ABCMdb

ABCA3 p.Gly1221Ser

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Publications

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
77 Electron micrographs of lung tissue from Patient 21 (who was homozygous for the 4909+1G>A mutation) revealed lamellarbodies(Fig.3)thatweresmallerthanthose from control lung tissue, with more densely packed membranes and eccentrically placed, dense inclusion bodies, similar to those previously described in Patients 1 and 2, who were homozygous for the W1142X mutation.17 Similarly abnormal lamellar bodies were observed in lung tissue from Patient 8 (who was heterozygous for the G1221S and L982P mutations) and Patient 9 (who was homozygous for the L1553P mutation). Login to comment
85 The sequences of the puffer fish and the zebra fish are not complete, resulting in some gaps in this information in the case of L982P, G1221S, L1552P, L1553P, and L1580P. 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
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
76 Electron micrographs of lung tissue from Patient 21 (who was homozygous for the 4909+1G>A mutation) revealed lamellarbodies(Fig.3)thatweresmallerthanthose from control lung tissue, with more densely packed membranes and eccentrically placed, dense inclusion bodies, similar to those previously described in Patients 1 and 2, who were homozygous for the W1142X mutation.17 Similarly abnormal lamellar bodies were observed in lung tissue from Patient 8 (who was heterozygous for the G1221S and L982P mutations) and Patient 9 (who was homozygous for the L1553P mutation). Login to comment
84 The sequences of the puffer fish and the zebra fish are not complete, resulting in some gaps in this information in the case of L982P, G1221S, L1552P, L1553P, and L1580P. 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: 19861431 [PubMed] Park SK et al: "Identification and characterization of a novel ABCA3 mutation."

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
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
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
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

PMID: 18676873 [PubMed] Matsumura Y et al: "Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease."

No. Sentence Comment

23 For example, patients with type I homozygous ABCA3 mutations such as W1142X/W1142X or type I/type II compound heterozygous ABCA3 mutations such as L982P/G1221S die of surfactant deficiency within the neonatal period (27). 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
233 Patients with fatal surfactant deficiency carrying a type I homozygous ABCA3 mutation (W1142X/W1142X, L101P/ L101P, or L1553P/L1553P) or a type I/type II compound heterozygous mutation (L982P/G1221S or Ins1518/L1580P) die within the neonatal period (Table 1) (27). 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
18 For example, patients with type I homozygous ABCA3 mutations such as W1142X/W1142X or type I/type II compound heterozygous ABCA3 mutations such as L982P/G1221S die of surfactant deficiency within the neonatal period (27). 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

PMID: 17618459 [PubMed] Yokota T et al: "Heterozygous ABCA3 mutation associated with non-fatal evolution of respiratory distress."

No. Sentence Comment

40 All of the patients reported with fatal surfactant deficiency carrying type I/type II compound heterozygous ABCA3 mutation (L982P/G1221S or Ins1518fs/L1580P) died within the neonatal period [4], and ATP-hydrolysis activity was severely impaired in the case of the G1221S and L1580P mutants [2]. 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
5 In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. 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
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
160 The level of photoaffinity labeling of the G1221S mutant protein FIGURE 4. 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
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
178 ATP Hydrolysis of Site-directed Mutants of Gly-1221 in 11th Transmembrane Segment-Since transmembrane domains and NBDs are suggested to communicate during the ATP hydrolysis cycle (31), local environmental changes in the 11th transmembrane segment (TM-11) resulting from the G1221S mutation might allosterically impair ATP hydrolysis activity of the ABCA3 protein. Login to comment
181 In the G1221T mutant protein, which had hydroxyl-containing amino acids, vanadate-induced nucleotide trapping was decreased to 36% of that of wild-type protein, as also in G1221S mutant protein (Fig. 6, A, lanes 9-12, and B). Login to comment
201 A, 20,000 ϫ g membrane fraction prepared from HEK293 cells stably expressing wild-type (Wt) ABCA3-GFP (lanes 3 and 4), G1221A (lanes 5 and 6), G1221V (lanes 7 and 8), G1221T (lanes 9 and 10), G1221S (lanes 11 and 12), 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 werephotoaffinity-labeledwithUVirradiationafterremovalofunboundATP, electrophoresed on SDS-PAGE (5%), and transferred to a PVDF membrane. 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
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
234 *, p b0d; 0.01 versus wild type. Characterization and Classification of ABCA3 Mutants NOVEMBER 10, 2006ߦVOLUME 281ߦNUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 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
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
46 hABCA3-GFP/HEK293, GFP/HEK293, L101P-hABCA3-GFP/HEK293, and G1221S hABCA3-GFP/HEK293 stable cell lines were selected with 500 ␮g/ml G418 and maintained with 200 ␮g/ml G418 in growth medium. Login to comment
47 When L101P hABCA3-GFP/HEK293 and G1221S hABCA3-GFP/HEK293 cells were at 80% confluence, 1 mM 4-PBA was added and cells incubated for 24 h at 37 °C. 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
100 The construct containing mutation G1221S occasionally localized to lysosomes (Fig. 2A, j-l, arrows) and otherwise remained in the ER (Fig. 2B, j-l). 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
122 investigate whether trafficking of mutant hABCA3 could be restored, 4-PBA was applied to cells expressing the hABCA3-GFP missense mutants L101P hABCA3-GFP, and G1221S hABCA3-GFP and visualized by confocal microscopy. Login to comment
123 The addition of 1 mM 4-PBA to HEK239 cells stably transfected with G1221S hABCA3-GFP and L101P hABCA3-GFP markedly altered GFP localization from the ER to the membranes of punctate vesicles (Fig. 3, A and B, and supplemental Fig. 3), but it did not alter the localization of wild-type hABCA3-GFP (supplemental Fig. 4). Login to comment
124 Vesicles with G1221S hABCA3-GFP (Fig. 3B, b, d, and f)), but not L101P hABCA3-GFP (Fig. 3A, b, d, and f)), stained positively with LysoTracker Red. Login to comment
125 Western blots with GFP antibody showed that total hABCA3-GFP protein was increased in the presence of 4-PBA for both L101P and G1221S hABCA3-GFP (Fig. 3C) but that the ratio of 180/220 kDa protein bands increased only for the G1221S hABCA3-GFP mutant (Fig. 3D), providing further evidence that the lower molecular weight protein form correlates with lysosomal processing. Login to comment
139 4-PBA facilitates lysosomal targeting of G1221S-GFP but not L101P-GFP. Login to comment
140 A, 4-PBA treatment failed to restore L101P ABCA3-GFP trafficking to lysosomes. Login to comment
141 B, 4-PBA treatment facilitates G1221S ABCA3-GFP trafficking to lysosomes. Login to comment
143 Western blots with anti-GFP (top) and anti beta-actin (bottom) antibodies of cell lysates from L101P and G1221S hABCA3-GFP cells in the absence (-) or presence (ϩ) of 1 mM 4-PBA treatment. 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
195 The (G1221S) mutant in transmembrane domain 11 of ABCA3 is somewhat localized to lysosomes and in some of those lysosomes it appeared to transport NBD-PC. Login to comment
205 The ratio of 150- and 180-kDa protein for the G1221S mutant increases with 4-PBA treatment, FIGURE 6. Login to comment
218 Our finding that 4-PBA treatment partially restored trafficking of G1221S hABCA3-GFP to the lysosomal membrane suggests that an approach using chemical or pharmacological chaperones should be explored further as a potential treatment for lung surfactant deficiency caused by ABCA3 mutations (Fig. 3B). 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
121 ABCA3 Role in Lipid Transport and Lamellar Body Biogenesis 9794 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 14ߦAPRIL 7, 2006 investigate whether trafficking of mutant hABCA3 could be restored, 4-PBA was applied to cells expressing the hABCA3-GFP missense mutants L101P hABCA3-GFP, and G1221S hABCA3-GFP and visualized by confocal microscopy. Login to comment
138 4-PBA facilitates lysosomal targeting of G1221S-GFP but not L101P-GFP. Login to comment
142 Western blots with anti-GFP (top) and anti beta-actin (bottom) antibodies of cell lysates from L101P and G1221S hABCA3-GFP cells in the absence (afa;) or presence (af9;) of 1 mM 4-PBA treatment. 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
194 The (G1221S) mutant in transmembrane domain 11 of ABCA3 is somewhat localized to lysosomes and in some of those lysosomes it appeared to transport NBD-PC. Login to comment
204 The ratio of 150- and 180-kDa protein for the G1221S mutant increases with 4-PBA treatment, FIGURE 6. Silencing of ABCA3 reduces uptake of C12-NBD-phosphatidylcholine. Login to comment
216 Our finding that 4-PBA treatment partially restored trafficking of G1221S hABCA3-GFP to the lysosomal membrane suggests that an approach using chemical or pharmacological chaperones should be explored further as a potential treatment for lung surfactant deficiency caused by ABCA3 mutations (Fig. 3B). 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: 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
304 Although not restricted to any particular domain, functional characterization of a subgroup of ABCA3 mutations (including L101P, L982P, L1553P, Q1591P, G1221S) by transient or stable expression results in their total or partial retention in the ER (30, 103). Login to comment