ABCMdb

ABCA3 p.Gln215Lys

Predicted by SNAP2:	A: N (53%), C: N (53%), D: N (61%), E: N (72%), F: N (53%), G: N (57%), H: N (78%), I: N (66%), K: N (78%), L: N (61%), M: N (61%), N: N (72%), P: N (61%), R: N (78%), S: N (78%), T: N (78%), V: N (66%), W: D (71%), Y: N (57%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Respiratory syncytial virus potentiates ABCA3 muta... Hum Mol Genet. 2012 Jun 15;21(12):2793-806. Epub 2012 Mar 20. Kaltenborn E, Kern S, Frixel S, Fragnet L, Conzelmann KK, Zarbock R, Griese M
Respiratory syncytial virus potentiates ABCA3 mutation-induced loss of lung epithelial cell differentiation.
Hum Mol Genet. 2012 Jun 15;21(12):2793-806. Epub 2012 Mar 20., [PMID:22434821]

Abstract [show]
ATP-binding cassette transporter A3 (ABCA3) is a lipid transporter active in lung alveolar epithelial type II cells (ATII) and is essential for their function as surfactant-producing cells. ABCA3 mutational defects cause respiratory distress in newborns and interstitial lung disease (ILD) in children. The molecular pathomechanisms are largely unknown; however, viral infections may initiate or aggravate ILDs. Here, we investigated the impact of the clinically relevant ABCA3 mutations, p.Q215K and p.E292V, by stable transfection of A549 lung epithelial cells. ABCA3 mutations strongly impaired expression of the ATII differentiation marker SP-C and the key epithelial cell adhesion proteins E-cadherin and zonula occludens-1. Concurrently, cells expressing ABCA3 mutation acquired mesenchymal features as observed by increased expression of SNAI1, MMP-2 and TGF-beta1, and elevated phosphorylation of Src. Infection with respiratory syncytial virus (RSV), the most common viral respiratory pathogen in small children, potentiated the observed mutational effects on loss of epithelial and acquisition of mesenchymal characteristics. In addition, RSV infection of cells harboring ABCA3 mutations resulted in a morphologic shift to a mesenchymal phenotype. We conclude that ABCA3 mutations, potentiated by RSV infection, induce loss of epithelial cell differentiation in ATII. Loss of key epithelial features may disturb the integrity of the alveolar epithelium, thereby comprising its functionality. We suggest the impairment of epithelial function as a mechanism by which ABCA3 mutations cause ILD.

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No. Sentence Comment
4 Here, we investigated the impact of the clinically relevant ABCA3 mutations, p.Q215K and p.E292V, by stable transfection of A549 lung epithelial cells. Login to comment
38 For our study, we chose two clinically relevant ABCA3 mutations, namely p.Q215K and p.E292V. Login to comment
40 In vitro studies reported that ABCA3 p.E292V is properly localized to the LB but only has residual ATPase activity, therefore belonging to the class of functional mutations (20). Login to comment
41 The ABCA3 mutation p.Q215K has been observed in a neonate that died of respiratory distress. Login to comment
42 Immunohistochemical analysis of the lung tissue of this patient who was the compound heterozygote for ABCA3 p.Q215K and p.R288K showed no ABCA3 staining in ATII cells, and electron microscopy revealed the presence of many electron-dense bodies in the absence of LBs (9). Login to comment
43 Previous work from our group showed that ABCA3-p.Q215K caused mislocalization of the protein to the ER and its absence from the LB (21). Login to comment
45 RESULTS Stable expression of HA-tagged ABCA3 in A549 cells To study the effect of the two clinically relevant ABCA3 mutations p.Q215K and p.E292V on cellular homeostasis, A549 lung epithelial cells were stably transfected with HA-tagged ABCA3. Login to comment
46 In agreement with our previous report (21), the ABCA3 protein with the p.Q215K mutation is localized to the ER and is not, or only to a very small amount, processed and trafficked to the LBs (Fig. 1C). Login to comment
47 While ABCA3 harboring the p.E292V mutation is properly localized to LBs (Fig. 1C), it is functionally impaired (20). Login to comment
51 E-cadherin, a cellular adhesion protein found in adherens junctions and desmosomes, was decreased up to 4-fold in p.Q215K and p.E292V cells when compared with untransfected A549 and cells expressing wild-type (WT) ABCA3 (Fig. 1B, right). Login to comment
53 A549 cells expressing p.Q215K had approximately 2.5-fold decreased amounts of ZO-1. Login to comment
57 In p.Q215K and p.E292V cells, E-cadherin overall appeared to be less abundant, thus confirming the results obtained by western blotting. Login to comment
60 ZO-1 was less abundantly detected on the cell surface and more diffusely distributed in cells expressing p.Q215K compared with cells with ABCA3-WT (Fig. 1C, left). Login to comment
66 Therefore, we investigated MMP-2 expression and secretion in our type II cell model system. MMP-2 mRNA levels were significantly elevated more than 8-fold in p.Q215K cells when compared with ABCA3-WT cells (Fig. 2B, left). Login to comment
68 MMP-2 is secreted to the extracellular matrix (ECM) in tissues or into the cell culture supernatants in our model system. MMP-2 protein in supernatants of ABCA3-WT cells could not be detected, whereas p.Q215K-cells secreted large amounts of MMP-2 (Fig. 2C, left). Login to comment
71 Cells expressing the ABCA3 p.Q215K mutation produce increased amounts of TGF-b1 TGF-b1 is a protein controlling cellular proliferation and differentiation. Login to comment
73 TGF-b1 mRNA was significantly elevated in p.Q215K-expressing cells when compared with ABCA3-WT cells (Fig. 2B, right). Login to comment
74 Also, significantly elevated levels of secreted TGF-b1 were found in supernatants of p.Q215K cells when compared with supernatants from ABCA3-WT cells (Fig. 2C, right). Login to comment
75 TGF-b1 secretion was not increased in E292V cells. Login to comment
76 Expression of ABCA3 p.Q215K protein in A549 cells therefore induces synthesis and secretion of TGF-b1. Login to comment
77 Src kinase activation is increased in cells with ABCA3-Q215K mutation Next, we wanted to investigate whether phosphorylation of the tyrosine kinase Src, which has important signaling functions in cell proliferation, motility and cytoskeleton assembly (26), was upregulated in cells with ABCA3 mutations as well. Login to comment
78 As shown in Fig. 2D, increased amounts of phosphorylated Src kinase could be detected in cells expressing ABCA3 p.Q215K. Login to comment
80 The most pronounced effect was seen in ABCA3 p.Q215K cells. Login to comment
81 While non-infected p.Q215K cells showed a cell shape similar to untransfected A549 control Figure 1. Login to comment
103 2796 cells, p.Q215K cell morphology changed to a more elongated shape after virus infection. Login to comment
110 When these cells were infected with RSV, the arrangement of microtubules inside the cells was more organized and the tubulin fibers in p.Q215K-transfected cells almost had a parallel arrangement in order to support their elongated cell shape (Fig. 3B, right). Login to comment
120 Collagen type I mRNA increased significantly in p.Q215K cells after RSV infection when compared with non-infected cells. Login to comment
123 In RSV-infected p.Q215K cells, we detected significantly increased levels of SNAI1 mRNA when compared with the other cell lines (Fig. 5B). Login to comment
124 In addition to SNAI1 mRNA, we also found elevated amounts of SNAI2 mRNA in p.Q215K cells (Fig 5C). Login to comment
134 After infection with RSV, MMP-2 mRNA levels in p.Q215K and p.E292V cells increased even further when compared with ABCA3-WT, the change being more than 15-fold between p.Q215K and WT and more than 7-fold between p.E292V and WT (Fig. 6A, left and B). Login to comment
151 Therefore, MMP-2 protein in RSV-infected UV-inactivated samples was detectable only in p.Q215K supernatants (Fig. 6A, right). Login to comment
155 When both types of samples, non-infected and RSV-infected, were UV-inactivated and analyzed for TGF-b1 secretion, we found that RSV infection significantly induced TGF-b1 secretion in all cells, with p.Q215K samples having the significantly highest TGF-b1 amounts secreted (Fig. 6C and D, right). Login to comment
157 Even though Src phosphorylation was increased significantly in all cells after RSV infection, it was higher in p.Q215K cells when compared with all other cells (Fig. 7A and B). Login to comment
158 Additionally, increased phosphorylation of the mitogen-activated protein kinases (MAPKs) JNK1 and ERK1/2 was observed in p.Q215K cells when compared with ABCA3-WT cells after RSV infection (Fig. 7A, C, D). Login to comment
161 However, TGF-b1 secretion and phosphorylation of Src as well as MAPKs were elevated in cells harboring the ABCA3-p-Q215K mutation when compared with A549 cells transfected with ABCA3-WT. Login to comment
163 Phosphorylation of Src and MAPKs is induced by RSV infection and is increased in Q215K cells. Login to comment
179 Thus, ER stress does not seem to account for the changes in p.Q215K cell morphology and protein expression as observed after RSV infection when compared with A549 cells expressing ABCA3 WT or p.E292V. Login to comment
190 In addition to upregulation of SNAI1, we found increased phosphorylation of Src in cells with the p.Q215K mutation. Login to comment
194 ABCA3 mutations (especially p.Q215K) lead to downregulation of epithelial markers accompanied by acquisition of mesenchymal characteristics and activation of signaling molecules. Login to comment
206 Both TGF-b1 production and secretion were increased in cells expressing the ABCA3 p.Q215K mutation. Login to comment
219 Moreover, RSV infection also induced SNAI2 and collagen type I mRNA in cells harboring the ABCA3-p.Q215K mutation. Login to comment
229 Interestingly, we and others found decreased levels of mature SP-C protein in immunohistochemistry staining of lung tissue and in lavage samples from patients (compound) heterozygote for the ABCA3 mutations Q215K and E292V (data not shown), respectively (9). Login to comment
250 We found elevated MAPK phosphorylation in p.Q215K cells after RSV infection, supporting a role for MAPK signaling in ABCA3-mediated EMT. Login to comment
275 A549 cells were transfected with the pUB6-ABCA3-WT/Q215K/E292V vectors using ExGene 500 (Fermentas) according to the manufacturer`s protocol. Login to comment
44 Immunohistochemical analysis of the lung tissue of this patient who was the compound heterozygote for ABCA3 p.Q215K and p.R288K showed no ABCA3 staining in ATII cells, and electron microscopy revealed the presence of many electron-dense bodies in the absence of LBs (9). Login to comment
48 In agreement with our previous report (21), the ABCA3 protein with the p.Q215K mutation is localized to the ER and is not, or only to a very small amount, processed and trafficked to the LBs (Fig. 1C). Login to comment
55 A549 cells expressing p.Q215K had approximately 2.5-fold decreased amounts of ZO-1. Login to comment
59 In p.Q215K and p.E292V cells, E-cadherin overall appeared to be less abundant, thus confirming the results obtained by western blotting. Login to comment
62 ZO-1 was less abundantly detected on the cell surface and more diffusely distributed in cells expressing p.Q215K compared with cells with ABCA3-WT (Fig. 1C, left). Login to comment
70 MMP-2 is secreted to the extracellular matrix (ECM) in tissues or into the cell culture supernatants in our model system. MMP-2 protein in supernatants of ABCA3-WT cells could not be detected, whereas p.Q215K-cells secreted large amounts of MMP-2 (Fig. 2C, left). Login to comment
79 Src kinase activation is increased in cells with ABCA3-Q215K mutation Next, we wanted to investigate whether phosphorylation of the tyrosine kinase Src, which has important signaling functions in cell proliferation, motility and cytoskeleton assembly (26), was upregulated in cells with ABCA3 mutations as well. Login to comment
82 The most pronounced effect was seen in ABCA3 p.Q215K cells. Login to comment
83 While non-infected p.Q215K cells showed a cell shape similar to untransfected A549 control Figure 1. Login to comment
106 cells, p.Q215K cell morphology changed to a more elongated shape after virus infection. Login to comment
113 When these cells were infected with RSV, the arrangement of microtubules inside the cells was more organized and the tubulin fibers in p.Q215K-transfected cells almost had a parallel arrangement in order to support their elongated cell shape (Fig. 3B, right). Login to comment
126 In RSV-infected p.Q215K cells, we detected significantly increased levels of SNAI1 mRNA when compared with the other cell lines (Fig. 5B). Login to comment
127 In addition to SNAI1 mRNA, we also found elevated amounts of SNAI2 mRNA in p.Q215K cells (Fig 5C). Login to comment
137 After infection with RSV, MMP-2 mRNA levels in p.Q215K and p.E292V cells increased even further when compared with ABCA3-WT, the change being more than 15-fold between p.Q215K and WT and more than 7-fold between p.E292V and WT (Fig. 6A, left and B). Login to comment
154 Therefore, MMP-2 protein in RSV-infected UV-inactivated samples was detectable only in p.Q215K supernatants (Fig. 6A, right). Login to comment
160 Even though Src phosphorylation was increased significantly in all cells after RSV infection, it was higher in p.Q215K cells when compared with all other cells (Fig. 7A and B). Login to comment
164 However, TGF-b1 secretion and phosphorylation of Src as well as MAPKs were elevated in cells harboring the ABCA3-p-Q215K mutation when compared with A549 cells transfected with ABCA3-WT. Login to comment
166 Phosphorylation of Src and MAPKs is induced by RSV infection and is increased in Q215K cells. Login to comment
182 Thus, ER stress does not seem to account for the changes in p.Q215K cell morphology and protein expression as observed after RSV infection when compared with A549 cells expressing ABCA3 WT or p.E292V. Login to comment
193 In addition to upregulation of SNAI1, we found increased phosphorylation of Src in cells with the p.Q215K mutation. Login to comment
197 ABCA3 mutations (especially p.Q215K) lead to downregulation of epithelial markers accompanied by acquisition of mesenchymal characteristics and activation of signaling molecules. Login to comment
209 Both TGF-b1 production and secretion were increased in cells expressing the ABCA3 p.Q215K mutation. Login to comment
222 Moreover, RSV infection also induced SNAI2 and collagen type I mRNA in cells harboring the ABCA3-p.Q215K mutation. Login to comment
232 Interestingly, we and others found decreased levels of mature SP-C protein in immunohistochemistry staining of lung tissue and in lavage samples from patients (compound) heterozygote for the ABCA3 mutations Q215K and E292V (data not shown), respectively (9). Login to comment
253 We found elevated MAPK phosphorylation in p.Q215K cells after RSV infection, supporting a role for MAPK signaling in ABCA3-mediated EMT. Login to comment
278 A549 cells were transfected with the pUB6-ABCA3-WT/Q215K/E292V vectors using ExGene 500 (Fermentas) according to the manufacturer`s protocol. 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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No. Sentence Comment
34 Two ABCA3 point mutations Q215K and E292V were introduced into WT hABCA3 by site-directed mutagenesis (Stratagene). Login to comment
90 We used two ILD-related ABCA3 mutations (Supplementary Fig. 1): Q215K, a misfolding defect studied here for the first time, and E292V, a functional mutation [10], to prove that the ER retention in general, regardless of the cause (mutation as intrinsic cause or enforced processing impairment, Fig. 2) hinders the ABCA3 maturation. Login to comment
91 Localization studies in A549 cells stably expressing WT, Q215K or E292V proteins with C-terminal HA-tag showed that the E292V mutant colocalized properly with the MVB/LB protein LAMP3 (Fig. 4A) but not with the ER protein calnexin (Fig. 4B). Login to comment
92 Q215K mutant remained in the ER compartment, overlapping with calnexin (Fig. 4B) and showing no colocalization with LAMP3 (Fig. 4A). Login to comment
93 Immunodetection of WT, Q215K and E292V proteins via HA-tag demonstrated that the ER retained Q215K protein completely lacked the 150 kDa band, which was strongly present in the MVB/LB-localized WT Fig. 2. Login to comment
121 Moreover, strong ABCA3 ER retention caused by Q215K mutation resulted in vanishing of the lower band (Fig. 4), showing that the effect was not specific only for the chemical interference of the ABCA3 processing. Login to comment
124 The ER-retained Q215K mutant lacks the 150 kDa band. Login to comment
125 WT ABCA3 and two mutants E292V and Q215K (all C-terminal HA-tag) were stably expressed in A549 cells. Login to comment
126 HA-tag immunofluorescence of WT and E292V (green) showed their correct LAMP3-colocalization (A, red) while Q215K mutant (green) remained in the ER colocalizing with calnexin (B, red). Login to comment
127 (C) ABCA3 immunodetection in cell lysates via anti-HA-tag antibody showed absence of the 150 kDa band in the case of the Q215K protein. Login to comment
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
137 The ER retained mutation Q215K, which completely lacks the 150 kDa band, does not support the biogenesis of LAMP3-positive vesicles (Fig. 4, red signal) in A549 cells, while WT protein and even E292V mutant that has reduced ATP hydrolysis capacity [10] both do. Login to comment
138 Thus it appears that the presence of only the 190 kDa band (as for Q215K mutant) within A549 cells is not enough for ABCA3 function, and thus ABCA3 maturation is indispensible for its role. Login to comment

[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. Login to comment

[hide] ABCA3 protects alveolar epithelial cells against f... Biochim Biophys Acta. 2015 Jul;1851(7):987-95. doi: 10.1016/j.bbalip.2015.03.004. Epub 2015 Mar 25. Zarbock R, Kaltenborn E, Frixel S, Wittmann T, Liebisch G, Schmitz G, Griese M
ABCA3 protects alveolar epithelial cells against free cholesterol induced cell death.
Biochim Biophys Acta. 2015 Jul;1851(7):987-95. doi: 10.1016/j.bbalip.2015.03.004. Epub 2015 Mar 25., [PMID:25817392]

Abstract [show]
Diffuse parenchymal lung diseases (DPLDs) are characterized by chronic inflammation and fibrotic remodeling of the interstitial tissue. A small fraction of DPLD cases can be genetically defined by mutations in certain genes, with ABCA3 being the gene most commonly affected. However, the pathomechanisms underlying ABCA3-induced DPLD are far from clear. To investigate whether ABCA3 plays a role in cellular cholesterol homeostasis, phospholipids, free cholesterol, and cholesteryl esters were quantified in cells stably expressing ABCA3 using mass spectrometry. Cellular free cholesterol and lipid droplets were visualized by filipin or oil red staining, respectively. Expression of SREBP regulated genes was measured using qPCR. Cell viability was assessed using the XTT assay. We found that wild type ABCA3 reduces cellular free cholesterol levels, induces the SREBP pathway, and renders cells more resistant to loading with exogenous cholesterol. Moreover, ABCA3 mutations found in patients with DPLD interfere with this protective effect of ABCA3, resulting in free cholesterol induced cell death. We conclude that ABCA3 plays a previously unrecognized role in the regulation of cellular cholesterol levels. Accumulation of free cholesterol as a result of a loss of ABCA3 export function represents a novel pathomechanism in ABCA3-induced DPLD.

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35 Two clinically relevant mutations belonging to different categories were chosen for this study: p.Q215K leads to mistrafficking [20], while p.E292V causes limited functional impairment of ABCA3 transporter function [21]. Login to comment
42 Stable transfection of A549 cells with pUB6-ABCA3-WT/Q215K/ E292V vectors was carried out as previously described [22]. Login to comment
86 Free cholesterol was visualized by filipin staining in A549 cells stably transfected with ABCA3-WT, p.Q215K and p.E292V or mock transfected cells. Login to comment
105 A549 cells expressing wild type ABCA3 (ABCA3-WT) showed significantly reduced levels of FC and of PC compared to mock transfected A549 cells and also compared to cells expressing ABCA3-Q215K (Fig. 1A, D). Login to comment
107 The expression of both mutations resulted in significantly increased levels of CE compared to both, mock and ABCA3-WT transfected cells (Fig. 1B) Cellular levels of total phospholipids were elevated in ABCA3-Q215K cells compared to ABCA3-WT cells (Fig. 1C). Login to comment
111 Shown are mRNA levels of SREBPs and INSIGs in stably transfected A549 ABCA3-WT, ABCA3-Q215K and ABCA3-E292V cells and mock transfected cells. Login to comment
118 These vesicles are markedly smaller in ABCA3-E292V cells and completely absent in ABCA3-Q215K cells. Login to comment
121 This was also the case in cells expressing ABCA3-Q215K and ABCA3-E292V. Login to comment
135 Shown are mRNA levels of SREBP target genes in stably transfected A549 ABCA3-WT, ABCA3-Q215K and ABCA3-E292V cells and mock transfected cells. Login to comment
141 Unlike expression of ABCA3-WT, expression of mutated ABCA3 failed to induce expression of SREBPs and INSIG1; mRNA levels of all three proteins were similar to mock controls in cells expressing either ABCA3-Q215K or ABCA3-E292V (Fig. 4A-C). Login to comment
145 While abundant lipid droplets were seen in cells expressing ABCA3-Q215K and ABCA3-E292V and also in mock transfected cells, they were scarcely present in ABCA3-WT expressing cells (Fig. 6). Login to comment
153 In cells expressing ABCA3-Q215K and also in mock transfected cells, a change of cell morphology and membrane blebbing was noticed when compared to untreated control cells, indicating cell death (Fig. 7, arrowheads). Login to comment
155 While cell viability was almost unchanged in cells expressing wild type ABCA3 and only a slight reduction was seen in ABCA3 p.E292V and mock transfected cells, viability was reduced significantly in ABCA3-Q215K cells (Fig. 8A). Login to comment
156 In the case of ABCA3-Q215K and mock transfected cells, the effect of cholesterol loading on cell viability was partly reversed when cells were simultaneously treated with U18666A (10 bc;M) which inhibits egress of cholesterol from late endosomes (Fig. 8B). Login to comment
160 Cells expressing ABCA3-WT, p.Q215K and p.E292V and mock transfected cells were treated with free cholesterol-b2;- methylcyclodextrin complex. Login to comment
165 Lipid droplets in stably transfected A549 ABCA3-WT, p.Q215K and p.E292V cells and mock transfected cells were visualized by Oil Red O staining and fluorescence microscopy. Login to comment
169 Furthermore, we show that ABCA3 mutations affect the cells' capability to deal with exogenous cholesterol: while in cells expressing wild type ABCA3, FC loading had little effect on cell viability, FC loading proved to be toxic especially to cells expressing the mistrafficking mutation p.Q215K. Login to comment
197 (A) Cells expressing ABCA3-WT, p.Q215K and p.E292V and mock transfected cells were treated with free cholesterol-b2;- methylcyclodextrin complex before cell viability was assessed by XTT assay. Login to comment
217 Here we also delineate differentially the effect of two clinically relevant ABCA3 mutations, i.e. p.Q215K and p.E292V. Login to comment
218 While p.Q215K was associated with severe respiratory distress and early death [34], p.E292V seems to permit longer survival [3,13]. Login to comment
221 In contrast, expression of ABCA3-Q215K resulted in reduced viability not only compared to wild type ABCA3 but also compared to mock transfected cells. Login to comment