ABCMdb

ABCA3 p.Arg295Cys

Predicted by SNAP2:	A: D (75%), C: D (75%), D: D (75%), E: D (71%), F: D (75%), G: D (71%), H: D (59%), I: D (71%), K: N (57%), L: D (71%), M: D (71%), N: D (53%), P: D (71%), Q: D (59%), S: D (53%), T: D (53%), V: D (71%), W: D (85%), Y: D (71%),
Predicted by PROVEAN:	A: N, C: N, D: D, E: N, F: N, G: D, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: N, W: N, Y: N,

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[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
11 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. Login to comment
15 The ABCA3 protein containing the R295C mutation undergoes normal glycosylation and intracellular localization but has dramatically reduced ATP hydrolysis activity (12% of wild type). Login to comment
31 This mutation substitutes a cysteine for an arginine at amino acid position 295 in the first intracellular loop (ICL-1) of ABCA3. Login to comment
32 Functional analysis of this R295C mutation demonstrates that the mutation severely compromises the ability of the protein to hydrolyze ATP. Login to comment
49 The R295C mutant was initially generated from the pEGFPN1-ABCA3-green fluorescent protein (GFP) construct (14) with the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the following primers: forward 5=-AGGCTGAAG- GAGTACATGTGCATGATGGGGCTCAGCAG-3= and reverse 5=-CTGCTGAGCCCCATCATGCACATGTACTCCTTCAGCCT-3= (underlines indicate substituted nucleotides). Login to comment
50 A R295C-GFP construct in a pCAGIpuro vector was generated by inserting the coding region of ABCA3-R295C-GFP into the pCAGIpuro vector. Login to comment
51 Presence of the mutation in the pEGFN1-ABCA3-R295C-GFP and pCAGIpuro-ABCA3-R295C-GFP constructs was confirmed by sequencing. Login to 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
63 RESULTS Identification of a novel R295C mutation. Login to comment
80 Although the R295C variant had not been observed in previously characterized populations, it was unclear whether the R295C variant was a common polymorphism in Hmong individuals or a clinically significant mutation. Login to comment
81 To determine whether the R295C variant was a polymorphism or a mutation, the frequency of this variant in the Hmong population was examined. Login to comment
84 None of these individuals had the R295C variant, indicating that this variation is indeed a mutation and not a polymorphism. Login to comment
86 Effects of ABCA3 R295C mutation on function. Login to comment
87 The R295C mutation is located in the first ICL (ICL-1) of the protein (Fig. 2A) and is adjacent to the previously reported mutant E292V (2). Login to comment
88 The R295C mutation resides in a region that is conserved in different members of the ABCA subfamily (Fig. 2B) and across ABCA3 homologs in vertebrates (Fig. 2C). Login to comment
90 To examine whether the intracellular localization of the R295C mutant was altered, the glycosylation state of the R295C mutant was characterized. Login to comment
91 Membranes from HEK293 cells expressing wild-type ABCA3-GFP, the R295C-GFP mutant, or several previously characterized mutants were examined for sensitivity to the glycosidases Endo H and PNGase F. Login to comment
95 Schematic diagram of ATP-binding cassette protein A3 (ABCA3) and conservation of amino acids in the region of the R295C mutant. Login to comment
97 ଁ, Mutations reported previously; ૽, novel mutant R295C. 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
101 C: alignment of sequences surrounding the R295C mutation in human, rat, mouse, and chimpanzee. Login to comment
104 The R295C variant demonstrated a level of resistance to Endo H comparable to that of the wild-type protein (Fig. 3A, compare lanes 6 and 7 to lanes 2 and 3), suggesting that the variant protein has undergone normal glycosylation and resides in post-Golgi membranes. 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
107 To determine whether the R295C mutation affected the ATP hydrolysis activity of the R295C mutant, vanadate-induced nucleotide trapping with photoaffinity labeling of the trapped intermediate (3) was examined. Login to comment
109 As shown in Fig. 4A, the level of vanadate-induced nucleotide trapping in the R295C mutant was greatly reduced compared with that of the wild-type ABCA3 protein. 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
112 Quantitation of three independent experiments demonstrated that the degree of trapping in the R295C mutant was dramatically reduced to 12% of that of the wild type (Fig. 4B). Login to comment
113 These results indicate that the ability of the R295C mutant to hydrolyze ATP is severely impaired. 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
116 The E292V mutation is in ICL-1 only three amino acids from the R295C mutation. 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
131 The R295C mutation does not affect glycosylation and intracellular localization of the protein. Login to comment
134 Normal glycosylation and intracellular localization of the R295C mutant is indicated by the similar levels of sensitivity to Endo H and PNGase F observed for the wild-type ABCA3-GFP protein and the R295C mutant. Login to comment
135 The observation that a substantial portion of the R295C mutant protein is resistant to Endo H indicates that the mutation does not affect intracellular localization. Login to comment
144 One possibility is that an interaction between the R295C mutation and the patient`s prematurity resulted in the severe BPD observed. Login to comment
147 In conclusion, clinical management of a premature infant with severe BPD and chronic respiratory failure led to the discovery of the novel ABCA3 mutation R295C. Login to comment
5 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. Login to comment
9 The ABCA3 protein containing the R295C mutation undergoes normal glycosylation and intracellular localization but has dramatically reduced ATP hydrolysis activity (12% of wild type). Login to comment
25 This mutation substitutes a cysteine for an arginine at amino acid position 295 in the first intracellular loop (ICL-1) of ABCA3. Login to comment
26 Functional analysis of this R295C mutation demonstrates that the mutation severely compromises the ability of the protein to hydrolyze ATP. Login to comment
44 The R295C mutant was initially generated from the pEGFPN1-ABCA3-green fluorescent protein (GFP) construct (14) with the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the following primers: forward 5=-AGGCTGAAG- GAGTACATGTGCATGATGGGGCTCAGCAG-3= and reverse 5=-CTGCTGAGCCCCATCATGCACATGTACTCCTTCAGCCT-3= (underlines indicate substituted nucleotides). Login to comment
45 A R295C-GFP construct in a pCAGIpuro vector was generated by inserting the coding region of ABCA3-R295C-GFP into the pCAGIpuro vector. Login to comment
46 Presence of the mutation in the pEGFN1-ABCA3-R295C-GFP and pCAGIpuro-ABCA3-R295C-GFP constructs was confirmed by sequencing. 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
58 RESULTS Identification of a novel R295C mutation. Login to comment
75 Although the R295C variant had not been observed in previously characterized populations, it was unclear whether the R295C variant was a common polymorphism in Hmong individuals or a clinically significant mutation. Login to comment
76 To determine whether the R295C variant was a polymorphism or a mutation, the frequency of this variant in the Hmong population was examined. Login to comment
79 None of these individuals had the R295C variant, indicating that this variation is indeed a mutation and not a polymorphism. Login to comment
82 The R295C mutation is located in the first ICL (ICL-1) of the protein (Fig. 2A) and is adjacent to the previously reported mutant E292V (2). Login to comment
83 The R295C mutation resides in a region that is conserved in different members of the ABCA subfamily (Fig. 2B) and across ABCA3 homologs in vertebrates (Fig. 2C). Login to comment
85 To examine whether the intracellular localization of the R295C mutant was altered, the glycosylation state of the R295C mutant was characterized. Login to comment
92 ᜡ, Mutations reported previously; ɏd;, novel mutant R295C. Login to comment
96 C: alignment of sequences surrounding the R295C mutation in human, rat, mouse, and chimpanzee. Login to comment
102 To determine whether the R295C mutation affected the ATP hydrolysis activity of the R295C mutant, vanadate-induced nucleotide trapping with photoaffinity labeling of the trapped intermediate (3) was examined. Login to comment
108 These results indicate that the ability of the R295C mutant to hydrolyze ATP is severely impaired. 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
130 Normal glycosylation and intracellular localization of the R295C mutant is indicated by the similar levels of sensitivity to Endo H and PNGase F observed for the wild-type ABCA3-GFP protein and the R295C mutant. Login to comment
140 One possibility is that an interaction between the R295C mutation and the patient`s prematurity resulted in the severe BPD observed. Login to comment
143 In conclusion, clinical management of a premature infant with severe BPD and chronic respiratory failure led to the discovery of the novel ABCA3 mutation R295C. Login to comment