ABCMdb

PMID: 21214890

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., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 9 ABCA3 p.Arg43Leu R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. Login to comment 35 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu Initially we investigated intracellular localization of the WT and R43L, R280C and L101P transporters. Login to comment 88 ABCA3 p.Arg43Leu Similar was observed for the R43L mutant, which showed a vesicular signal that overlapped with LAMP3 fluorescence (Figure 1A). Login to comment 89 ABCA3 p.Arg43Leu For both WT and R43L mutant, very little to no colocalization was detected with calnexin (Figure 1B). Login to comment 90 ABCA3 p.Arg43Leu WT and R43L might colocalize with the ER-resident protein calnexin during their folding in the ER as expected during the protein maturation process. Login to comment 91 ABCA3 p.Arg280Cys R280C protein colocalized frequently with LAMP3, however the colocalization was not absolute, showing often cytoplasmic distribution that overlapped with the fluorescence of calnexin (Figure 1A, B). Login to comment 92 ABCA3 p.Leu101Pro 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 ABCA3 p.Leu101Pro ABCA3 p.Arg43Leu 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 94 ABCA3 p.Arg280Cys Dual localization of R280C protein might be a sign of hindrance in the processing and folding of this mutant which slows down but does not abolish its progress through the ER, Golgi and toward LAMP3+ vesicles. Login to comment 95 ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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). Login to comment 97 ABCA3 p.Leu101Pro 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu YFP fluorescence of ABCA3-YFP fusions (green) was used to detect ABCA3 WT and R43L, R280C and L101P proteins. Login to comment 103 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 104 ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys (C) Partial ER localization of HA-tagged R280C in A549 cells transfected with pUB6/HA-R280C plasmid confirms the partial R280C ER retention as independent on the type of the plasmid or protein tag. Login to comment 106 ABCA3 p.Leu101Pro 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 107 ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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. Login to comment 109 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro 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 111 ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu ABCA3 p.Arg43Leu However, R43L and R280C mutation are mostly (R280C) or completely (R43L) correctly localized and potentially functional (Figure 1A, B). Login to comment 113 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro L101P mutant was used to monitor the situation with nonfunctional ABCA3 protein. Login to comment 116 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu (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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu (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 119 ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu Figure 3 Function of the R43L and R280C transporters. Login to comment 122 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu HA-tag was used to detect ABCA3 WT and R43L, R280C and L101P by immunofluorescence (red). Login to comment 123 ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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. Login to comment 125 ABCA3 p.Leu101Pro 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 133 ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu Similar colocalization of NBD fluorescence with ABCA3-HA vesicles was extremely rarely observed in cells expressing R43L and R280C mutations (Figure 3A, B). Login to comment 134 ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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. Login to comment 137 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 139 ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu Decreased uptake of NBD fluorescence in lamellar bodies and impact on lamellar body biogenesis together suggest functional impairment of the R43L and R280C proteins. Login to comment 140 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys 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 143 ABCA3 p.Arg43Leu No significant BiP increase was detectable between WT and correctly localized R43L mutation. Login to comment 145 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys (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 ABCA3 p.Leu101Pro 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu ABCA3 p.Arg43Leu (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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys (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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro Figure 6 Apoptosis in A549 cells expressing L101P and R280 mutations. Login to comment 188 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys In contrast, cleaved caspase 4 in R280C and L101P mutants increased significantly in comparison to WT. Login to comment 197 ABCA3 p.Arg43Leu Changes measured between R43L and WT were not significant (Figure 7C). Login to comment 198 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys (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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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 210 ABCA3 p.Arg280Cys and calnexin+ ER compartment (Figure 1A, B and 1C), indicating less efficient but not abolished R280C trafficking. Login to comment 212 ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu 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. Login to comment 213 ABCA3 p.Leu101Pro 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 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu While ER retention of L101P excludes ABCA3 function, the function of R43L and R280C transporters was studied additionally. Login to comment 217 ABCA3 p.Leu101Pro 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 220 ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys ABCA3 p.Arg43Leu ABCA3 p.Arg43Leu In the case of R43L and R280C mutations such colocalization was rarely observed suggesting functional impairment of R43L and R280C proteins. Login to comment 223 ABCA3 p.Leu101Pro 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 ABCA3 p.Leu101Pro 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 231 ABCA3 p.Arg280Cys ABCA3 p.Arg280Cys The ER stress caused by R280C mutation was slightly lower and surface staining with annexin V, as an early apoptosis sign, was the only apoptotic marker detected in R280C cells. Login to comment 233 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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 234 ABCA3 p.Arg43Leu 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). Login to comment 237 ABCA3 p.Leu101Pro ABCA3 p.Arg280Cys 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