Probably because of the robustness of the method, finer differences between WT and R43L and R280C were not observable, and the effect was measurable only in the case of the L101P mutant with the strongest protein defect (Figure 5A, D). Login to comment
161 (A) XBP1 splicing in untransfected A549 cells with and without tunicamycin (TM) treatment (10 μg/ml, 14 h) and in A549 cells with R43L, R280C and L101P mutations. Login to comment
168 Lower effect of L101P mutation on XBP1 splicing in A549 cells and no effect in A549 with WT and R43L and R280C mutations were observed. Login to comment
169 (B, E) TM treatment (10 μg/ml, 14 h) strongly induced XBP1 splicing (disappearance of unspliced bands u1 and u2) in all A549 cells expressing ABCA3 mutations, with the most significant increase in A549 expressing R280C and L101P mutations (increase in spliced band s). Login to comment
172 However, after exposure to tunicamycin XBP1 splicing was considerably more pronounced in R280C and L101P mutations if compared to A549 cells with WT and R43L mutations (Figure 5B, E). Login to comment
173 Obviously, although XBP1 splicing was measurable only for the strongest L101P defect under non-stimulated condition, cells with L101P and R280C mutations were significantly more prone to further elevation of ER stress upon exposure to an external stressor. Login to comment
174 L101P and to a lesser extent R280C mutation induce apoptosis of A549 cells Since prolonged ER stress can activate apoptosis, we analyzed if ABCA3 mutations can induce early and late apoptotic markers in A549 cells. Login to comment
178 Flow cytometry assay of Cy5-coupled Annexin V surface binding showed an increase in the number of annexin V+ /PI- cells in transfected YFP+ cells in the case of R280C and L101P mutations when compared to WT and R43L indicating an early apoptotic state of those cells (Figure 6A). Login to comment
181 Intracellular GSH level, measured by flow cytometry of monochlorobimane binding to GSH and generation of a fluorescent adduct, was decreased in the YFP+ cells with L101P protein compared to YFP+ WT, R43L and also compared to R280C (Figure 6B). Login to comment
185 Via flow cytometry assay of intracellular active caspase 3 we found an increase in caspase 3 activation in cells expressing ER retained L101P mutant in comparison to the WT and R43L cells (Figure 6C). Login to comment
186 Figure 6 Apoptosis in A549 cells expressing L101P and R280 mutations. Login to comment
188 Elevated early and late apoptotic markers were detectable in cells expressing L101P mutation, and one early marker (Annexin V) in cells expressing R280C mutation. Login to comment
190 In summary, while R43L mutation did not raise apoptotic signaling above the A549 or WT level, R280C mutation increased one early apoptotic marker and ER-localized L101P mutations significantly elevated early and late apoptotic markers, indicating injury of the cells with L101P protein. Login to comment
191 Prolonged ER stress leads to apoptosis through caspase 4 activation in cells expressing R280C and L101P mutations To examine if initiation of apoptosis in cells with ABCA3 mutations is indeed a consequence of the ER stress signaling, we assessed activation of caspase 4, which is activated by apoptotic stimuli that cause ER stress, but not other apoptotic stimuli [28,37]. Login to comment
195 Immunoblotting of whole cell lysates from the cells expressing WT or R43L, R280C or L101P mutations showed insignificant changes in pro-caspase 4 level between WT and mutations, but the level of pro-caspase 4 was somewhat higher in transfected cells then in A549 (Figure 7B). Login to comment
196 In contrast, cleaved caspase 4 in R280C and L101P mutants increased significantly in comparison to WT. Login to comment
198 This shows that caspase 4 is involved in apoptotic signaling in cells with L101P and R280C mutations and that activation of the apoptotic pathway can be a consequence of the ER stress caused by complete or partial ER retention of the ABCA3 protein. Login to comment
201 In this study we investigated the influence of three ABCA3 mutations, R43L, R280C and L101P, on intracellular stress and induction of apoptosis in cultured lung epithelial A549 cells. Login to comment
202 All three mutations were found in children with ABCA3-associated lung disease being either fatal neonatal respiratory distress syndrome (L101P and R43L [10,14]) or chronic ILD (R280C; own unpublished data, [19]). Login to comment
203 While cell biology of R43L and R280C mutations was studied here for the first time, L101P mutation was used as a known example of the trafficking/folding defect leading to the ER retention of ABCA3 with no information on ER stress [6,20]. Login to comment
205 We showed correct localization of WT and R43L proteins in LAMP3+ vesicles and dual localization of R280C protein in LAMP3+ vesicles Figure 7 Apoptotic signaling in cells with L101P and R280C mutations is activated by ER stress. Login to comment
206 (A) Immunoblotting on whole cell lysates from A549 cells transfected with pEYFP-N1/ABCA3 and densitometric analyses of (B) pro-caspase 4 and (C) cleaved caspase 4 demonstrated increased caspase 4 cleavage in cells expressing L101P and R280C mutations, as well as after TNFa treatment of A549 (25 ng/ml, 16 h) in comparison to WT and untreated A549. Login to comment
207 No significant changes in the pro-caspase 4 level in transfected cells with WT, R43L, R280C and L101P mutations were detected but pro-caspase 4 was slightly increased in transfected cells compared to A549 (B). Login to comment
213 L101P protein remained in the ER, having therefore no complex sugars, and no smaller 180 kDa protein form (Figure 1B and 2A, B) [6,20]. Login to comment
214 While ER retention of L101P excludes ABCA3 function, the function of R43L and R280C transporters was studied additionally. Login to comment
217 In contrast to the WT, expression of ABCA3 mutations, especially L101P, impaired biogenesis of LAMP3+ vesicles by reducing their number and size (Figure 3D). Login to comment
223 The uptake and number of NBD-vesicles observed in the cytoplasm was similar for both phospholipids in A549 cells and in transfected A549, and was also independent of the ABCA3 mutation, including L101P. Login to comment
230 L101P protein which accumulates in the ER, caused significant increase of the ER stress and early and late apoptosis markers in the A549 cells (Figure 4, 5 and 6). Login to comment
233 The connection between ER stress and apoptosis induction was established through the upregulation of caspase 4 in the case of both L101P and R280C mutations (Figure 7). Login to comment
237 The cells with mutations R280C and L101P, which impair ABCA3 trafficking, were more prone to further XBP1 splicing than the WT or A549. Login to comment

[hide] The surfactant lipid transporter ABCA3 is N-termin... FEBS Lett. 2010 Oct 22;584(20):4306-12. Epub 2010 Sep 21. Engelbrecht S, Kaltenborn E, Griese M, Kern S
The surfactant lipid transporter ABCA3 is N-terminally cleaved inside LAMP3-positive vesicles.
FEBS Lett. 2010 Oct 22;584(20):4306-12. Epub 2010 Sep 21., [PMID:20863830]

Abstract [show]
ABCA3 mutations cause fatal surfactant deficiency and interstitial lung disease. ABCA3 protein is a lipid transporter indispensible for surfactant biogenesis and storage in lamellar bodies (LB). The protein folds in endoplasmic reticulum and is glycosylated in Golgi en route to the membrane of mature LB and their precursor multivesicular bodies (MVB). In immunoblots, C-terminally labeled ABCA3 appears as two protein bands of 150 and 190 kDa. Using N- and C-terminal protein tags and hindering ABCA3 processing we show that the 150 kDa protein represents the mature ABCA3 whose N-terminus is cleaved by a cysteine protease inside MVB/LB.

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134 complete absence of the lower band we noticed only in the case of Q215K and other ER retained ABCA3 mutations (e.g. L101P; own data, [8,9]). 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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89 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
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
85 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
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] Haplotype analysis of ABCA3: association with resp... Ann Med. 2008;40(1):56-65. Karjalainen MK, Haataja R, Hallman M
Haplotype analysis of ABCA3: association with respiratory distress in very premature infants.
Ann Med. 2008;40(1):56-65., [PMID:18246475]

Abstract [show]
BACKGROUND: Adenosine triphosphate (ATP)-binding cassette transporter A3 (ABCA3) gene mutations cause fatal respiratory failure in term infants, but common ABCA3 polymorphisms have remained uncharacterized at the population level. AIM: To define a subset of tagging single-nucleotide polymorphisms (tSNPs) which capture most of the variation within the ABCA3 gene, and to assess ABCA3 as a novel candidate gene for susceptibility to respiratory distress syndrome (RDS) in preterm infants. METHODS: Based on an initial screen, nine tSNPs were selected. These 9 tSNPs and a length variation, representing > 90% of haplotypic variation of the gene, and 5 nonsynonymous coding SNPs were genotyped in 267 preterm infants. SNP rs13332514 was genotyped in an additional 48 infants. RESULTS: The fourth common haplotype was overrepresented in very premature infants with RDS, being accounted for by SNP rs13332514 (F353F), with an increased minor allele frequency in RDS. Furthermore, rs13332514 associated significantly with chronic lung disease defined as a requirement for supplemental O2 at 28 postnatal days in very premature infants. CONCLUSIONS: The results are suggestive of an association of a synonymous SNP in the ABCA3 gene with a prolonged course of respiratory distress syndrome in very premature infants and serve as a reference for further population-based studies of ABCA3.

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90 SNP rs149532 (corresponding to residue S1372S) was rare (MAF50.02) in our population, and it was thus excluded from further analysis, together with the nonpolymorphic cSNPs (rs28936412, 875AwT, rs13332760, rs28936690, and rs28936691, corresponding to residues L101P, E292V, V839F, L1552P, and Q1591P, respectively). Login to comment
94 SNP No. or position a Alleles (major/minor) Position b Location Affected residue Minor allele frequency tSNPs c Best pairwise r2 value and the corresponding tSNP d rs28936412 T/C 2316029 Exon 5 L101P 0 - 320-17GwA G/A 2314550 Intron 5 0.055 tSNP1 875AwT A/T 2307765 Exon 9 E292V 0 - rs13332547 C/T 2307425 Intron 9 0.093 tSNP2, r2 51 rs13332514 C/T 2307337 Exon 10 F353F 0.093 tSNP2 rs323069 G/C 2304052 Intron 10 0.271 tSNP3 rs323073 C/T 2298761 Intron 10 0.283 tSNP3, r2 50.831 rs323074 G/A 2298314 Intron 11 0.201 tSNP5, r2 50.848 rs323033 A/G 2296546 Intron 11 0.259 tSNP4 rs323040 G/A 2290527 Intron 12 0.168 tSNP5 rs170447 A/G 2289372 Intron 14 0.396 tSNP6, r2 50.832 rs2240523 T/C 2288658 Intron 14 0.381 tSNP6, r2 50.849 rs17183533 A/G 2285389 Intron 18 0.420 tSNP7, r2 50.986 rs13332760 G/T 2279621 Exon 20 V839F 0 - rs313909 C/T 2277994 Intron 21 0.373 tSNP6 rs2014467 A/G 2276395 Intron 22 0.424 tSNP7 rs2238464 G/A 2272578 Intron 26 0.420 tSNP8 rs149532 T/C 2271431 Exon 27 S1372S 0.020 - rs150926 G/C 2270170 Intron 28 0.372 tSNP6, r2 50.975 rs150928 C/T 2268651 Intron 29 0.226 tSNP9 rs28936690 T/C 2268353 Exon 30 L1552P 0 - rs28936691 A/C 2268018 Exon 31 Q1591P 0 - a rs numbers are shown for SNPs with entries in dbSNP. SNPs without entries in dbSNP are numbered from start codon as advised in (34). Login to comment
212 Of these, two (corresponding to L101P and Q1591P) have been previously identified in term infants with fatal surfactant deficiency or chronic lung disease (13). 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
121 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 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
131 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
154 To examine ATP hydrolysis of the mutant retained to the ER, cells stably expressing L101P mutant protein were established. Login to comment
155 L101P mutant protein was mainly localized to the ER, consistent with the result in transiently expressing cells (data not shown). Login to comment
156 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
164 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 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
176 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 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
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
223 The large C-terminal deletion of the ABCA3 protein (Ins1518fs and W1142X) also might hamper this process. Login to comment
224 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
266 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
271 It is possible that the E292V mutation causes less severe disruption of intracellular trafficking or ATP hydrolysis activity. Login to comment
273 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
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
264 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

[hide] Functional and trafficking defects in ATP binding ... J Biol Chem. 2006 Apr 7;281(14):9791-800. Epub 2006 Jan 16. Cheong N, Madesh M, Gonzales LW, Zhao M, Yu K, Ballard PL, Shuman H
Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome.
J Biol Chem. 2006 Apr 7;281(14):9791-800. Epub 2006 Jan 16., [PMID:16415354]

Abstract [show]
Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures.

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43 hABCA3 missense mutants (L101P, N568D, and G1221S) were generated using the PCR method with the following primers: L101P (forward primer, 5Ј-AGACAGTGCGCAGGGCACCTGTGATCAACATGCG- AG-3Ј; reverse primer, 5Ј-CTCGCATGTTGATCACAGGTGCCCT- GCGCACTGTCT-3Ј); N568D (forward primer, 5Ј-ATCACCGTCCT- GCTGGGCCACGACGGTGCCGGGAAGAC-3Ј; reverse primer, 5Ј- GTCTTCCCGGCACCGTCGTGGCCCAGCAGGACGGTGAT-3Ј); and G1221S (forward primer, 5Ј-ATCTTCAACATCCTGTCAGCCA- TCGCCACCTTCCTG-3Ј; reverse primer, 5Ј-CAGGAAGGTGGCG- AGGCCTGACAGGATGTTGAAGAT-3Ј), where the mutated nucleotides are underlined. Login to comment
46 hABCA3-GFP/HEK293, GFP/HEK293, L101P-hABCA3-GFP/HEK293, and G1221S hABCA3-GFP/HEK293 stable cell lines were selected with 500 ␮g/ml G418 and maintained with 200 ␮g/ml G418 in growth medium. Login to comment
47 When L101P hABCA3-GFP/HEK293 and G1221S hABCA3-GFP/HEK293 cells were at 80% confluence, 1 mM 4-PBA was added and cells incubated for 24 h at 37 °C. Login to comment
96 The first was in extracellular loop 1 (L101P), the second in the Walker A motif or P-loop (phosphate binding loop) of the N-terminal nucleotide binding domain (N568D), and the third in transmembrane domain 11 (G1221S) of hABCA3. Login to comment
99 The construct containing mutation L101P of ABCA3-GFP failed to target the lysosomal membrane (Fig. 2A, d-f) and mainly remained in the ER (Fig. 2B, d-f)). Login to comment
103 Western blotting with GFP antibody revealed that mutant protein is expressed at a lower overall level than wild-type protein and that wild-type and mutant fusion proteins (L101P, N568D, and G1221S) are processed differently (Fig. 2C). Login to comment
106 Densitometry analysis of a 180/220-kDa ratio of Western blot (Fig. 2C) was 0.85 for the wild-type protein, 0.45 for the N568D mutant, 0.3 for the G1221S mutant, and essentially 0.0 for the L101P mutant. Login to comment
117 LysoTracker Red (A) and ERTracker Red (B) staining of wild-type (a-c) and missense mutations of hABCA3 (L101P (d-f), N568D (g-i), and G1221S (j-l)) linked to GFP in transfected A549 cells. Login to comment
119 The wild-type, L101P, N568D, and G1221S hABCA3-GFP membrane fractions were incubated in the absence (-) or presence (ϩ) of 250 units of PNGase F at 37 °C for 1 h. Login to comment
122 investigate whether trafficking of mutant hABCA3 could be restored, 4-PBA was applied to cells expressing the hABCA3-GFP missense mutants L101P hABCA3-GFP, and G1221S hABCA3-GFP and visualized by confocal microscopy. Login to comment
123 The addition of 1 mM 4-PBA to HEK239 cells stably transfected with G1221S hABCA3-GFP and L101P hABCA3-GFP markedly altered GFP localization from the ER to the membranes of punctate vesicles (Fig. 3, A and B, and supplemental Fig. 3), but it did not alter the localization of wild-type hABCA3-GFP (supplemental Fig. 4). Login to comment
124 Vesicles with G1221S hABCA3-GFP (Fig. 3B, b, d, and f)), but not L101P hABCA3-GFP (Fig. 3A, b, d, and f)), stained positively with LysoTracker Red. Login to comment
125 Western blots with GFP antibody showed that total hABCA3-GFP protein was increased in the presence of 4-PBA for both L101P and G1221S hABCA3-GFP (Fig. 3C) but that the ratio of 180/220 kDa protein bands increased only for the G1221S hABCA3-GFP mutant (Fig. 3D), providing further evidence that the lower molecular weight protein form correlates with lysosomal processing. Login to comment
139 4-PBA facilitates lysosomal targeting of G1221S-GFP but not L101P-GFP. Login to comment
140 A, 4-PBA treatment failed to restore L101P ABCA3-GFP trafficking to lysosomes. Login to comment
143 Western blots with anti-GFP (top) and anti beta-actin (bottom) antibodies of cell lysates from L101P and G1221S hABCA3-GFP cells in the absence (-) or presence (ϩ) of 1 mM 4-PBA treatment. Login to comment
192 The (L101P) mutant in extracellular loop 1 of ABCA3 had the most severe trafficking defect. Login to comment
223 Scale bars represent 10 ␮m. lowing 4-PBA treatment in the L101P mutant (Fig. 3A). Login to comment
116 LysoTracker Red (A) and ERTracker Red (B) staining of wild-type (a-c) and missense mutations of hABCA3 (L101P (d-f), N568D (g-i), and G1221S (j-l)) linked to GFP in transfected A549 cells. Login to comment
118 The wild-type, L101P, N568D, and G1221S hABCA3-GFP membrane fractions were incubated in the absence (afa;) or presence (af9;) of 250 units of PNGase F at 37 &#b0;C for 1 h. Login to comment
121 ABCA3 Role in Lipid Transport and Lamellar Body Biogenesis 9794 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 14ߦAPRIL 7, 2006 investigate whether trafficking of mutant hABCA3 could be restored, 4-PBA was applied to cells expressing the hABCA3-GFP missense mutants L101P hABCA3-GFP, and G1221S hABCA3-GFP and visualized by confocal microscopy. Login to comment
138 4-PBA facilitates lysosomal targeting of G1221S-GFP but not L101P-GFP. Login to comment
142 Western blots with anti-GFP (top) and anti beta-actin (bottom) antibodies of cell lysates from L101P and G1221S hABCA3-GFP cells in the absence (afa;) or presence (af9;) of 1 mM 4-PBA treatment. Login to comment
191 The (L101P) mutant in extracellular loop 1 of ABCA3 had the most severe trafficking defect. Login to comment
221 Scale bars represent 10 òe;m. ABCA3 Role in Lipid Transport and Lamellar Body Biogenesis APRIL 7, 2006ߦVOLUME 281ߦNUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9799 lowing 4-PBA treatment in the L101P mutant (Fig. 3A). Login to comment

[hide] Disruption of N-linked glycosylation promotes prot... Am J Physiol Lung Cell Mol Physiol. 2013 Dec;305(12):L970-80. doi: 10.1152/ajplung.00184.2013. Epub 2013 Oct 18. Beers MF, Zhao M, Tomer Y, Russo SJ, Zhang P, Gonzales LW, Guttentag SH, Mulugeta S
Disruption of N-linked glycosylation promotes proteasomal degradation of the human ATP-binding cassette transporter ABCA3.
Am J Physiol Lung Cell Mol Physiol. 2013 Dec;305(12):L970-80. doi: 10.1152/ajplung.00184.2013. Epub 2013 Oct 18., [PMID:24142515]

Abstract [show]
The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the endoplasmic reticulum (ER) folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild-type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility (Mr) and reduced the expression level of wild-type ABCA3 in a dose-dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that although single motif mutants exhibited a vesicular distribution pattern similar to wild-type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan-deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability, and its disruption results in protein destabilization and proteasomal degradation.

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228 Firstly, expression of the leucine-to-proline mutation at residue 101 (L101P) results in ER-retained, unprocessed ABCA3 product that induces the unfolded proteins response and ER stress (10, 37, 56), suggesting that this mutation causes gross misfolding of ABCA3. Login to comment

[hide] Mutations in the ABCA3 gene are associated with ca... Invest Ophthalmol Vis Sci. 2014 Nov 18;55(12):8031-43. doi: 10.1167/iovs.14-14098. Chen P, Dai Y, Wu X, Wang Y, Sun S, Xiao J, Zhang Q, Guan L, Zhao X, Hao X, Wu R, Xie L
Mutations in the ABCA3 gene are associated with cataract-microcornea syndrome.
Invest Ophthalmol Vis Sci. 2014 Nov 18;55(12):8031-43. doi: 10.1167/iovs.14-14098., [PMID:25406294]

Abstract [show]
PURPOSE: Cataract-microcornea syndrome (CCMC) is an autosomal dominant inherited disease characterized by the association of congenital cataract and microcornea without any other systemic anomaly or dysmorphism. Although mutations of several genes have been shown to cause dominant CCMC, in many patients the causative gene has not yet been identified. Our aim was to identify the disease-associated gene in Chinese patients with CCMC. METHODS: The CCMC patients from two unrelated Chinese families and 26 sporadic patients were enrolled. All the patients were screened by Sanger sequencing with no identified mutations. Genetic variations were screened by whole-exome sequencing and then validated using Sanger sequencing. RESULTS: By sequencing the whole exome of three patients in a Chinese four-generation dominant CCMC family (Family A), three heterozygous missense mutation (c.115C>G, c.277G>A, and c.4393G>A) were identified in ATP-binding cassette protein A3 (ABCA3). At highly conserved positions, changes (c.115C>G and c.4393G>A) were predicted to have functional impacts and completely cosegregated with the phenotype. We further confirmed our finding by identifying another heterozygous missense mutation, c.2408C>T, in ABCA3 in an additional dominant CCMC family (Family B), which also cosegregated with the phenotype. Moreover, four heterozygous mutations, two missense mutations (c.4253A>T, c.2069A>T) and two splice site mutations (c.4053+2T>C, c.2765-1G>T) were identified from the sporadic patients. The ABCA3 protein was expressed in human lens capsule, choroid-retinal pigment epithelium and retinal pigment epithelial cells. CONCLUSIONS: Mutations in the human ABCA3 gene were associated with lethal respiratory distress. Our study showed, for the first time to our knowledge, that mutations in ABCA3 were associated with CCMC, warranting further investigations on the pathogenesis of this disorder.

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293 Genetic Variants Identified in ABCA3 in Patients With Surfactant Metabolism Dysfunction-3 (SMDP3) dbSNP rs# Cluster ID Codons Substitution Mutation Type Mutation Mode rs121909181 c.3426G>A W1142X Missense Homozygosity rs121909182 c.301T>C L101P Missense Homozygosity rs121909183 c.4657T>C L1553P Missense Homozygosity rs28936691 c.4772A>C Q1591P Missense Heterozygosity rs121909184 c.1702G>A N568D Missense Heterozygosity - c.4909&#fe;1G>A - Splice site Homozygosity rs121909185 c.977T>C L326P Missense Homozygosity 19. 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