ABCA3 p.Arg43Leu
| Predicted by SNAP2: | A: D (71%), C: D (71%), D: D (63%), E: D (66%), F: D (71%), G: D (63%), H: D (53%), I: D (71%), K: N (66%), L: D (71%), M: D (71%), N: N (61%), P: D (63%), Q: D (59%), S: N (53%), T: N (53%), V: D (71%), W: D (85%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D, |
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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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No. Sentence Comment
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.
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ABCA3 p.Arg43Leu 21567408:155:406
status: NEW[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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No. Sentence Comment
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.
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ABCA3 p.Arg43Leu 21214890:4:143
status: NEW9 R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling.
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ABCA3 p.Arg43Leu 21214890:9:0
status: NEW35 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.
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ABCA3 p.Arg43Leu 21214890:35:226
status: NEW39 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).
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ABCA3 p.Arg43Leu 21214890:39:29
status: NEW40 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`.
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ABCA3 p.Arg43Leu 21214890:40:37
status: NEWX
ABCA3 p.Arg43Leu 21214890:40:83
status: NEW82 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.
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ABCA3 p.Arg43Leu 21214890:82:36
status: NEWX
ABCA3 p.Arg43Leu 21214890:82:208
status: NEW83 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.
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ABCA3 p.Arg43Leu 21214890:83:22
status: NEW84 Initially we investigated intracellular localization of the WT and R43L, R280C and L101P transporters.
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ABCA3 p.Arg43Leu 21214890:84:67
status: NEW88 Similar was observed for the R43L mutant, which showed a vesicular signal that overlapped with LAMP3 fluorescence (Figure 1A).
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ABCA3 p.Arg43Leu 21214890:88:29
status: NEW89 For both WT and R43L mutant, very little to no colocalization was detected with calnexin (Figure 1B).
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ABCA3 p.Arg43Leu 21214890:89:16
status: NEW90 WT and R43L might colocalize with the ER-resident protein calnexin during their folding in the ER as expected during the protein maturation process.
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ABCA3 p.Arg43Leu 21214890:90:7
status: NEW93 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.
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ABCA3 p.Arg43Leu 21214890:93:49
status: NEW95 B) Processing and maturation In immunoblots with anti-GFP antibody on cell lysates from transfected A549 cells expressing YFP labeled WT, R43L and R280C proteins, two protein bands of 180 kDa (150 kDa ABCA3 plus 30 kDa YFP) and 220 kDa (190 kDa ABCA3 plus 30 kDa YFP) were detected (Figure 2A).
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ABCA3 p.Arg43Leu 21214890:95:138
status: NEW99 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.
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ABCA3 p.Arg43Leu 21214890:99:146
status: NEW101 YFP fluorescence of ABCA3-YFP fusions (green) was used to detect ABCA3 WT and R43L, R280C and L101P proteins.
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ABCA3 p.Arg43Leu 21214890:101:78
status: NEW103 WT and R43L localized in LAMP3+ vesicles, R280C partially in LAMP3+ vesicles and partially in the ER and L101P completely in the ER.
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ABCA3 p.Arg43Leu 21214890:103:7
status: NEW107 Both sugar types were present in WT, R43L and R280C proteins, as visible by the resistance of a portion of the 220 kDa band to the EndoH treatment.
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ABCA3 p.Arg43Leu 21214890:107:37
status: NEW109 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.
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ABCA3 p.Arg43Leu 21214890:109:106
status: NEW111 However, R43L and R280C mutation are mostly (R280C) or completely (R43L) correctly localized and potentially functional (Figure 1A, B).
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ABCA3 p.Arg43Leu 21214890:111:9
status: NEWX
ABCA3 p.Arg43Leu 21214890:111:67
status: NEW113 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.
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ABCA3 p.Arg43Leu 21214890:113:228
status: NEW116 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.
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ABCA3 p.Arg43Leu 21214890:116:198
status: NEW117 (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.
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ABCA3 p.Arg43Leu 21214890:117:150
status: NEW118 (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.
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ABCA3 p.Arg43Leu 21214890:118:265
status: NEW119 Figure 3 Function of the R43L and R280C transporters.
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ABCA3 p.Arg43Leu 21214890:119:25
status: NEW122 HA-tag was used to detect ABCA3 WT and R43L, R280C and L101P by immunofluorescence (red).
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ABCA3 p.Arg43Leu 21214890:122:39
status: NEW123 Both C12-NBD-PC and C12-NBD-PE fluorescence (green) frequently colocalized with the ring-like ABCA3 WT signal as well as within the ABCA3-WT vesicles and almost never with the R43L and R280C vesicles.
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ABCA3 p.Arg43Leu 21214890:123:176
status: NEW127 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).
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ABCA3 p.Arg43Leu 21214890:127:115
status: NEW133 Similar colocalization of NBD fluorescence with ABCA3-HA vesicles was extremely rarely observed in cells expressing R43L and R280C mutations (Figure 3A, B).
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ABCA3 p.Arg43Leu 21214890:133:116
status: NEW134 This probably indicates the ability of WT-ABCA3-HA vesicles to take up and accumulate both fluorescent lipids, while R280C-ABCA3-HA and R43L-ABCA3-HA vesicles did not show such ability.
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ABCA3 p.Arg43Leu 21214890:134:136
status: NEW137 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).
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ABCA3 p.Arg43Leu 21214890:137:14
status: NEW139 Decreased uptake of NBD fluorescence in lamellar bodies and impact on lamellar body biogenesis together suggest functional impairment of the R43L and R280C proteins.
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ABCA3 p.Arg43Leu 21214890:139:141
status: NEW143 No significant BiP increase was detectable between WT and correctly localized R43L mutation.
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ABCA3 p.Arg43Leu 21214890:143:78
status: NEW156 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).
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ABCA3 p.Arg43Leu 21214890:156:83
status: NEW161 (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.
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ABCA3 p.Arg43Leu 21214890:161:135
status: NEW168 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.
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ABCA3 p.Arg43Leu 21214890:168:96
status: NEW172 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).
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ABCA3 p.Arg43Leu 21214890:172:153
status: NEW178 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).
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ABCA3 p.Arg43Leu 21214890:178:211
status: NEW181 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).
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ABCA3 p.Arg43Leu 21214890:181:199
status: NEW185 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).
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ABCA3 p.Arg43Leu 21214890:185:177
status: NEW190 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.
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ABCA3 p.Arg43Leu 21214890:190:18
status: NEW195 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).
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ABCA3 p.Arg43Leu 21214890:195:69
status: NEW197 Changes measured between R43L and WT were not significant (Figure 7C).
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ABCA3 p.Arg43Leu 21214890:197:25
status: NEW201 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.
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ABCA3 p.Arg43Leu 21214890:201:70
status: NEW202 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]).
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ABCA3 p.Arg43Leu 21214890:202:147
status: NEW203 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].
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ABCA3 p.Arg43Leu 21214890:203:22
status: NEW205 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.
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ABCA3 p.Arg43Leu 21214890:205:41
status: NEW207 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).
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ABCA3 p.Arg43Leu 21214890:207:80
status: NEW212 R43L and R280C proteins showed WT-processing with two protein bands (Figure 2A) and presence of complex oligosaccharides (Figure 2B) confirming their ability to proceed from the ER to the Golgi.
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ABCA3 p.Arg43Leu 21214890:212:0
status: NEW214 While ER retention of L101P excludes ABCA3 function, the function of R43L and R280C transporters was studied additionally.
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ABCA3 p.Arg43Leu 21214890:214:69
status: NEW220 In the case of R43L and R280C mutations such colocalization was rarely observed suggesting functional impairment of R43L and R280C proteins.
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ABCA3 p.Arg43Leu 21214890:220:15
status: NEWX
ABCA3 p.Arg43Leu 21214890:220:116
status: NEW234 Correctly localized R43L mutation, despite its influence on the ABCA3 function and lamellar body biogenesis, had almost no impact on stress and apoptosis under any conditions above the range of the WT values (Figure 4, 5, 6 and 7).
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ABCA3 p.Arg43Leu 21214890:234:20
status: NEW[hide] Unexplained neonatal respiratory distress due to c... J Pediatr. 2007 Jun;150(6):649-53, 653.e1. Somaschini M, Nogee LM, Sassi I, Danhaive O, Presi S, Boldrini R, Montrasio C, Ferrari M, Wert SE, Carrera P
Unexplained neonatal respiratory distress due to congenital surfactant deficiency.
J Pediatr. 2007 Jun;150(6):649-53, 653.e1., [PMID:17517255]
Abstract [show]
Genetic abnormalities of pulmonary surfactant were identified by DNA sequence analysis in 14 (12 full-term, 2 preterm) of 17 newborn infants with fatal respiratory distress of unknown etiology. Deficiency of adenosine triphosphate-binding cassette protein, member A3 (n = 12) was a more frequent cause of this phenotype than deficiency of surfactant protein B (n = 2).
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No. Sentence Comment
51 Characteristics of patients n GA (W) BW (G) Sex Familial Therapies Age at death Gene/mutation Histology Immunostaining Electron microscopy 1 40 3400 M No MV, surfactant, HFOV 4 hours No mutations NA NA NA 2 40 3700 F No MV 2 hours No mutations NA NA NA 3 37 3110 M No Corticosteroids, MV, surfactant, prostacyclin, HFOV 3 days No mutations HMD ϩ SP-B - proSP-C, alveolar epithelium NA 4 40 3050 F Yes Corticosteroids, MV, surfactant, prostacyclin, iNO, HFOV 28 days SFTPB mutations 121ins2/ 122delC PAP Absent SP-B and proSP-B PAP material pro-SP-C ϩ NA 5 39 3200 F Yes MV, surfactant 38 days SFTPB mutations 121ins2/ 122delC PAP Absent SP-B and proSP-B PAP material pro-SP-C ϩ NA 6 38 3650 F Yes MV, surfactant 27 days ABCA3 mutations 4240delC/ W165X DIP ϩ SP-B ϩ proSP-C, alveolar epithelium NA 7 39 2850 M No MV 2 days ABCA3 mutation R280C/wt NA NA NA 8 35 3000 M No MV, surfactant 2 days ABCA3 mutation E292V/wt NA NA NA 9 40 3700 F No MV, surfactant 37 days ABCA3 mutations R208W/ T1423I DIP ϩ SP-B ϩ proSP-C, alveolar epithelium Numerous small LBs with dense cores 10 40 3220 M Yes MV, surfactant 30 days ABCA3 mutations 3997delAG/3997delAG DIP ϩ SP-B ϩ proSP-C, alveolar epithelium Few small LBs with dense cores 11 38 2700 F Yes MV, surfactant, corticosteroids 13 days ABCA3 mutations R155Q/ R155Q DIP ϩ SP-B ϩ proSP-C, alveolar epithelium 12 40 3050 M No MV, surfactant, iNO, prostacyclin 30 days ABCA3 mutations R43L/ R1482W DIP ϩ SP-B ϩ proSP-C, alveolar epithelium Numerous small LBs with dense cores 13 41 3420 F No MV, surfactant 180 days ABCA3 mutation S341N/wt NA NA NA 14 39 3150 M Yes MV, surfactant 64 days ABCA3 mutations P248L/ P248L DIP ϩ SP-B ϩ proSP-C, alveolar epithelium NA 15 38 3280 M Yes MV, surfactant, HFOV, corticosteroids 66 days ABCA3 mutations 4240delC/ W165X NA NA NA 16 41 3000 F No MV, surfactant, HFOV 50 days ABCA3 mutations R208W/ 4296_4301delATCACG NA NA NA 17 33 1750 M No MV, surfactant, HFOV 206 days ABCA3 mutations P147L/ R155Q DIP ϩ SP-B ϩ proSP-C, alveolar epithelium Numerous small LBs with dense cores GA, gestational age; BW, birth weight; MV, mechanical ventilation; HFOV, high-frequency oscillatory ventilation; iNO, inhaled nitric oxide; wt, wild type; LB, lamellar body; HMD, hyaline membrane disease; PAP, pulmonary alveolar proteinosis; DIP, desquamative intersititial pneumonia; HMD, hyaline membrane disease; NA, not available.
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ABCA3 p.Arg43Leu 17517255:51:1488
status: NEW48 Characteristics of patients n GA (W) BW (G) Sex Familial Therapies Age at death Gene/mutation Histology Immunostaining Electron microscopy 40 3400 M No MV, surfactant, HFOV 4 hours No mutations NA NA NA 2 40 3700 F No MV 2 hours No mutations NA NA NA 3 37 3110 M No Corticosteroids, MV, surfactant, prostacyclin, HFOV 3 days No mutations HMD af9; SP-B afa; proSP-C, alveolar epithelium NA 4 40 3050 F Yes Corticosteroids, MV, surfactant, prostacyclin, iNO, HFOV 28 days SFTPB mutations 121ins2/ 122delC PAP Absent SP-B and proSP-B PAP material pro-SP-C af9; NA 5 39 3200 F Yes MV, surfactant 38 days SFTPB mutations 121ins2/ 122delC PAP Absent SP-B and proSP-B PAP material pro-SP-C af9; NA 6 38 3650 F Yes MV, surfactant 27 days ABCA3 mutations 4240delC/ W165X DIP af9; SP-B af9; proSP-C, alveolar epithelium NA 7 39 2850 M No MV 2 days ABCA3 mutation R280C/wt NA NA NA 8 35 3000 M No MV, surfactant 2 days ABCA3 mutation E292V/wt NA NA NA 9 40 3700 F No MV, surfactant 37 days ABCA3 mutations R208W/ T1423I DIP af9; SP-B af9; proSP-C, alveolar epithelium Numerous small LBs with dense cores 10 40 3220 M Yes MV, surfactant 30 days ABCA3 mutations 3997delAG/3997delAG DIP af9; SP-B af9; proSP-C, alveolar epithelium Few small LBs with dense cores 11 38 2700 F Yes MV, surfactant, corticosteroids 13 days ABCA3 mutations R155Q/ R155Q DIP af9; SP-B af9; proSP-C, alveolar epithelium 12 40 3050 M No MV, surfactant, iNO, prostacyclin 30 days ABCA3 mutations R43L/ R1482W DIP af9; SP-B af9; proSP-C, alveolar epithelium Numerous small LBs with dense cores 13 41 3420 F No MV, surfactant 180 days ABCA3 mutation S341N/wt NA NA NA 14 39 3150 M Yes MV, surfactant 64 days ABCA3 mutations P248L/ P248L DIP af9; SP-B af9; proSP-C, alveolar epithelium NA 15 38 3280 M Yes MV, surfactant, HFOV, corticosteroids 66 days ABCA3 mutations 4240delC/ W165X NA NA NA 16 41 3000 F No MV, surfactant, HFOV 50 days ABCA3 mutations R208W/ 4296_4301delATCACG NA NA NA 17 33 1750 M No MV, surfactant, HFOV 206 days ABCA3 mutations P147L/ R155Q DIP af9; SP-B af9; proSP-C, alveolar epithelium Numerous small LBs with dense cores GA, gestational age; BW, birth weight; MV, mechanical ventilation; HFOV, high-frequency oscillatory ventilation; iNO, inhaled nitric oxide; wt, wild type; LB, lamellar body; HMD, hyaline membrane disease; PAP, pulmonary alveolar proteinosis; DIP, desquamative intersititial pneumonia; HMD, hyaline membrane disease; NA, not available.
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ABCA3 p.Arg43Leu 17517255:48:1493
status: NEW[hide] Alteration of the pulmonary surfactant system in f... Am J Respir Crit Care Med. 2006 Sep 1;174(5):571-80. Epub 2006 May 25. Brasch F, Schimanski S, Muhlfeld C, Barlage S, Langmann T, Aslanidis C, Boettcher A, Dada A, Schroten H, Mildenberger E, Prueter E, Ballmann M, Ochs M, Johnen G, Griese M, Schmitz G
Alteration of the pulmonary surfactant system in full-term infants with hereditary ABCA3 deficiency.
Am J Respir Crit Care Med. 2006 Sep 1;174(5):571-80. Epub 2006 May 25., [PMID:16728712]
Abstract [show]
RATIONALE: ABCA3 mutations are known to cause fatal surfactant deficiency. OBJECTIVE: We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis. METHODS: Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting. RESULTS: ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively. CONCLUSION: Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.
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123 MUTATIONS OF THE ABCA3 GENE IN THE STUDY GROUP AND ABCA3 PROTEIN EXPRESSION IN TYPE II PNEUMOCYTES IN INDEX PATIENTS ABCA3 Protein Expression in Type II Pneumocytes in Index Patients Family Localization* Nucleotide Deviation Structural Relevance Affected Domain (immunohistochemical score)† 1 Exon 15 c1755C Ͼ G Silent polymorphism - Weak (1) Exon 15 c1814G Ͼ A R605Q (Arg Ͼ Gln) NBD 1 Exon 31 c4877-8delAG Frameshift/Stop C-terminus 2 Exon 10 c1058C Ͼ T Silent polymorphism - Weak (1) Intron 15 c1897-1G Ͼ C Acceptor splice-site mutation NBD 1 3 Exon 8 c643C Ͼ A Q215K (Gln Ͼ Lys) First extracellular loop Absent (0) Exon 8 c863G Ͼ A R288K (Arg Ͼ Lys) First extracellular loop 4 Intron 21 c3005-1G Ͼ A Acceptor splice-site mutation Second half-size transporter Weak (1) 5 Exon 5 c128G Ͼ T (het) R43L (Arg Ͼ Leu) First extracellular loop Absent (0) Exon 8 c863G Ͼ A (het) R288K (Arg Ͼ Lys) First extracellular loop Exon 15 c1755C Ͼ G (het) Silent polymorphism - Exon 31 c4751delT (het) Frameshift/Stop C-terminus 6 Exon 14 c1736T Ͼ C (het) L579P (Leu Ͼ Pro) NBD 1 Weak (1) Exon 25 c3812delG (het) Frameshift/Stop Last extracellular loop, C-terminus 7 Exon 30 c4681 C Ͼ T R1561X (Arg Ͼ Stop) C-terminus Weak (1) 8 Exon 19 c2429-30delTT Frameshift/Stop Second half-size transporter Weak (1) Definition of abbreviation: NBD ϭ nucleotide-binding domain.
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ABCA3 p.Arg43Leu 16728712:123:869
status: NEW[hide] Surfactant composition and function in patients wi... Pediatr Res. 2006 Jun;59(6):801-5. Epub 2006 Apr 26. Garmany TH, Moxley MA, White FV, Dean M, Hull WM, Whitsett JA, Nogee LM, Hamvas A
Surfactant composition and function in patients with ABCA3 mutations.
Pediatr Res. 2006 Jun;59(6):801-5. Epub 2006 Apr 26., [PMID:16641205]
Abstract [show]
Mutations in the gene encoding the ATP binding cassette transporter member A3 (ABCA3) are associated with fatal surfactant deficiency. ABCA3 lines the limiting membrane of lamellar bodies within alveolar type-II cells, suggesting a role in surfactant metabolism. The objective of this study was to determine the surfactant phospholipid composition and function in patients with mutations in the ABCA3 gene. Bronchoalveolar lavage (BAL) fluid was analyzed from three groups of infants: 1) Infants with ABCA3 mutations, 2) infants with inherited surfactant protein-B deficiency (SP-B), and 3) patients without parenchymal lung disease (CON). Surfactant phospholipid profile was determined using two-dimensional thin-layer chromatography, and surface tension was measured with a pulsating bubble surfactometer. Phosphatidylcholine comprised 41 +/- 19% of the total phospholipid in the BAL fluid of the ABCA3 group compared with 78 +/- 3% and 68 +/- 18%, p = 0.008 and 0.05, of the CON and SP-B groups, respectively. Surface tension was 31.5 +/- 9.3 mN/m and was significantly greater than CON but no different from SP-B. We conclude that mutations in ABCA3 are associated with surfactant that is deficient in phosphatidylcholine and has decreased function, suggesting that ABCA3 plays an important role in pulmonary surfactant phospholipid homeostasis.
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66 ABCA3 patient data Patient Sex Age at sample acquisition (mo) Allele 1 Allele 2 Histopathologic diagnosis PC/lipid (%) Surface tension (mN/m) Lipid/ protein SP-A (g)* SP-B (ng)* 1 M 5 R194G delF1203 Chronic pneumonitis of infancy 59.6 28 0.76 533 448 2 F 3 L1595P D253Y Interstitial pneumonitis/alveolar hypoplasia NA NA NA 21.3 212 3 F 5 1112-20 GϾA 3163del10 Pulmonary alveolar proteinosis/interstitial pneumonitis 25.4 37.8 0.33 60.8 66.5 4 F 2 M1I delE203 Interstitial pneumonitis 31.2 34.6 0.39 68 24 5 M 15 1126ins15 1866del25 Interstitial fibrosis/interstitial pneumonitis 23.8 28.5 0.43 NA 18 6 F 3 P766S-L960F 1729delTC Pulmonary alveolar proteinosis NA NA NA 135 193 7 F 4 W179C 1382delTG Interstitial pneumonitis/dysmature alveolar segments 78.1 11.1 0.48 51.9 59.1 8 F 4 R43L P264R Chronic interstitial pneumonitis 39.8 34.3 0.25 22.7 40 NA, not available.
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ABCA3 p.Arg43Leu 16641205:66:796
status: NEW[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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229 In contrast, a second group of lung disease-associated mutations that are proximal to N-glycan sites and/or that could profoundly alter the structural makeup of the loop such as P147L (23) and R43L (6, 20) may adversely influence glycosylation and subsequent ABCA3 biosynthesis.
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ABCA3 p.Arg43Leu 24142515:229:193
status: NEW[hide] Genotype-phenotype correlations for infants and ch... Am J Respir Crit Care Med. 2014 Jun 15;189(12):1538-43. doi: 10.1164/rccm.201402-0342OC. Wambach JA, Casey AM, Fishman MP, Wegner DJ, Wert SE, Cole FS, Hamvas A, Nogee LM
Genotype-phenotype correlations for infants and children with ABCA3 deficiency.
Am J Respir Crit Care Med. 2014 Jun 15;189(12):1538-43. doi: 10.1164/rccm.201402-0342OC., [PMID:24871971]
Abstract [show]
RATIONALE: Recessive mutations in the ATP-binding cassette transporter A3 (ABCA3) cause lethal neonatal respiratory failure and childhood interstitial lung disease. Most ABCA3 mutations are private. OBJECTIVES: To determine genotype-phenotype correlations for recessive ABCA3 mutations. METHODS: We reviewed all published and unpublished ABCA3 sequence and phenotype data from our prospective genetic studies of symptomatic infants and children at Washington and Johns Hopkins Universities. Mutations were classified based on their predicted disruption of protein function: frameshift and nonsense mutations were classified as "null," whereas missense, predicted splice site mutations, and insertion/deletions were classified as "other." We compared age of presentation and outcomes for the three genotypes: null/null, null/other, and other/other. MEASUREMENTS AND MAIN RESULTS: We identified 185 infants and children with homozygous or compound heterozygous ABCA3 mutations and lung disease. All of the null/null infants presented with respiratory failure at birth compared with 75% of infants with null/other or other/other genotypes (P = 0.00011). By 1 year of age, all of the null/null infants had died or undergone lung transplantation compared with 62% of the null/other and other/other children (P < 0.0001). CONCLUSIONS: Genotype-phenotype correlations exist for homozygous or compound heterozygous mutations in ABCA3. Frameshift or nonsense ABCA3 mutations are predictive of neonatal presentation and poor outcome, whereas missense, splice site, and insertion/deletions are less reliably associated with age of presentation and prognosis. Counseling and clinical decision making should acknowledge these correlations.
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109 Seven unrelated individuals from diverse ethnic and geographic origins had a mutation in codon 43 (p.R43C, p.R43H, and p.R43L, Subjects 92 and 93 [siblings], 101, 104, 105, 118, 173, 174) and these mutations have been reported in other ABCA3-deficient patients from diverse geographic locations (12, 28, 29), suggesting that this codon may be particularly susceptible to mutation.
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ABCA3 p.Arg43Leu 24871971:109:121
status: NEW[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.
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ABCA3 p.Arg43Leu 26186947:267:2550
status: NEW