ABCMdb

PMID: 16415354

Cheong N, Madesh M, Gonzales LW, Zhao M, Yu K, Ballard PL, Shuman H
Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome.
J Biol Chem. 2006 Apr 7;281(14):9791-800. Epub 2006 Jan 16., [PubMed]

Sentences

No. Mutations Sentence Comment

43 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser 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 ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser 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 ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser 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 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser 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 99 ABCA3 p.Leu101Pro The construct containing mutation L101P of ABCA3-GFP failed to target the lysosomal membrane (Fig. 2A, d-f) and mainly remained in the ER (Fig. 2B, d-f)). Login to comment 100 ABCA3 p.Gly1221Ser 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 101 ABCA3 p.Asn568Asp 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 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser 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 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser 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 116 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser 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 117 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser 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 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser 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 119 ABCA3 p.Leu101Pro ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser 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 121 ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser 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 122 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser 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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser 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 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser 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 ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser 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 138 ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser 4-PBA facilitates lysosomal targeting of G1221S-GFP but not L101P-GFP. Login to comment 139 ABCA3 p.Leu101Pro ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser 4-PBA facilitates lysosomal targeting of G1221S-GFP but not L101P-GFP. Login to comment 140 ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser A, 4-PBA treatment failed to restore L101P ABCA3-GFP trafficking to lysosomes. Login to comment 141 ABCA3 p.Gly1221Ser B, 4-PBA treatment facilitates G1221S ABCA3-GFP trafficking to lysosomes. Login to comment 142 ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser 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 143 ABCA3 p.Leu101Pro ABCA3 p.Gly1221Ser 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 160 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser 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 161 ABCA3 p.Asn568Asp ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser ABCA3 p.Gly1221Ser 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 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser 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 191 ABCA3 p.Leu101Pro The (L101P) mutant in extracellular loop 1 of ABCA3 had the most severe trafficking defect. Login to comment 192 ABCA3 p.Leu101Pro The (L101P) mutant in extracellular loop 1 of ABCA3 had the most severe trafficking defect. Login to comment 194 ABCA3 p.Gly1221Ser 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 195 ABCA3 p.Gly1221Ser 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 196 ABCA3 p.Asn568Asp 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 197 ABCA3 p.Asn568Asp 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 204 ABCA3 p.Gly1221Ser 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 205 ABCA3 p.Gly1221Ser The ratio of 150- and 180-kDa protein for the G1221S mutant increases with 4-PBA treatment, FIGURE 6. Login to comment 216 ABCA3 p.Gly1221Ser 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 218 ABCA3 p.Gly1221Ser 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 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser B, A549 cells with N568D (a-c) and G1221S (d-f) hABCA3-DsRed incubated with C12-NBD-PC-labeled liposomes. Login to comment 221 ABCA3 p.Leu101Pro Scale bars represent 10 òe;m. ABCA3 Role in Lipid Transport and Lamellar Body Biogenesis APRIL 7, 2006ߦVOLUME 281ߦNUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9799 lowing 4-PBA treatment in the L101P mutant (Fig. 3A). Login to comment 222 ABCA3 p.Asn568Asp ABCA3 p.Gly1221Ser B, A549 cells with N568D (a-c) and G1221S (d-f) hABCA3-DsRed incubated with C12-NBD-PC-labeled liposomes. Login to comment 223 ABCA3 p.Leu101Pro Scale bars represent 10 ␮m. lowing 4-PBA treatment in the L101P mutant (Fig. 3A). Login to comment