ABCMdb

ABCA3 p.Glu690Lys

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

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[hide] An intronic ABCA3 mutation that is responsible for... Pediatr Res. 2012 Jun;71(6):633-7. doi: 10.1038/pr.2012.21. Epub 2012 Feb 15. Agrawal A, Hamvas A, Cole FS, Wambach JA, Wegner D, Coghill C, Harrison K, Nogee LM
An intronic ABCA3 mutation that is responsible for respiratory disease.
Pediatr Res. 2012 Jun;71(6):633-7. doi: 10.1038/pr.2012.21. Epub 2012 Feb 15., [PMID:22337229]

Abstract [show]
INTRODUCTION: Member A3 of the ATP-binding cassette family of transporters (ABCA3) is essential for surfactant metabolism. Nonsense, missense, frameshift, and splice-site mutations in the ABCA3 gene (ABCA3) have been reported as causes of neonatal respiratory failure (NRF) and interstitial lung disease. We tested the hypothesis that mutations in noncoding regions of ABCA3 may cause lung disease. METHODS: ABCA3-specific cDNA was generated and sequenced from frozen lung tissue from a child with fatal lung disease with only one identified ABCA3 mutation. ABCA3 was sequenced from genomic DNA prepared from blood samples obtained from the proband, parents, and other children with NRF. RESULTS: ABCA3 cDNA from the proband contained sequences derived from intron 25 that would be predicted to alter the structure and function of the ABCA3 protein. Genomic DNA sequencing revealed a heterozygous C>T transition in intron 25 trans to the known mutation, creating a new donor splice site. Seven additional infants with an ABCA3-deficient phenotype and inconclusive genetic findings had this same variant, which was not found in 2,132 control chromosomes. DISCUSSION: These findings support that this variant is a disease-causing mutation that may account for additional cases of ABCA3 deficiency with negative genetic studies.

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No. Sentence Comment
30 To determine whether the aberrantly spliced transcripts were derived from the allele with the c.2068 G>A (p.E690K) mutation, primers were designed to amplify either the transcripts containing the intronic sequence or those that were normally spliced, by using a 3' primer located in the intronic sequence, or one that spanned exons 25 and 26. Login to comment
33 Electrophoretic analysis of the restriction products demonstrated that the relevant Ban II restriction fragment derived from the aberrantly spliced transcripts was smaller than that derived from the normally spliced products, indicating the presence of the G allele (wild type) in those amplicons containing the intronic sequences, and the A allele (p.E690K mutation) in those containing normal splicing. Login to comment
42 The mother was found to be heterozygous GA at position c.2068 (site of the p.E690K mutation) but was homozygous CC at position IVS25-98. Login to comment
46 2068 G>A, (p.E690K) 17 23 24 TAT A GC GGG CTG ACA TCC 252627 Figure 1. ABCA3 exons 25-26 aberrant splicing. Login to comment
52 2068 A (p.E690K) eliminates Ban II site Size markers Ban II: + + Retained intron-specific amplicon Normal splice-specific amplicon Larger (645 bp) product indicates A at position 2068 586 bp 645 bp G A BanII sites 1872 bp 1865 bp P-intron P3-WT-span P1 P1 Figure 2. Transcript-specific RT-PCR and Ban II restriction endonuclease digest. Login to comment
53 ABCA3 cDNA showing allele with inserted intronic sequence corresponding to the non-p.E690K allele. Login to comment
54 Using a BanII restriction digest, the known p.E690K mutation eliminates a restriction site in the -normally spliced amplicon, generating a larger product (645 bp vs. 586 bp), -confirming the intronic insertion is in trans to the previously identified mutation. Login to comment
80 However, the mechanism to account for this larger aberrant transcript with an additional 249 bases Table 1. Characteristics of subjects with one ABCA3 mutation Patient Ethnicity Presentation Allele 1 mutation Allele 2 mutation Findings consistent with ABCA3 deficiency Outcome A Caucasian Newborn, RDS p.E690K IVS25-98T Case patient; lung histopathology and EM Died B Caucasian RDS p.L941P IVS25-98T Family history of sibling with fatal RDS Died C Caucasian 8 y/old, ILD L212M ? Login to comment
116 Genetic testing performed through the Johns Hopkins University DNA Diagnostic Lab revealed normal SFTPB and SFTPC gene sequences but a heterozygous mutation in ABCA3 (Nt2068G->A, p.E690K) that had previously been identified as disease causing (22,23). Login to comment
135 Specific primers were created to generate amplicons expanding from the previously known mutation (p.E690K at exon 17) to the area of interest in intron 25. Login to comment

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

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

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No. Sentence Comment
99 Type I mutations include L101P, L982P, L1553P, and Q1591P; type II mutations include E292V, N568D, E690K, T1114, G1221S, and L1580P (13, 14). Login to comment
94 Type I mutations include L101P, L982P, L1553P, and Q1591P; type II mutations include E292V, N568D, E690K, T1114, G1221S, and L1580P (13, 14). Login to comment

[hide] Aberrant catalytic cycle and impaired lipid transp... Am J Physiol Lung Cell Mol Physiol. 2008 Oct;295(4):L698-707. Epub 2008 Aug 1. Matsumura Y, Ban N, Inagaki N
Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease.
Am J Physiol Lung Cell Mol Physiol. 2008 Oct;295(4):L698-707. Epub 2008 Aug 1., [PMID:18676873]

Abstract [show]
The ATP-binding cassette transporter ABCA3 mediates uptake of choline-phospholipids into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have shown previously that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[(32)P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.

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No. Sentence Comment
12 E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Login to comment
13 Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Login to comment
24 On the other hand, patients with the common missense mutation E292V and a second, specific mutation such as E690K or T1114M develop pediatric interstitial lung disease (pILD), the phenotype of which is milder than that of fatal surfactant deficiency, suggesting that the E292V ABCA3 mutation is responsible for the development of pILD (4). Login to comment
26 In this study, we characterized E292V, E690K, and T1114M mutant ABCA3 proteins identified in pILD. Login to comment
29 The plasmid pEGFPN1-ABCA3 (17), which encodes ABCA3 protein fused with enhanced green fluorescent protein (GFP) at the COOH terminus (ABCA3-GFP), and its mutants containing pILD mutations (E292V, E690K, and T1114M) and other site-directed mutations (E292D, E292K, T1114S, E690D, and E690R) were generated as described previously and used for transient transfection experiment. Login to comment
90 The E292V, E690K, and T1114M mutations identified in pILD (4) are located at intracellular loop 1 (ICL-1), adjacent to the Walker B motif in NBD-1 and the 8th putative transmembrane segment (TM-8), respectively (Fig. 1A). Login to comment
91 When E292V, E690K, and T1114M mutant ABCA3-GFP proteins were transiently expressed in HEK-293 cells, most of the GFP fluorescence was located at intracellular vesicles, as with the wild-type protein (Fig. 1B). Login to comment
95 In E690K mutant protein, the amount of the 180-kDa cleaved form was increased compared with that of wild-type protein. Login to comment
97 In the E292V, E690K, and T1114M mutant proteins, 50-60% of the 220-kDa protein remained as Endo H-insensitive complex-type protein (Fig. 1E), indicating that intracellular trafficking and processing of oligosaccharide of these mutant proteins are largely preserved and that these mutations are not type I mutations. Login to comment
107 B: intracellular localization of GFP, wild-type ABCA3-GFP protein (WT), and its mutants (E292V, E690K, and T1114M) transiently expressed in human embryonic kidney HEK-293 cells were determined by confocal microscopy. Login to comment
117 The level of LAMP3 in E292V transfectant was comparable to that in wild-type transfectant, whereas those in E690K and T1114M transfectants were lower than in wild-type transfectant (Fig. 2A and Supplemental Fig. 1A). Login to comment
123 The levels of choline-phospholipids were significantly increased 1.38- and 1.13-fold in wild-type and E292V transfectants, respectively, compared with the level in HEK-293 cells, whereas levels in E690K and T1114M transfectants were similar to that in HEK-293 cells (Fig. 2B). Login to comment
131 In E690K transfectant, the distribution of choline-phospholipid content was similar to that in HEK-293 cells. Login to comment
132 In T1114M transfectant, choline-phospholipid content in fractions 2-4 was somewhat higher than that in HEK-293 cells, but the difference was not statistically significant (n ϭ 4, Supplemental Fig. 1C). Login to comment
133 Considered together, these results suggest that although the lipid transport function of the E292V mutant protein is moderate, those of the E690K and T1114M mutant proteins are severely impaired. Login to comment
136 Lipids transport function of wild-type ABCA3-GFP and pILD mutant proteins. A: Immunoblot analysis of the level of ABCA3-GFP, LAMP3, and GRP78 in HEK-293 cells stably expressing WT ABCA3-GFP, E292V, E690K, T1114M, or untransfected HEK-293 cells. Login to comment
152 On the other hand, in the E690K mutant protein, it was increased to 200% of that of wild-type protein (Fig. 3A, lanes 7 and 8, and B). Login to comment
157 However, in E690K mutant protein, photoaffinity labeling of the 220-kDa protein was similar to that of wild type protein regardless of decreased noncleaved form protein, and photoaffinity labeling of the 180-kDa protein was dramatically enhanced even when the increased levels of the cleaved form of the protein were taken into consideration. Login to comment
158 When normalized to the level of ABCA3-GFP proteins (total 220-kDa noncleaved form plus 180-kDa cleaved form), photoaffinity labeling of the E690K mutant protein was increased to 250% of that of wild-type protein. Login to comment
166 A: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4), E292V (lanes 5 and 6), E690K (lanes 7 and 8), T1114M (lanes 9 and 10), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 ␮M 8-azido-[␣-32 P]ATP in the absence (-) or presence (ϩ) of 0.4 mM orthovanadate (Vi) and 3 mM MgCl2 for 10 min at 37°C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Login to comment
171 C: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP, E292V, E690K, T1114M, or untransfected HEK-293 cells was incubated with 40 ␮M 8-azido-[␥-32 P] and 3 mM MgCl2 for 10 min at 0°C. Protein was photoaffinity labeled with UV irradiation, immunoprecipitated with anti-human ABCA3 antibody, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by FLA-5000 (top) and IB using anti-GFP antibody (bottom). Login to comment
181 To clarify this, we substituted Thr1114 with Ser and examined vanadate-induced nucleotide trapping. Login to comment
184 Interaction of E690K mutant ABCA3-GFP protein with nucleotides. Login to comment
185 To investigate the mechanism of enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in E690K mutant protein, we performed the trap- Fig. 4. Login to comment
201 In this procedure, a posthydrolyzed trapped nucleotide should not be detected by vanadate-induced trapping because of hydrolytic loss of [␥-32 P]PO4. Indeed, in the presence of orthovanadate, photoaffinity labeling of wild-type and E690K mutant proteins with 8-azido-[␥-32 P]ATP was barely detectable (Fig. 6A). Login to comment
202 These data combined with vanadate-induced trapping using 8-azido- [␣-32 P]ATP indicate that E690K mutant protein hydrolyzes 8-azido-[␣-32 P]ATP and releases ␥-phosphate and that the nucleotide trapped by E690K mutant protein is mostly in the ADP form. Login to comment
203 In E690K mutant protein, ATP binding determined by photoaffinity labeling with 8-azido-[␥-32 P]ATP was dramatically increased compared with that of wild-type protein (see Fig. 3, C and D). Login to comment
204 To determine whether the E690K mutation alters ADP binding, we performed labeling experiments using 8-azido-[␣-32 P]ADP at 0°C. Login to comment
205 In this condition, photoaffinity labeling of E690K mutant protein was comparable to that of wild-type ABCA3-GFP protein (Fig. 6, B and C), suggesting that ␥-phosphate of 8-azido-[␥-32 P]ATP contributes to enhanced photoaffinity labeling in E690K mutant protein. Login to comment
206 Since some mutations in the Glu residues following the Walker B motif have been reported to interfere with the ATP-hydrolysis cycle including the ADP release step (5, 24, 25, 33), we examined release of trapped nucleotides from wild-type and E690K mutant ABCA3-GFP proteins. A 20,000-g membrane fraction was first incubated for 10 min at 37°C with 8-azido-[␣-32 P]ATP in the presence of orthovanadate, and unbound nucleotides were removed by washing. Login to comment
209 In contrast, in E690K mutant protein, trapped nucleotides were more slowly released than in wild-type protein, and photoaffinity labeling at 5 min was 73% of that at 0 min, suggesting that E690K mutant protein forms a more stable inhibitory intermediate after hydrolysis of 8-azido-[␣-32 P]ATP than wild-type protein. Login to comment
210 Thus these data further suggest abnormal interaction with nucleotides in E690K mutant protein. Login to comment
212 To further clarify the enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in E690K mutant, we performed mutational analyses of the Glu690 residue adjacent to the Walker B motif in NBD-1. Login to comment
213 Because Glu and Lys are negatively and positively charged amino acids, respectively, alteration of charge at the 690th amino acid residue could well be responsible for the abnormal interaction with nucleotides in the E690K mutant. Login to comment
214 Accordingly, Glu690 was substituted with Asp and Arg, which are negatively and positively charged, respectively. Login to comment
216 Interaction of E690K mutant ABCA3-GFP protein with nucleotides. Login to comment
217 A: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4) E690K (lanes 5 and 6), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 ␮M 8-azido-[␥-32 P] ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37°C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by autoradiography (top) and IB using anti-GFP antibody (bottom). Login to comment
218 B: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP, E690K, or untransfected HEK-293 cells was incubated with 40 ␮M 8-azido-[␣-32 P]ADP and 3 mM MgCl2 for 10 min at 0°C. Protein was photoaffinity labeled with UV irradiation, immunoprecipitated with anti-human ABCA3 antibody, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by FLA-5000 (top) and IB using anti-GFP antibody (bottom). Login to comment
221 D: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP or E690K was first incubated for 10 min at 37°C with 10 ␮M 8-azido-[␣-32 P]ATP in the presence of orthovanadate, and unbound nucleotides were removed by washing. Login to comment
228 These results show that both negative charge and side chain length of Glu690 are important for ATP hydrolysis of wild-type ABCA3 protein and that not only positive charge but also side chain length of Lys690 contribute to enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in the E690K mutant protein. Login to comment
230 Although E292V, E690K, and T1114M mutant proteins were found to traffic to intracellular vesicles, the lipid transport function of E292V mutant protein was partially impaired, and those of E690K and T1114M mutant protein were severely impaired, accompanied by an aberrant catalytic cycle. Login to comment
232 Accordingly, E292V, E690K, and T1114M are type II mutations. Login to comment
234 On the other hand, patients carrying a type II/type II ABCA3 mutation (E292V/T1114M or E292V/E690K) exhibit pILD (4), suggesting that the type II/type II ABCA3 mutation produces a milder phenotype. Login to comment
245 In E690K mutant Fig. 7. Login to comment
247 A: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4), E690K (lanes 5 and 6), E690D (lanes 7 and 8), E690R (lanes 9 and 10), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 ␮M 8-azido-[␣-32 P]ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37°C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by autoradiography (top) and IB using anti-GFP antibody (bottom). Login to comment
252 Genotype-phenotype correlation for ABCA3 mutation ABCA3 Mutation Age of Symptoms Phenotype Ref. W1142X W1142X Neonate FSD 27 L101P L101P Neonate FSD 27 L1553P L1553P Neonate FSD 27 Ins1518 L1580P Neonate FSD 27 L982P G1221S Neonate FSD 27 E292V T1114M Neonate pILD 4 E292V E690K 5 or 7 yr pILD 4 W1148X T1114A 12 mo pILD 37 Type I and type II ATP binding cassette A3 (ABCA3) mutations are shown in italics and roman, respectively. Login to comment
255 Furthermore, mutational analysis of Glu690 indicates that side chain length of Lys690 contributes to enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in the E690K mutant protein. Login to comment
256 To clarify the origin of the abnormal interaction with nucleotides in E690K mutant protein, we modeled the structure of NBD-1 of ABCA3 based on the crystal structure of E. coli MalK using SWISS-MODEL (Supplemental Fig. 2). Login to comment
258 On the other hand, in the model of E690K mutant, the distance from side chain nitrogen of Lys690 to ␥-phosphate oxygen of ATP is ϳ3.6 Å. Login to comment
259 One possible interpretation of these biochemical results and modeling is that ionic interaction of Lys690 and ␥-phosphate of ATP in the E690K mutant protein may tighten the binding of ATP in NBD-1, resulting in delayed ADP release after ATP hydrolysis, probably in NBD-2. Login to comment
261 Analysis using purified ABCA3 protein would further clarify the aberrant catalytic cycle and impaired lipid transport in E690K mutant protein. Login to comment
265 Interestingly, in E690K mutant protein, the amount of 180-kDa cleaved form was increased compared with that of wild-type protein. Login to comment
266 The increased level of 180-kDa cleaved form E690K mutant protein might be due to the abnormal nucleotide-bound conformation of E690K mutant protein being preferentially cleaved by enzymes within intracellular vesicles, compared with that in wild-type protein. Login to comment
7 E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Login to comment
8 Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Login to comment
19 On the other hand, patients with the common missense mutation E292V and a second, specific mutation such as E690K or T1114M develop pediatric interstitial lung disease (pILD), the phenotype of which is milder than that of fatal surfactant deficiency, suggesting that the E292V ABCA3 mutation is responsible for the development of pILD (4). Login to comment
21 In this study, we characterized E292V, E690K, and T1114M mutant ABCA3 proteins identified in pILD. Login to comment
86 The E292V, E690K, and T1114M mutations identified in pILD (4) are located at intracellular loop 1 (ICL-1), adjacent to the Walker B motif in NBD-1 and the 8th putative transmembrane segment (TM-8), respectively (Fig. 1A). Login to comment
87 When E292V, E690K, and T1114M mutant ABCA3-GFP proteins were transiently expressed in HEK-293 cells, most of the GFP fluorescence was located at intracellular vesicles, as with the wild-type protein (Fig. 1B). Login to comment
93 In the E292V, E690K, and T1114M mutant proteins, 50-60% of the 220-kDa protein remained as Endo H-insensitive complex-type protein (Fig. 1E), indicating that intracellular trafficking and processing of oligosaccharide of these mutant proteins are largely preserved and that these mutations are not type I mutations. Login to comment
103 B: intracellular localization of GFP, wild-type ABCA3-GFP protein (WT), and its mutants (E292V, E690K, and T1114M) transiently expressed in human embryonic kidney HEK-293 cells were determined by confocal microscopy. Login to comment
113 The level of LAMP3 in E292V transfectant was comparable to that in wild-type transfectant, whereas those in E690K and T1114M transfectants were lower than in wild-type transfectant (Fig. 2A and Supplemental Fig. 1A). Login to comment
119 The levels of choline-phospholipids were significantly increased 1.38- and 1.13-fold in wild-type and E292V transfectants, respectively, compared with the level in HEK-293 cells, whereas levels in E690K and T1114M transfectants were similar to that in HEK-293 cells (Fig. 2B). Login to comment
127 In E690K transfectant, the distribution of choline-phospholipid content was similar to that in HEK293 cells. Login to comment
129 Considered together, these results suggest that although the lipid transport function of the E292V mutant protein is moderate, those of the E690K and T1114M mutant proteins are severely impaired. Login to comment
148 On the other hand, in the E690K mutant protein, it was increased to 200% of that of wild-type protein (Fig. 3A, lanes 7 and 8, and B). Login to comment
153 However, in E690K mutant protein, photoaffinity labeling of the 220-kDa protein was similar to that of wild type protein regardless of decreased noncleaved form protein, and photoaffinity labeling of the 180-kDa protein was dramatically enhanced even when the increased levels of the cleaved form of the protein were taken into consideration. Login to comment
154 When normalized to the level of ABCA3-GFP proteins (total 220-kDa noncleaved form plus 180-kDa cleaved form), photoaffinity labeling of the E690K mutant protein was increased to 250% of that of wild-type protein. Login to comment
162 A: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4), E292V (lanes 5 and 6), E690K (lanes 7 and 8), T1114M (lanes 9 and 10), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 òe;M 8-azido-[ॷ-32 P]ATP in the absence (afa;) or presence (af9;) of 0.4 mM orthovanadate (Vi) and 3 mM MgCl2 for 10 min at 37&#b0;C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Login to comment
167 C: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP, E292V, E690K, T1114M, or untransfected HEK-293 cells was incubated with 40 òe;M 8-azido-[ॹ-32 P] and 3 mM MgCl2 for 10 min at 0&#b0;C. Protein was photoaffinity labeled with UV irradiation, immunoprecipitated with anti-human ABCA3 antibody, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by FLA-5000 (top) and IB using anti-GFP antibody (bottom). Login to comment
180 Interaction of E690K mutant ABCA3-GFP protein with nucleotides. Login to comment
198 Indeed, in the presence of orthovanadate, photoaffinity labeling of wild-type and E690K mutant proteins with 8-azido-[ॹ-32 P]ATP was barely detectable (Fig. 6A). Login to comment
199 These data combined with vanadate-induced trapping using 8-azido- [ॷ-32 P]ATP indicate that E690K mutant protein hydrolyzes 8-azido-[ॷ-32 P]ATP and releases ॹ-phosphate and that the nucleotide trapped by E690K mutant protein is mostly in the ADP form. Login to comment
200 In E690K mutant protein, ATP binding determined by photoaffinity labeling with 8-azido-[ॹ-32 P]ATP was dramatically increased compared with that of wild-type protein (see Fig. 3, C and D). Login to comment
207 Thus these data further suggest abnormal interaction with nucleotides in E690K mutant protein. Login to comment
215 B: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP, E690K, or untransfected HEK-293 cells was incubated with 40 òe;M 8-azido-[ॷ-32 P]ADP and 3 mM MgCl2 for 10 min at 0&#b0;C. Protein was photoaffinity labeled with UV irradiation, immunoprecipitated with anti-human ABCA3 antibody, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by FLA-5000 (top) and IB using anti-GFP antibody (bottom). Login to comment
225 These results show that both negative charge and side chain length of Glu690 are important for ATP hydrolysis of wild-type ABCA3 protein and that not only positive charge but also side chain length of Lys690 contribute to enhanced production of a photoaffinity-labeled intermediate during ATP hydrolysis in the E690K mutant protein. Login to comment
227 Although E292V, E690K, and T1114M mutant proteins were found to traffic to intracellular vesicles, the lipid transport function of E292V mutant protein was partially impaired, and those of E690K and T1114M mutant protein were severely impaired, accompanied by an aberrant catalytic cycle. Login to comment
229 Accordingly, E292V, E690K, and T1114M are type II mutations. Login to comment
231 On the other hand, patients carrying a type II/type II ABCA3 mutation (E292V/T1114M or E292V/E690K) exhibit pILD (4), suggesting that the type II/type II ABCA3 mutation produces a milder phenotype. Login to comment
242 In E690K mutant Fig. 7. Login to comment
244 A: 20,000-g membrane fraction prepared from HEK-293 cells stably expressing the WT ABCA3-GFP (lanes 3 and 4), E690K (lanes 5 and 6), E690D (lanes 7 and 8), E690R (lanes 9 and 10), or untransfected HEK-293 cells (lanes 1 and 2) was incubated with 10 òe;M 8-azido-[ॷ-32 P]ATP in the absence or presence of 0.4 mM Vi and 3 mM MgCl2 for 10 min at 37&#b0;C. Protein was photoaffinity labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE, and transferred to a PVDF membrane. Membrane was analyzed by autoradiography (top) and IB using anti-GFP antibody (bottom). Login to comment
249 Genotype-phenotype correlation for ABCA3 mutation ABCA3 Mutation Age of Symptoms Phenotype Ref. W1142X W1142X Neonate FSD 27 L101P L101P Neonate FSD 27 L1553P L1553P Neonate FSD 27 Ins1518 L1580P Neonate FSD 27 L982P G1221S Neonate FSD 27 E292V T1114M Neonate pILD 4 E292V E690K 5 or 7 yr pILD 4 W1148X T1114A 12 mo pILD 37 Type I and type II ATP binding cassette A3 (ABCA3) mutations are shown in italics and roman, respectively. Login to comment
253 To clarify the origin of the abnormal interaction with nucleotides in E690K mutant protein, we modeled the structure of NBD-1 of ABCA3 based on the crystal structure of E. coli MalK using SWISS-MODEL (Supplemental Fig. 2). Login to comment
262 Interestingly, in E690K mutant protein, the amount of 180-kDa cleaved form was increased compared with that of wild-type protein. Login to comment
263 The increased level of 180-kDa cleaved form E690K mutant protein might be due to the abnormal nucleotide-bound conformation of E690K mutant protein being preferentially cleaved by enzymes within intracellular vesicles, compared with that in wild-type protein. Login to comment

[hide] ABCA3 mutations associated with pediatric intersti... Am J Respir Crit Care Med. 2005 Oct 15;172(8):1026-31. Epub 2005 Jun 23. Bullard JE, Wert SE, Whitsett JA, Dean M, Nogee LM
ABCA3 mutations associated with pediatric interstitial lung disease.
Am J Respir Crit Care Med. 2005 Oct 15;172(8):1026-31. Epub 2005 Jun 23., [PMID:15976379]

Abstract [show]
RATIONALE: ABCA3 is a member of the ATP-binding cassette family of proteins that mediate the translocation of a wide variety of substrates, including lipids, across cellular membranes. Mutations in the gene encoding ABCA3 were recently identified in full-term neonates with fatal surfactant deficiency. OBJECTIVE: To test the hypothesis that ABCA3 mutations are not always associated with fatal neonatal lung disease but are a cause of pediatric interstitial lung disease. METHODS: DNA samples were obtained from 195 children with chronic lung disease of unknown etiology. The 30 coding exons of the ABCA3 gene were sequenced in four unrelated children with a referring diagnosis of desquamative interstitial pneumonitis and who were older than 10 years at the time of enrollment. RESULTS: Three of four patients (ages 16, 23, and 11 years) with desquamative interstitial pneumonitis had ABCA3 mutations identified on both alleles. All three had the same missense mutation (E292V) and a second unique mutation. The E292V mutation was not found on 200 control alleles from adults without lung disease, but seven additional patients of the remaining study patients had the E292V mutation on one allele. Immunohistochemical analysis of surfactant protein expression in three patients revealed a specific staining pattern for surfactant protein-B, which was the same pattern observed in several infants with fatal lung disease due to ABCA3 mutations. CONCLUSION: ABCA3 mutations cause some types of interstitial lung disease in pediatric patients.

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105 In addition to the E292V mutation, five additional missense variants (N1076K, G1302E, P1301L, T1114M, E690K) and three splice junction site mutations (c3704-1 GϾT, c1742-9 GϾA, c1612-2 AϾG) that would likely alter RNA splicing were identified. Login to comment
121 Some of this variability may reflect different stages of the disease and/or patchy distribution of disease within the lung, as well as TABLE 2. CHARACTERISTICS OF CHILDREN WITH CHRONIC LUNG DISEASE WHO SCREENED POSITIVE FOR E292V MUTATION Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Race White White White White White Hispanic White Age of symptoms Neonate Neonate Neonate 7 yr 5 yr Neonate Neonate Sex Female Female Male Male Male Male Male Lung biopsy Not done Not done Not done IP/PF Not done BO/fibrosis Normal Current age NA 11 yr 11 yr 11 yr 9 yr NA 6 yr Family history Yes No Yes Yes Yes No No Outcome Died at 11 yr Alive Alive Alive Alive Died at 6 mo Alive ABCA3 mutations E292V/c1612-2 E292V/G1302E E292V/T1114M E292V/E690K E292V/E690K E292V/none E292V/none AϾG/P1301L identified identified Definition of abbreviations: BO ϭ bronchiolitis obliterans; IP ϭ interstitial pneumonitis; NA ϭ not available; PF ϭ pulmonary fibrosis. Login to comment
124 However, both siblings with the E292V/E690K mutations reportedly did not develop symptoms of lung disease until 5 to 7 years of age. Login to comment

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

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

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

[hide] Structural Features of the ATP-Binding Cassette (A... Int J Mol Sci. 2015 Aug 19;16(8):19631-44. doi: 10.3390/ijms160819631. Paolini A, Baldassarre A, Del Gaudio I, Masotti A
Structural Features of the ATP-Binding Cassette (ABC) Transporter ABCA3.
Int J Mol Sci. 2015 Aug 19;16(8):19631-44. doi: 10.3390/ijms160819631., [PMID:26295388]

Abstract [show]
In this review we reported and discussed the structural features of the ATP-Binding Cassette (ABC) transporter ABCA3 and how the use of bioinformatics tools could help researchers to obtain a reliable structural model of this important transporter. In fact, a model of ABCA3 is still lacking and no crystallographic structures (of the transporter or of its orthologues) are available. With the advent of next generation sequencing, many disease-causing mutations have been discovered and many more will be found in the future. In the last few years, ABCA3 mutations have been reported to have important pediatric implications. Thus, clinicians need a reliable structure to locate relevant mutations of this transporter and make genotype/phenotype correlations of patients affected by ABCA3-related diseases. In conclusion, we strongly believe that the model preliminarily generated by these novel bioinformatics tools could be the starting point to obtain more refined models of the ABCA3 transporter.

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111 In particular, we focused on the following mutations: p.E292V, p.E690K, p.R1333G, p.W1142X, p.Y1515X, and p.L1553P. Login to comment
114 p.E690K reside in the NBD1, whereas p.Y1515X and p.L1553P are localized in the NBD2. Login to comment