ABCMdb

ABCA3 p.Asn568Asp

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Publications

PMID: 21586796 [PubMed] Beers MF et al: "A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments."

No. Sentence Comment

167 Most prominent of these was L101P, which, when transfected into similar cell lines, produced profound ER retention. Login to comment
168 In addition to this trafficking mutant, another mutant, N568D, was in fact trafficked to lysosomes but failed to concentrate NBD-phosphaty- dilcholine in this compartment. Login to comment

PMID: 15044640 [PubMed] Shulenin S et al: "ABCA3 gene mutations in newborns with fatal surfactant deficiency."

No. Sentence Comment

44 Family History/ Consanguinity Outcome Histologic Findings ABCA3 Mutation 1 White F 1 Yes/Yes Death within 3 mo after birth DIP, PAP W1142X/W1142X 2 White F 1 Yes/Yes Death during neonatal period DIP, PAP W1142X/W1142X 3 Black M 2 Yes/Yes Death during neonatal period NA L101P/L101P 4 Black M 2 Yes/Yes Death during neonatal period NA L101P/L101P 5 White F 3 Yes/No Death during neonatal period NA 4552insT/L1580P 6 White F 3 Yes/No Death during neonatal period NA 4552insT/L1580P 7 White M 4 Yes/No Death within 3 mo after birth PAP G1221S/L982P 8 White M 4 Yes/No Death during neonatal period PAP G1221S/L982P 9 Middle Eastern M 5 Yes/Yes Death during neonatal period DIP, PAP L1553P/L1553P 10 Middle Eastern M 5 Yes/Yes Death during neonatal period NA L1553P/L1553P 11 White M 6 Yes/No Recovery from RDS NA None found 12 White M 6 Yes/No Recovery from RDS NA None found 13 Middle Eastern M 7 No/Yes Unknown NA 1644delC/1644delC 14 Middle Eastern M 8 Yes/No Death during neonatal period DIP, PAP R106X/R106X 15 Asian F 9† Yes/Yes Death during neonatal period NA 4909+1G>A/4909+1G>A 16 White M 10 Yes/Yes Death during neonatal period NA None found 17 White M 11 No/No Recovery from RDS NA None found 18 White F 12 No/No Death during neonatal period NA None found 19 White M 13 Yes/No Chronic lung disease CPI, DIP Q1591P/-‡ 20 Hispanic M 14 No/No Death after lung transplantation PAP N568D/-‡ 21 Asian F 9† Yes/Yes Death during neonatal period PAP 4909+1G>A/4909+1G>A entorganisms,weusedthededucedaminoacidse- quence of ABCA3 (GenBank accession number NP_001080) to search the sequence data base using the BLAST program (http://www.ncbi.nlm.nih. Login to comment
59 Seven missense muta- tionswereidentifiedinconservedaminoacids(Fig. 2), including homozygous substitutions of proline for leucine in codons 101 and 1553 (L101P and L1553P, respectively) and heterozygous substitutions of aspartic acid for asparagine at position 568 (N568D), proline for leucine at position 982 (L982P), serine for glycine at position 1221 (G1221S), proline for leucine at position 1580 (L1580P), and proline for glutamine at position 1591 (Q1591P). Login to comment
87 Human Mouse Rat Puffer fish ETVRRALVIN ETVKREFMIK EAVRREFMIK EDVRGKLELS QDVQQNLVRG L101P NGAGKTT NGAGKTT NGAGKTT NGAGKTT NGAGKTT N568D VARRLL VARRLL VARRLL VARRLL L1553P Q1591P T301C(L101P) C316T(R106X) A1702G(N568D) 1644delC G3426A(W1142X) G3661A(G1221S) T4657C(L1553P) 4552insT A4771C(Q1591P) 4909+1G>A G1221S LSGIAT LSGIAT LSGIAT ATP-binding domains L982P QQLSEHL QQLSENL QQLSEHL T2945C(L982P) ECEALC LAIMVQGQFKC ECEALC ECEALC L1580P T4739C(L1580P) Nonsense MissenseSpliceFrameshift LAIMVQGQFKC LAIMVQGQFKC LAVMVNGQFKC Zebra fish LAVMVNGQFKC tified occur in residues that are highly conserved (Fig.2).Theaminoacidalignmentwasusedtopro- duce a phylogenetic tree of the ABCA3-related proteins showing the relation of the proteins from different organisms (see Supplementary Appendix 2, available with the full text of this article at www. nejm.org).ThefishABCA3proteinsclusterwiththe mammalian ABCA3 proteins and are distinct from other, more distant ABCA-family proteins, such as the mouse Abca14, Abca15, and Abca16 proteins and the sea-urchin ABCA proteins (see Supplementary Appendix 2. Login to comment
97 In the case of the L101P and L1553P mutations, each of which affected one pair of siblings from two different families, the two pairs of siblings were both homozygous for the variant and homozygous for all other polymorphisms that we found in the gene - findings that are consistent with the occurrence of a recessive mutation in these consanguineous families. Login to comment
98 The N568D mutation is in a highly conserved residue in the ATP-binding domain, and it almost certainly disrupts the function of the protein. Login to comment
106 Nucleotide Affected* Site Affected or Outcome SNP No.† Mutation Exon 5 3, 4 T301C L101P‡ Exon 5 14 C316T R106X‡ Exon 14 20 A1702G N568D Exon 14 13 1644delC Frameshift‡ Exon 21 7, 8 T2945C L982P Exon 23 1, 2 G3426A W1142X‡ Exon 24 7, 8 G3661A G1221S Exon 30 9, 10 T4657C L1553P Exon 30 5, 6 4552insT Frameshift Exon 31 15, 21 4909+1G>A Splice site‡ Exon 31 5, 6 T4739C L1580P Exon 31 19 A4772C Q1591P Polymorphism Exon 5 Multiple Exon 5+50A/G Intron rs46725 Exon 6 20 393C/T A131A Exon 6 18 Exon 6+119G/A Intron rs323059 Exon 7 19 Exon 7-14C/G Intron Exon 8 14 681C/T A227A Exon 10 Multiple Exon 10-105C/A Intron rs323066 Exon 10 Multiple Exon 10-20C/T Intron Exon 10 Multiple 1058C/T F353F Exon 14 Multiple Exon 14+33G/A Intron rs170447 Exon 15 Multiple 1755C/G P585P rs323043 Exon 18 13 Exon 18-17G/A Intron Exon 18 1 2340C/T H780H Exon 21 Multiple Exon 21-20C/G Intron rs313908 Exon 21 Multiple Exon 21+34C/T Intron rs313909 Exon 27 Multiple 4116C/T S1372S rs149532 Exon 32 11 4944C/T V1648V In two patients, a mutation was identified on only one allele. Login to comment
43 Family History/ Consanguinity Outcome Histologic Findings ABCA3 Mutation 1 White F 1 Yes/Yes Death within 3 mo after birth DIP, PAP W1142X/W1142X 2 White F 1 Yes/Yes Death during neonatal period DIP, PAP W1142X/W1142X 3 Black M 2 Yes/Yes Death during neonatal period NA L101P/L101P 4 Black M 2 Yes/Yes Death during neonatal period NA L101P/L101P 5 White F 3 Yes/No Death during neonatal period NA 4552insT/L1580P 6 White F 3 Yes/No Death during neonatal period NA 4552insT/L1580P 7 White M 4 Yes/No Death within 3 mo after birth PAP G1221S/L982P 8 White M 4 Yes/No Death during neonatal period PAP G1221S/L982P 9 Middle Eastern M 5 Yes/Yes Death during neonatal period DIP, PAP L1553P/L1553P 10 Middle Eastern M 5 Yes/Yes Death during neonatal period NA L1553P/L1553P 11 White M 6 Yes/No Recovery from RDS NA None found 12 White M 6 Yes/No Recovery from RDS NA None found 13 Middle Eastern M 7 No/Yes Unknown NA 1644delC/1644delC 14 Middle Eastern M 8 Yes/No Death during neonatal period DIP, PAP R106X/R106X 15 Asian F 9ߤ Yes/Yes Death during neonatal period NA 4909+1G>A/4909+1G>A 16 White M 10 Yes/Yes Death during neonatal period NA None found 17 White M 11 No/No Recovery from RDS NA None found 18 White F 12 No/No Death during neonatal period NA None found 19 White M 13 Yes/No Chronic lung disease CPI, DIP Q1591P/-ߥ 20 Hispanic M 14 No/No Death after lung transplantation PAP N568D/-ߥ 21 Asian F 9ߤ Yes/Yes Death during neonatal period PAP 4909+1G>A/4909+1G>A entorganisms,weusedthededucedaminoacidse- quence of ABCA3 (GenBank accession number NP_001080) to search the sequence data base using the BLAST program (http://www.ncbi.nlm.nih. Login to comment
58 Seven missense muta- tionswereidentifiedinconservedaminoacids(Fig. 2), including homozygous substitutions of proline for leucine in codons 101 and 1553 (L101P and L1553P, respectively) and heterozygous substitutions of aspartic acid for asparagine at position 568 (N568D), proline for leucine at position 982 (L982P), serine for glycine at position 1221 (G1221S), proline for leucine at position 1580 (L1580P), and proline for glutamine at position 1591 (Q1591P). Login to comment
86 Human Mouse Rat Puffer fish ETVRRALVIN ETVKREFMIK EAVRREFMIK EDVRGKLELS QDVQQNLVRG L101P NGAGKTT NGAGKTT NGAGKTT NGAGKTT NGAGKTT N568D VARRLL VARRLL VARRLL VARRLL L1553P Q1591P T301C(L101P) C316T(R106X) A1702G(N568D) 1644delC G3426A(W1142X) G3661A(G1221S) T4657C(L1553P) 4552insT A4771C(Q1591P) 4909+1G>A G1221S LSGIAT LSGIAT LSGIAT ATP-binding domains L982P QQLSEHL QQLSENL QQLSEHL T2945C(L982P) ECEALC LAIMVQGQFKC ECEALC ECEALC L1580P T4739C(L1580P) Nonsense Missense Splice Frameshift LAIMVQGQFKC LAIMVQGQFKC LAVMVNGQFKC Zebra fish LAVMVNGQFKC tified occur in residues that are highly conserved (Fig.2).Theaminoacidalignmentwasusedtopro- duce a phylogenetic tree of the ABCA3-related proteins showing the relation of the proteins from different organisms (see Supplementary Appendix 2, available with the full text of this article at www. nejm.org).ThefishABCA3proteinsclusterwiththe mammalian ABCA3 proteins and are distinct from other, more distant ABCA-family proteins, such as the mouse Abca14, Abca15, and Abca16 proteins and the sea-urchin ABCA proteins (see Supplementary Appendix 2. Login to comment
105 Nucleotide Affected* Site Affected or Outcome SNP No.ߤ Mutation Exon 5 3, 4 T301C L101Pߥ Exon 5 14 C316T R106Xߥ Exon 14 20 A1702G N568D Exon 14 13 1644delC Frameshiftߥ Exon 21 7, 8 T2945C L982P Exon 23 1, 2 G3426A W1142Xߥ Exon 24 7, 8 G3661A G1221S Exon 30 9, 10 T4657C L1553P Exon 30 5, 6 4552insT Frameshift Exon 31 15, 21 4909+1G>A Splice siteߥ Exon 31 5, 6 T4739C L1580P Exon 31 19 A4772C Q1591P Polymorphism Exon 5 Multiple Exon 5+50A/G Intron rs46725 Exon 6 20 393C/T A131A Exon 6 18 Exon 6+119G/A Intron rs323059 Exon 7 19 Exon 7-14C/G Intron Exon 8 14 681C/T A227A Exon 10 Multiple Exon 10-105C/A Intron rs323066 Exon 10 Multiple Exon 10-20C/T Intron Exon 10 Multiple 1058C/T F353F Exon 14 Multiple Exon 14+33G/A Intron rs170447 Exon 15 Multiple 1755C/G P585P rs323043 Exon 18 13 Exon 18-17G/A Intron Exon 18 1 2340C/T H780H Exon 21 Multiple Exon 21-20C/G Intron rs313908 Exon 21 Multiple Exon 21+34C/T Intron rs313909 Exon 27 Multiple 4116C/T S1372S rs149532 Exon 32 11 4944C/T V1648V In two patients, a mutation was identified on only one allele. Login to comment

PMID: 21873242 [PubMed] Aung T et al: "Exosomal evasion of humoral immunotherapy in aggressive B-cell lymphoma modulated by ATP-binding cassette transporter A3."

No. Sentence Comment

96 As a control, enforced expression of a nonfunctional ABCA3 mutated in the ATP-binding site (N568D) was not sufficient to induce such effects (Fig. 6 D-F) (16). Login to comment
163 To analyze ABCA3 function in exosome release, both silencing of ABCA3 with two independent shABCA3 constructs (PLKO.1-shRNA.38 and .39) and enforced gene expression with an ABCA3 wild-type plasmid and a nonfunctional mutant (pABCA3 N568D) were applied. Login to comment
92 As a control, enforced expression of a nonfunctional ABCA3 mutated in the ATP-binding site (N568D) was not sufficient to induce such effects (Fig. 6 D-F) (16). Login to comment
159 To analyze ABCA3 function in exosome release, both silencing of ABCA3 with two independent shABCA3 constructs (PLKO.1-shRNA.38 and .39) and enforced gene expression with an ABCA3 wild-type plasmid and a nonfunctional mutant (pABCA3 N568D) were applied. Login to comment

PMID: 19861431 [PubMed] Park SK et al: "Identification and characterization of a novel ABCA3 mutation."

No. Sentence Comment

53 Transient transfections of HEK293 cells with wild-type ABCA3-GFP and ABCA3 mutants L101P-GFP, N568D-GFP, and L982P-GFP (14), as well as the new pEGFPN1 construct for R295C-GFP, were performed with FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) as previously described (14). Login to comment
99 Type I mutations include L101P, L982P, L1553P, and Q1591P; type II mutations include E292V, N568D, E690K, T1114, G1221S, and L1580P (13, 14). Login to comment
100 B: alignment of sequences surrounding the R295C mutation in ICL-1 in various members of the ABCA subfamily. Login to comment
105 As reported previously, the N568D variant shows resistance to Endo H (Fig. 3A, lanes 8 and 9) at a level similar to that of the wild-type protein; however, the L101P and L982P variants (Fig. 3A, lanes 4 and 5 and lanes 10 and 11, respectively) show no Endo H resistance, indicating that these mutants have not left the endoplasmic reticulum (14). Login to comment
110 The level of the ABCA3-R295C-GFP mutant protein was comparable to that of wild-type ABCA3-GFP as demonstrated in the anti-GFP immunoblot. Login to comment
111 Vanadate-induced nucleotide trapping was also decreased in the N568D mutant as reported previously (14). Login to comment
114 DISCUSSION The results presented here demonstrate that R295C is a novel mutation that results in severely impaired ATP hydrolysis activity as indicated by the dramatic reduction in vanadate-induced nucleotide trapping. Login to comment
115 Other mutations in the ABCA3 protein also result in impaired ATP hydrolysis, including E292V, N568D, G1221S, L1580P, and T1114M (13, 14). Login to comment
119 A: 20 ␮g of membrane fraction from untransfected HEK293 cells (lanes 1 and 2), HEK293 cells stably expressing WT ABCA3-GFP (lanes 3 and 4), ABCA3-GFP mutants N568D (lanes 5 and 6), and R295C (lanes 7 and 8) were incubated with 20 ␮M 8-azido-[␣-32 P]ATP in the absence (-) or presence (ϩ) of 0.4 mM orthovanadate (Vi) and 3 mM MgCl2 as described under MATERIALS AND METHODS. Login to comment
126 A: 20 ␮g of membrane fraction from HEK293 cells transiently transfected with WT ABCA3-GFP (lanes 2 and 3) or with ABCA3-GFP mutants L101P (lanes 4 and 5), R295C (lanes 6 and 7), N568D (lanes 8 and 9), and L982P (lanes 10 and 11) were treated without (-) or with (ϩ) endoglycosidase H (Endo H) and analyzed by 5% SDS-PAGE followed by immunoblotting with anti-GFP antibody. Lane 1, immunoblotting of untransfected HEK293 cells. Login to comment
127 B: WT ABCA3-GFP (lanes 2 and 3) or ABCA3-GFP mutants L101P (lanes 4 and 5), R295C (lanes 6 and 7), N568D (lanes 8 and 9), and L982P (lanes 10 and 11) were treated without (-) or with (ϩ) peptide N-glycosidase F (PNGase F) and were then analyzed by 5% SDS-PAGE followed by immunoblotting with anti-GFP antibody. Lane 1, immunoblotting of untransfected HEK293 cells. Login to comment
48 Transient transfections of HEK293 cells with wild-type ABCA3-GFP and ABCA3 mutants L101P-GFP, N568D-GFP, and L982P-GFP (14), as well as the new pEGFPN1 construct for R295C-GFP, were performed with FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) as previously described (14). Login to comment
94 Type I mutations include L101P, L982P, L1553P, and Q1591P; type II mutations include E292V, N568D, E690K, T1114, G1221S, and L1580P (13, 14). Login to comment
106 Vanadate-induced nucleotide trapping was also decreased in the N568D mutant as reported previously (14). Login to comment
122 A: 20 òe;g of membrane fraction from HEK293 cells transiently transfected with WT ABCA3-GFP (lanes 2 and 3) or with ABCA3-GFP mutants L101P (lanes 4 and 5), R295C (lanes 6 and 7), N568D (lanes 8 and 9), and L982P (lanes 10 and 11) were treated without (afa;) or with (af9;) endoglycosidase H (Endo H) and analyzed by 5% SDS-PAGE followed by immunoblotting with anti-GFP antibody. Lane 1, immunoblotting of untransfected HEK293 cells. Login to comment
123 B: WT ABCA3-GFP (lanes 2 and 3) or ABCA3-GFP mutants L101P (lanes 4 and 5), R295C (lanes 6 and 7), N568D (lanes 8 and 9), and L982P (lanes 10 and 11) were treated without (afa;) or with (af9;) peptide N-glycosidase F (PNGase F) and were then analyzed by 5% SDS-PAGE followed by immunoblotting with anti-GFP antibody. Lane 1, immunoblotting of untransfected HEK293 cells. Login to comment

PMID: 19880777 [PubMed] Chapuy B et al: "ABC transporter A3 facilitates lysosomal sequestration of imatinib and modulates susceptibility of chronic myeloid leukemia cell lines to this drug."

No. Sentence Comment

32 Enforced expression and small interfering RNA of ABCA3 pEGFP-N1-ABCA3 wild-type or pEGFP-N1-ABCA3 N568D mutant plasmids16 as well as the pre-designed short interfering RNA (siRNA) against ABCA3 and scrambled siRNA (Qiagen) were delivered by electroporation as previously described.15 Using siRNA knock-down, the nadir of ABCA3 mRNA and ABCA3 protein expression was documented 72 h after electroporation, as tested by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting (Online Supplementary Appendix Figures S2). Login to comment
91 normal BM non-SP SP Hoechst red Hoechstblu ABCA3abundance [foldofnon-SP] CML normal CD34+ CD34- CD34+ CD34- CML normal 0 100 250 500 1000 0 100 250 500 1000 0 100 250 500 10000 1 5 10 0 0.5 2.5 5 0 0.5 2.5 5 scrambled siRNAABCA3 siRNA scrambled siRNA ABCA3 siRNA Imatinib [µM] p-Crkl protein Imatinib [µM] Imatinib [µM] Imatinib[nM] Colonies[fractionofcontroll] Colonies[fractionofcontroll] Viability[fractionofcontroll] K562 K562 K562 LAMA84 LAMA84 BV173 LAMA84 Imatinib [µM] 0 1 5 10 ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3-N568D ABCA3-wt ABCA3-N568D ABCA3-wt 0 100 250 500 1000 0 100 250 500 1000 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 0 250 500 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 n=6 n=19 n=11 p=0.0255 0 1023 01023 ABCA3positive[%ofall] transcriptabundance 80 60 40 20 0 108 107 106 105 104 103 102 101 100 10-1 10-2 10-3 40 30 20 10 0 BC+AP CP BM CD34+ CD34- CML BM A A B C D B D C significantly increased the susceptibility of all three cell lines to the suppressive effects of imatinib. Login to comment
106 B C D A -1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 35 30 25 20 15 10 5 0 35 30 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 β-Hexosaminidase [14 C]-Imatinib [%ofall][%ofall][%ofall] Activity[%ofall] Fraction LAMP1 EEA1 ABCA3-GFP NaKATPase HEK293 + ABCA3 HEK293 LAMA84 K562 Activity[%ofall]Activity[%ofall]Activity[%ofall] [14 C] Imatinib β-Hexosaminidase [14 C] Imatinib β-Hexosaminidase [14 C] Imatinib β-Hexosaminidase [14 C] Imatinib β-Hexosaminidase [%ofall] 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 [14 C]-Imatinib [14 C]-Imatinib Free Membrane bound Cytosol/Organelles Nucleus LL+ LL+ 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 LAMA84 K562 HEK293-ABCA3 HEK293 HEK293-ABCA3 HEK293 HEK293-ABCA3 HEK293 with ectopic expression of wild-type ABCA3 and cells expressing the non-functional ABCA3-N568D variant carrying a mutation at the Walker A motif of the transporter (Figure 2C). Login to comment
92 normal BM non-SP SP Hoechst red Hoechst blu ABCA3 abundance [fold of non-SP] CML normal CD34+ CD34- CD34+ CD34- CML normal 0 100 250 500 1000 0 100 250 500 1000 0 100 250 500 1000 0 1 5 10 0 0.5 2.5 5 0 0.5 2.5 5 scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA Imatinib [&#b5;M] p-Crkl protein Imatinib [&#b5;M] Imatinib [&#b5;M] Imatinib [nM] Colonies [fraction of controll] Colonies [fraction of controll] Viability [fraction of controll] K562 K562 K562 LAMA84 LAMA84 BV173 LAMA84 Imatinib [&#b5;M] 0 1 5 10 ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3-N568D ABCA3-wt ABCA3-N568D ABCA3-wt 0 100 250 500 1000 0 100 250 500 1000 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 0 250 500 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 n=6 n=19 n=11 p=0.0255 0 1023 0 1023 ABCA3 positive [% of all] transcript abundance 80 60 40 20 0 108 107 106 105 104 103 102 101 100 10-1 10-2 10-3 40 30 20 10 0 BC+AP CP BM CD34 + CD34 - CML BM A A B C D B D C significantly increased the susceptibility of all three cell lines to the suppressive effects of imatinib. Login to comment
107 B C D A -1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 35 30 25 20 15 10 5 0 35 30 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 b2;-Hexosaminidase [14 C]-Imatinib [% of all] [% of all] [% of all] Activity [% of all] Fraction LAMP1 EEA1 ABCA3-GFP NaKATPase HEK293 + ABCA3 HEK293 LAMA84 K562 Activity [% of all] Activity [% of all] Activity [% of all] [14 C] Imatinib b2;-Hexosaminidase [14 C] Imatinib b2;-Hexosaminidase [14 C] Imatinib b2;-Hexosaminidase [14 C] Imatinib b2;-Hexosaminidase [% of all] 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 [14 C]-Imatinib [14 C]-Imatinib Free Membrane bound Cytosol/Organelles Nucleus LL+ LL+ 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 LAMA84 K562 HEK293-ABCA3 HEK293 HEK293-ABCA3 HEK293 HEK293-ABCA3 HEK293 with ectopic expression of wild-type ABCA3 and cells expressing the non-functional ABCA3-N568D variant carrying a mutation at the Walker A motif of the transporter (Figure 2C). Login to comment

PMID: 18463677 [PubMed] Chapuy B et al: "Intracellular ABC transporter A3 confers multidrug resistance in leukemia cells by lysosomal drug sequestration."

No. Sentence Comment

21 The pEGFP-N1-ABCA3 wild-type (wt) and pEGFP-N1-ABCA3 N568D mutant plasmids were described previously.10 The monoclonal mouse antibody to early endosomal antigen 1 (EEA1) was obtained commercially (Transduction Laboratories), the antibodies to lysosomal-associated membrane protein 1 (LAMP1, code H4A3) and LAMP2 (code H4B4) were from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA). Login to comment
62 Oldendorf, Germany) with 10 mg of either pmax-GFP, pEGFP- N1-ABCA3 wt or pEGFP-N1-ABCA3 N568D, pulsed in an EasyjecT (Equi Bio, Middlesex, UK) electroporator (300 V, 1050 mF, 99 O, t: 104 ms) and incubated for 3 h in 400 ml OptiMEM before further expansion with 1.5 ml of complete growth medium overnight. Login to comment
139 A nonfunctional, mutated variant N568D-eGFP of ABCA3 does not induce drug resistance, and specific siRNA against ABCA3 increases the cytotoxic efficacy of DNR, etoposide and Ara-C (Supplementary Figure 2C, D). Login to comment

PMID: 17574245 [PubMed] Matsumura Y et al: "ABCA3-mediated choline-phospholipids uptake into intracellular vesicles in A549 cells."

No. Sentence Comment

21 Establishment of stable cell line A549 cells stably expressing GFP-tagged Walker A motif mutant (N568D) ABCA3 protein (ABCA3-GFP) were established as previously described [10]. Login to comment
54 Results 3.1. Expression of wild-type ABCA3-GFP enlarges LAMP3-positive vesicles in A549 cells We previously established lung adenocarcinoma A549/ABCA3WT cells, which stably express GFP-tagged wild-type Fig. 1. Expression of wild-type and N568D ABCA3-GFP in A549 cells. Login to comment
62 Similarly, GFP-tagged expression plasmid of ABCA3 containing naturally occurring mutation N568D in the Walker A motif, which exhibits impaired ATP-binding and ATP-hydrolysis activities [10], was transfected in A549 cells to generate A549/ABCA3N568D cells. Login to comment
104 Discussion In this study, to clarify the mechanism of lipid transport mediated by ABCA3, GFP-tagged wild-type and N568D mutant ABCA3, which exhibits impaired ATP-binding and ATP-hydrolysis activities [10], were stably expressed in A549 Fig. 3. Login to comment
55 Results 3.1. Expression of wild-type ABCA3-GFP enlarges LAMP3-positive vesicles in A549 cells We previously established lung adenocarcinoma A549/ABCA3WT cells, which stably express GFP-tagged wild-type Fig. 1. Expression of wild-type and N568D ABCA3-GFP in A549 cells. Login to comment
63 Similarly, GFP-tagged expression plasmid of ABCA3 containing naturally occurring mutation N568D in the Walker A motif, which exhibits impaired ATP-binding and ATP-hydrolysis activities [10], was transfected in A549 cells to generate A549/ABCA3N568D cells. Login to comment
105 Discussion In this study, to clarify the mechanism of lipid transport mediated by ABCA3, GFP-tagged wild-type and N568D mutant ABCA3, which exhibits impaired ATP-binding and ATP-hydrolysis activities [10], were stably expressed in A549 Fig. 3. Login to comment

PMID: 16959783 [PubMed] Matsumura Y et al: "Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency."

No. Sentence Comment

3 Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Login to comment
4 Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. Login to comment
35 Partial cDNA fragments containing various fatal surfactant deficiency mutations (L101P, N568D, L982P, G1221S, L1553P, L1580P, Q1591P, W1142X, and Ins1518fs (abbreviation of Ins1518fs/ter1519 in this study), see Fig. 1A), were generated with PCR methods and replaced with the corresponding fragment of pEGFPN1-ABCA3. Login to comment
98 To examine the effect of the mutations found in fatal surfactant deficiency patients on subcellular localization of ABCA3, wild-type and mutant ABCA3-GFP (seven missense mutations L101P, N568D, L982P, G1221S, L1553P, L1580P, and Q1591P, and one nonsense mutation, Ins1518fs) were transiently expressed in HEK293 cells (Fig. 1A). Login to comment
99 The N568D, G1221S, and L1580P mutant proteins were mainly localized to the LAMP3-positive intracellular vesicle membrane (Fig. 2B, panels a-c, d-f, and g-i), similar to wild-type ABCA3 protein (Fig. 2A, panels e-h). Login to comment
102 In another cell line, mouse lung epithelial MLE12 cells, transiently expressed GFP-tagged wild-type and N568D, G1221S, and L1580P mutant proteins were mainly localized at the intracellular vesicle membrane, whereas the L101P, L982P, L1553P, Q1591P, and Ins1518fs mutant proteins were mainly localized to the ER (data not shown), confirming defective intracellular sorting of L101P, L982P, L1553P, Q1591P, and Ins1518fs ABCA3 mutant proteins. Login to comment
105 In the N568D, G1221S, and L1580P mutant proteins, which were mainly localized to the intracellular vesicle membrane, both the 220-kDa noncleaved form and the 180-kDa cleaved form were detected, similar to wild-type protein (Fig. 3A). Login to comment
121 A merged image of panels i-k is shown in panel l. B, HEK293 cells transiently expressing mutant ABCA3-GFP proteins (panels a, d, g, and j) were processed for immunofluorescence labeling of LAMP3 (panels b, e, h, and k): N568D (panels a-c), G1221S (panels d-f), L1580P (panels g-i), and L101P (panels j-l). Login to comment
124 The scale bar represents 5 ␮m. kDa wild-type ABCA3-GFP and the seven missense mutant (L101P, N568D, L982P, G1221S, L1553P, L1580P, and Q1591P) proteins to produce a 210-kDa deglycosylated protein (Fig. 3B). Login to comment
128 In the N568D, G1221S, and L1580P mutant proteins, about 30-40% of the 220-kDa protein remained as Endo H-insensitive complex-type protein (Fig. 3, C and D, band I), indicating that processing of oligosaccharide from high mannose type to complex type is largely preserved in these mutants. Login to comment
131 These results indicate that the N568D, G1221S, and L1580P mutant proteins are mainly localized at the intracellular vesicle membrane accompanied by processing of oligosaccharide from high mannose type to complex type, whereas the four missense mutant (L101P, L982D, L1553P, and Q1591P) and one nonsense mutant (Ins1518fs) proteins remain localized at the ER, with impaired processing of oligosaccharide. Login to comment
132 ATP Hydrolysis of ABCA3-GFP and Mutants-To investigate the mechanism of loss of function of the N568D, G1221S, and L1580P mutant proteins that are trafficked to intracellular vesicles accompanied by processing of sugar chains as is wild-type ABCA3 protein, we examined ATP hydrolysis of wild-type ABCA3-GFP and the mutant proteins. Login to comment
143 Band I shows Endo H-insensitive 220-kDa ABCA3-GFP proteins (wild-type, N568D, L982P, and L1553P) containing complex-type sugar chains. Login to comment
148 *, p Ͻ 0.05; **, p Ͻ 0.005 versus wild type. N.S., not significant. Characterization and Classification of ABCA3 Mutants 34508 expressing wild-type or mutant (N568D, G1221S, and L1580P) ABCA3-GFP fusion proteins were established. Login to comment
152 In the N568D, G1221S, and L1580P mutant proteins, vanadate-induced nucleotide trapping was significantly decreased to 12, 33, and 9% of that of the wild-type protein, respectively (Fig. 4, A, lanes 5-10, and C). Login to comment
157 ATP Binding of ABCA3-GFP and Mutants-To clarify the mechanism of loss of ATP hydrolysis activity of the N568D, G1221S, and L1580P mutant proteins, we examined ATP binding of wild-type ABCA3-GFP and the mutant proteins. Login to comment
162 A, 20,000 ϫ g membrane fraction prepared from HEK293 cells stably expressing the wild-type (Wt) ABCA3-GFP (lanes 3 and 4), N568D (lanes 5 and 6), G1221S (lanes 7 and 8), L1580P (lanes 9 and 10), or untransfected HEK293 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. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ATP, electrophoresed on SDS-PAGE (5%), and transferred to a PVDF membrane. Login to comment
170 A, 20,000 ϫ g membrane fraction prepared from HEK293 cells stably expressing wild-type (Wt) ABCA3-GFP, N568D, G1221S, L101P, or untransfected HEK293 cells was incubated with 20 ␮M 8-azido-[␥-32 P]ATP and 3 mM MgCl2 for 10 min at 0 °C. Proteins were photoaffinity-labeled with UV irradiation, immunoprecipitated using anti-human ABCA3 antibody, electrophoresed on SDS-PAGE (5%), and transferred to a PVDF membrane. Login to comment
175 However, the levels of photoaffinity labeling of N568D and L1580P mutant proteins were moderately decreased to 60 and 54% of that of wild-type protein, respectively. Login to comment
177 These results suggest that decreased ATP binding contributes to impaired ATP hydrolysis in the N568D, L1580P, and L101P mutants but not in the G1221S mutant. Login to comment
226 In contrast, the N568D, G1221S, and L1580P mutant proteins were localized to intracellular vesicle membrane accompanied by processing of oligosaccharide from high mannose type to complex type as found in wild-type ABCA3 protein. Login to comment
227 However, vanadate-induced nucleotide trapping analysis revealed ATP hydrolysis activity to be significantly decreased in N568D, G1221S, and L1580P mutant ABCA3 proteins compared with wild type. Login to comment
236 homozygous type II ABCA3 mutations have not been reported, patients with type I/type II compound heterozygous ABCA3 mutations (L982P/G1221S and Ins1518fs/L1580P) died of surfactant deficiency during the neonatal period, and the lamellar bodies of lung tissue from a patient with L982P/G1221S were reported to be smaller than those from normal lung tissue (12). Login to comment
238 Type II mutations lie in various locations as follows: N568D in the Walker A motif of NBD-1, G1221S in TM-11, and L1580P in NBD-2 (Fig. 1A), and all of these amino acids are conserved in the ABCA subfamily (Fig. 1, B-D). Login to comment
249 Characterization and Classification of ABCA3 Mutants 34512 from the crystal structure of other ABC transporters (40), both ATP binding and ATP hydrolysis activities should be impaired in the N568D mutant protein. Login to comment
271 It is possible that the E292V mutation causes less severe disruption of intracellular trafficking or ATP hydrolysis activity. Login to comment
273 Very recently, Cheong et al. (19) reported that L101P, G1221S, and N568D mutant proteins have the most severe, moderate, and the least severe trafficking and processing defects. Login to comment
274 In this study, the difference in degree of defect between N568D and G1221S mutant proteins is slight, if any. Login to comment
276 With regard to the function of the ABCA3 mutant proteins, their findings indicating impaired co-localization of fluorescence-labeled phosphatidylcholine with ABCA3-positive vesicles in G1221S and N568D may well correlate with our findings indicating impaired ATP hydrolysis activities of these mutants. Login to comment
225 In contrast, the N568D, G1221S, and L1580P mutant proteins were localized to intracellular vesicle membrane accompanied by processing of oligosaccharide from high mannose type to complex type as found in wild-type ABCA3 protein. Login to comment
247 Characterization and Classification of ABCA3 Mutants 34512 from the crystal structure of other ABC transporters (40), both ATP binding and ATP hydrolysis activities should be impaired in the N568D mutant protein. Login to comment
272 In this study, the difference in degree of defect between N568D and G1221S mutant proteins is slight, if any. Login to comment

PMID: 16415354 [PubMed] Cheong N et al: "Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome."

No. Sentence Comment

43 hABCA3 missense mutants (L101P, N568D, and G1221S) were generated using the PCR method with the following primers: L101P (forward primer, 5Ј-AGACAGTGCGCAGGGCACCTGTGATCAACATGCG- AG-3Ј; reverse primer, 5Ј-CTCGCATGTTGATCACAGGTGCCCT- GCGCACTGTCT-3Ј); N568D (forward primer, 5Ј-ATCACCGTCCT- GCTGGGCCACGACGGTGCCGGGAAGAC-3Ј; reverse primer, 5Ј- GTCTTCCCGGCACCGTCGTGGCCCAGCAGGACGGTGAT-3Ј); and G1221S (forward primer, 5Ј-ATCTTCAACATCCTGTCAGCCA- TCGCCACCTTCCTG-3Ј; reverse primer, 5Ј-CAGGAAGGTGGCG- AGGCCTGACAGGATGTTGAAGAT-3Ј), where the mutated nucleotides are underlined. Login to comment
96 The first was in extracellular loop 1 (L101P), the second in the Walker A motif or P-loop (phosphate binding loop) of the N-terminal nucleotide binding domain (N568D), and the third in transmembrane domain 11 (G1221S) of hABCA3. Login to comment
101 The construct containing mutation N568D often localized to the lysosomal membrane (Fig. 2A, g-i) and partially remained in the ER (Fig. 2B, g-i). Login to comment
103 Western blotting with GFP antibody revealed that mutant protein is expressed at a lower overall level than wild-type protein and that wild-type and mutant fusion proteins (L101P, N568D, and G1221S) are processed differently (Fig. 2C). Login to comment
106 Densitometry analysis of a 180/220-kDa ratio of Western blot (Fig. 2C) was 0.85 for the wild-type protein, 0.45 for the N568D mutant, 0.3 for the G1221S mutant, and essentially 0.0 for the L101P mutant. Login to comment
117 LysoTracker Red (A) and ERTracker Red (B) staining of wild-type (a-c) and missense mutations of hABCA3 (L101P (d-f), N568D (g-i), and G1221S (j-l)) linked to GFP in transfected A549 cells. Login to comment
119 The wild-type, L101P, N568D, and G1221S hABCA3-GFP membrane fractions were incubated in the absence (-) or presence (ϩ) of 250 units of PNGase F at 37 °C for 1 h. Login to comment
161 Because N568D and G1221S hABCA3-GFP protein partially localized to the lysosomal membrane, we examined whether those lysosomes would take up NBD-lipid. Login to comment
162 C12-NBD-PC did not colocalize with the N568D hABCA3-DsRed (Fig. 7B, a-c); however, a fraction of C12- NBD-PC colocalized with G1221S hABCA3-DsRed (Fig. 7B, d-f, arrows). Login to comment
197 The (N568D) mutant in the N-terminal P-loop of ABCA3 had the least severe trafficking defect but did not transport NBD-lipid. Login to comment
222 B, A549 cells with N568D (a-c) and G1221S (d-f) hABCA3-DsRed incubated with C12-NBD-PC-labeled liposomes. Login to comment
116 LysoTracker Red (A) and ERTracker Red (B) staining of wild-type (a-c) and missense mutations of hABCA3 (L101P (d-f), N568D (g-i), and G1221S (j-l)) linked to GFP in transfected A549 cells. Login to comment
118 The wild-type, L101P, N568D, and G1221S hABCA3-GFP membrane fractions were incubated in the absence (afa;) or presence (af9;) of 250 units of PNGase F at 37 &#b0;C for 1 h. Login to comment
160 Because N568D and G1221S hABCA3-GFP protein partially localized to the lysosomal membrane, we examined whether those lysosomes would take up NBD-lipid. Login to comment
196 The (N568D) mutant in the N-terminal P-loop of ABCA3 had the least severe trafficking defect but did not transport NBD-lipid. Login to comment
220 B, A549 cells with N568D (a-c) and G1221S (d-f) hABCA3-DsRed incubated with C12-NBD-PC-labeled liposomes. Login to comment

PMID: 24730976 [PubMed] Campo I et al: "A large kindred of pulmonary fibrosis associated with a novel ABCA3 gene variant."

No. Sentence Comment

273 In accordance with these results it was also shown previously that phosphatidylcholine accumulation in cells expressing ABCA3 with the walker A motif mutant N568D was diminished [27]. Login to comment

PMID: 25406294 [PubMed] Chen P et al: "Mutations in the ABCA3 gene are associated with cataract-microcornea syndrome."

No. Sentence Comment

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

PMID: 26186947 [PubMed] Mulugeta S et al: "Lost after translation: insights from pulmonary surfactant for understanding the role of alveolar epithelial dysfunction and cellular quality control in fibrotic lung disease."

No. Sentence Comment

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

PMID: 26295388 [PubMed] Paolini A et al: "Structural Features of the ATP-Binding Cassette (ABC) Transporter ABCA3."

No. Sentence Comment

49 One of these in vitro studies demonstrated that the p.N568D mutation in ABCA3 impairs choline-phospholipids uptake into intracellular vesicles in lung adenocarcinoma A549 cells [25] and primary AT2 cells [26]. Login to comment
50 Moreover, in WT cells` ultrastructural images confirmed the regular formation of lamellar bodies, whereas in the p.N568D mutant the formation of these multilamellar vesicles has not been observed. Login to comment