ABCMdb

PMID: 19861431

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., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys 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 11 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys Functional analysis of this R295C mutation demonstrates that the mutation severely compromises the ability of the protein to hydrolyze ATP. Login to comment 31 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys Functional analysis of this R295C mutation demonstrates that the mutation severely compromises the ability of the protein to hydrolyze ATP. Login to comment 44 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys Presence of the mutation in the pEGFN1-ABCA3-R295C-GFP and pCAGIpuro-ABCA3-R295C-GFP constructs was confirmed by sequencing. Login to comment 48 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Leu982Pro ABCA3 p.Arg295Cys 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 49 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys Presence of the mutation in the pEGFN1-ABCA3-R295C-GFP and pCAGIpuro-ABCA3-R295C-GFP constructs was confirmed by sequencing. Login to comment 53 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Leu982Pro ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys RESULTS Identification of a novel R295C mutation. Login to comment 63 ABCA3 p.Arg295Cys RESULTS Identification of a novel R295C mutation. Login to comment 75 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys None of these individuals had the R295C variant, indicating that this variation is indeed a mutation and not a polymorphism. Login to comment 80 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 82 ABCA3 p.Glu292Val ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys 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 84 ABCA3 p.Arg295Cys None of these individuals had the R295C variant, indicating that this variation is indeed a mutation and not a polymorphism. Login to comment 85 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys To examine whether the intracellular localization of the R295C mutant was altered, the glycosylation state of the R295C mutant was characterized. Login to comment 86 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys Effects of ABCA3 R295C mutation on function. Login to comment 87 ABCA3 p.Glu292Val ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys 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 92 ABCA3 p.Arg295Cys ᜡ, Mutations reported previously; ɏd;, novel mutant R295C. Login to comment 94 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Glu292Val ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro ABCA3 p.Glu690Lys 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 95 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys Schematic diagram of ATP-binding cassette protein A3 (ABCA3) and conservation of amino acids in the region of the R295C mutant. Login to comment 96 ABCA3 p.Arg295Cys C: alignment of sequences surrounding the R295C mutation in human, rat, mouse, and chimpanzee. Login to comment 97 ABCA3 p.Arg295Cys ଁ, Mutations reported previously; ૽, novel mutant R295C. Login to comment 99 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Glu292Val ABCA3 p.Gly1221Ser ABCA3 p.Leu1553Pro ABCA3 p.Leu1580Pro ABCA3 p.Gln1591Pro ABCA3 p.Leu982Pro ABCA3 p.Glu690Lys ABCA3 p.Arg295Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Leu982Pro ABCA3 p.Arg295Cys B: alignment of sequences surrounding the R295C mutation in ICL-1 in various members of the ABCA subfamily. Login to comment 101 ABCA3 p.Arg295Cys C: alignment of sequences surrounding the R295C mutation in human, rat, mouse, and chimpanzee. Login to comment 102 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 104 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Leu982Pro ABCA3 p.Arg295Cys 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 106 ABCA3 p.Asn568Asp Vanadate-induced nucleotide trapping was also decreased in the N568D mutant as reported previously (14). Login to comment 107 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys These results indicate that the ability of the R295C mutant to hydrolyze ATP is severely impaired. Login to comment 109 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Asn568Asp ABCA3 p.Glu292Val ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro ABCA3 p.Arg295Cys ABCA3 p.Thr1114Met 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 ABCA3 p.Asn568Asp ABCA3 p.Glu292Val ABCA3 p.Arg295Cys Vanadate-induced nucleotide trapping was also decreased in the N568D mutant as reported previously (14). Login to comment 112 ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys These results indicate that the ability of the R295C mutant to hydrolyze ATP is severely impaired. Login to comment 114 ABCA3 p.Asn568Asp ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Asn568Asp ABCA3 p.Glu292Val ABCA3 p.Gly1221Ser ABCA3 p.Leu1580Pro ABCA3 p.Thr1114Met Other mutations in the ABCA3 protein also result in impaired ATP hydrolysis, including E292V, N568D, G1221S, L1580P, and T1114M (13, 14). Login to comment 116 ABCA3 p.Glu292Val ABCA3 p.Arg295Cys The E292V mutation is in ICL-1 only three amino acids from the R295C mutation. Login to comment 119 ABCA3 p.Asn568Asp ABCA3 p.Arg295Cys 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 122 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Leu982Pro ABCA3 p.Arg295Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Leu982Pro ABCA3 p.Arg295Cys 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 126 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Leu982Pro ABCA3 p.Arg295Cys 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 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Leu982Pro ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 130 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 131 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys The R295C mutation does not affect glycosylation and intracellular localization of the protein. Login to comment 134 ABCA3 p.Arg295Cys ABCA3 p.Arg295Cys 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 ABCA3 p.Arg295Cys 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 140 ABCA3 p.Arg295Cys One possibility is that an interaction between the R295C mutation and the patient`s prematurity resulted in the severe BPD observed. Login to comment 142 ABCA3 p.Glu292Val Interestingly, the frequency of individuals heterozygous for the E292V mutation is elevated in a cohort of children with RDS, suggesting that a mutation in this region might impart increased genetic risk for respiratory insufficiency, even in heterozygotes (9). Login to comment 143 ABCA3 p.Arg295Cys 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 144 ABCA3 p.Arg295Cys One possibility is that an interaction between the R295C mutation and the patient`s prematurity resulted in the severe BPD observed. Login to comment 146 ABCA3 p.Glu292Val Interestingly, the frequency of individuals heterozygous for the E292V mutation is elevated in a cohort of children with RDS, suggesting that a mutation in this region might impart increased genetic risk for respiratory insufficiency, even in heterozygotes (9). Login to comment 147 ABCA3 p.Arg295Cys 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