Functional and Trafficking Defects in ATP Binding Cassette A3 Mutants Associated with Respiratory Distress Syndrome
2006; Elsevier BV; Volume: 281; Issue: 14 Linguagem: Inglês
10.1074/jbc.m507515200
ISSN1083-351X
AutoresNaeun Cheong, Muniswamy Madesh, Linda W. Gonzales, Ming Zhao, Kevin Yu, Philip L. Ballard, Henry Shuman,
Tópico(s)Neonatal Health and Biochemistry
ResumoMembers of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures. Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures. ATP binding cassette (ABC) 2The abbreviations used are: ABC, ATP binding cassette; ABCA, ABC subfamily A; ATII, alveolar epithelial type II; RDS, respiratory distress syndrome; LAMP, lysosome-associated membrane protein; 4-PBA, 4-phenylbutyric acid; siRNA, small interfering RNA; RT, reverse transcription; PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine; GFP, green fluorescent protein; DCI, dexamethasone/8-bromine-cAMP/isobutylmethylxanthine; ER, endoplasmic reticulum; SP-B, surfactant protein B; NBD, 7-nitrobenzo-2-oxa-1,3-diazolyl. transporters are a superfamily of highly conserved membrane proteins that transport a wide variety of substrates across cell membranes (1Dean M. Rzhetsky A. Allikmets R. Genome Res. 2001; 11: 1156-1166Crossref PubMed Scopus (1530) Google Scholar). Among the several subfamilies, the ABCA subclass has received considerable attention, because mutations of the ABCA1 gene cause Tangier disease and mutations of the ABCA4 gene cause Stargardt macular dystrophy in humans (2Peelman F. Labeur C. Vanloo B. Roosbeek S. Devaud C. Duverger N. Denefle P. Rosier M. Vandekerckhove J. Rosseneu M. J. Mol. Biol. 2003; 325: 259-274Crossref PubMed Scopus (109) Google Scholar, 3Oram J.F. Biochim. Biophys. Acta. 2000; 1529: 321-330Crossref PubMed Scopus (208) Google Scholar, 4Sun H. Smallwood P.M. Nathans J. Nat. Genet. 2000; 26: 242-246Crossref PubMed Scopus (171) Google Scholar, 5Maugeri A. Klevering B.J. Rohrschneider K. Blankenagel A. Brunner H.G. Deutman A.F. Hoyng C.B. Cremers F.P. Am. J. Hum. Genet. 2000; 67: 960-966Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). ABCA1 and ABCA4 are proposed to be transmembrane transporters for intracellular cholesterol/phospholipids and N-retinylidene phosphatidylethanolamine, respectively (3Oram J.F. Biochim. Biophys. Acta. 2000; 1529: 321-330Crossref PubMed Scopus (208) Google Scholar, 4Sun H. Smallwood P.M. Nathans J. Nat. Genet. 2000; 26: 242-246Crossref PubMed Scopus (171) Google Scholar, 5Maugeri A. Klevering B.J. Rohrschneider K. Blankenagel A. Brunner H.G. Deutman A.F. Hoyng C.B. Cremers F.P. Am. J. Hum. Genet. 2000; 67: 960-966Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). ABCA3, a member of the ABCA subfamily with unknown function (6Shulenin S Nogee L.M. Annilo T. Wert S.E. Whitsett J.A. Dean M. N. Engl. J. Med. 2004; 350: 10-17Crossref Scopus (576) Google Scholar, 7Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (239) Google Scholar, 8Mulugeta S Gray J.M. Notarfrancesco K.L. Gonzales L.W. Koval M. Feinstein S.I. Ballard P.L. Fisher A.B. Shuman H. J. Biol. Chem. 2002; 277: 22147-22155Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 9Nagata K. Yamamoto A. Ban N. Tanaka A.R. Matsuo M. Kioka N. Inagaki N. Ueda K. Biochim. Biophys. Res. Commun. 2004; 324: 262-268Crossref PubMed Scopus (68) Google Scholar, 10Bullard J.E Wert S.E Whitsett J.A Dean M. Nogee L.M. Am. J. Respir. Crit. Care Med. 2005; 172: 1026-1031Crossref PubMed Scopus (273) Google Scholar), is predominantly expressed in the lung and localized to the limiting membrane of lamellar bodies in alveolar epithelial type II cells (ATII) in both humans and rats (7Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (239) Google Scholar, 8Mulugeta S Gray J.M. Notarfrancesco K.L. Gonzales L.W. Koval M. Feinstein S.I. Ballard P.L. Fisher A.B. Shuman H. J. Biol. Chem. 2002; 277: 22147-22155Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). In the lung, development of structures for effective pulmonary gas exchange and production of pulmonary surfactant are necessary for successful adaptation to extrauterine life in the newborn infant. These key processes in lung maturation require differentiation of epithelium into ATII cells, the cellular source for surfactant. Pulmonary surfactant is a complex mixture of lipids, primarily phosphatidylcholine (60-70% of which is dipalmitoylphosphatidylcholine) and specific proteins that line the alveolar surface of the lung, reducing surface tension at the air-liquid interface and preventing collapse of the lung on expiration (11Dobbs L.G. Annu. Rev. Med. 1989; 40: 431-446Crossref PubMed Scopus (83) Google Scholar). Surfactant is assembled and stored in lamellar bodies, the secretory organelles of ATII cells (11Dobbs L.G. Annu. Rev. Med. 1989; 40: 431-446Crossref PubMed Scopus (83) Google Scholar, 12Rooney S.A. Young S.L. Mendelson C.R. FASEB J. 1994; 8: 957-967Crossref PubMed Scopus (310) Google Scholar, 13Schmitz G. Muller G. J. Lipid Res. 1991; 32: 1539-1570Abstract Full Text PDF PubMed Google Scholar). Two other members of the ABCA subfamily, ABCA1 and ABCA4, have been implicated in lipid transport leading to the hypothesis that ABCA3 transports lipid into the lamellar bodies of ATII cells (7Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (239) Google Scholar, 8Mulugeta S Gray J.M. Notarfrancesco K.L. Gonzales L.W. Koval M. Feinstein S.I. Ballard P.L. Fisher A.B. Shuman H. J. Biol. Chem. 2002; 277: 22147-22155Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 9Nagata K. Yamamoto A. Ban N. Tanaka A.R. Matsuo M. Kioka N. Inagaki N. Ueda K. Biochim. Biophys. Res. Commun. 2004; 324: 262-268Crossref PubMed Scopus (68) Google Scholar). Recently, it has been reported that mutations in ABCA3 are associated with defective assembly of lamellar bodies and fatal respiratory distress syndrome (RDS) in the newborn infant and interstitial lung disease (6Shulenin S Nogee L.M. Annilo T. Wert S.E. Whitsett J.A. Dean M. N. Engl. J. Med. 2004; 350: 10-17Crossref Scopus (576) Google Scholar, 10Bullard J.E Wert S.E Whitsett J.A Dean M. Nogee L.M. Am. J. Respir. Crit. Care Med. 2005; 172: 1026-1031Crossref PubMed Scopus (273) Google Scholar). To study the potential role of ABCA3 in RDS, we examined the subcellular trafficking and substrate specificity of ABCA3 in hATII cells and mammalian cell lines using green fluorescent protein (GFP)-tagged protein and fluorescent lipid analogs. Morphological and functional changes secondary to both loss- and gain-of-function experiments demonstrate that ABCA3 selectively transports phosphatidylcholine, sphingomyelin, and cholesterol to lamellar bodies in hATII cells. Our findings indicate that lipid trafficking by ABCA3 across lamellar body membranes is necessary for lamellar body biogenesis as a key step in assembly of lung surfactant in hATII cells. Reagent—PNGase F was obtained from New England Biolabs (Beverly, MA). Sodium 4-phenylbutyrate (SBP11) was purchased from Scandinavian Formulas, (Sellersville, PA). LysoTracker Red, ERTracker Red, and Nile-Red were obtained from Molecular Probes. Rabbit anti-surfactant protein B (SP-B) and mouse anti-actin antibodies were purchased from Chemicon International. Mouse anti-GFP antibody was purchased from BD Biosciences. DC-LAMP antibody was purchased from Immunotech (Beckman Coulter, Inc). The LAMP-1 (H4A3) and LAMP-2 (H4B4) monoclonal antibodies developed by J. T. August and J. E. K. Hildreth were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA. C12-NBD-phosphatidylcholine, C12-NBD-sphingomyelin, C12-NBD-phosphatidylethanolamine, and NBD-cholesterol were purchased from Avanti Polar Lipids, and other lipids were purchased from Sigma. All other reagents were electrophoretic grade and obtained from either Sigma or Invitrogen. DNA Construct—For hABCA3-GFP or hABCA3-DsRed construction, a DNA fragment containing a full-length hABCA3 construct was generated using the PCR method with 5′-XhoI primer (CTCGAGCGATGGCTGTGCTCAGGCAG) and 3′-EcoRI primer (GAATTCCGTGTCGCCCCTCCTCTGC). The PCR fragments were subcloned to XhoI-EcoRI-digested pEGFP-N1 or pDsRed-N1 (Clontech) vectors. hABCA3 missense mutants (L101P, N568D, and G1221S) were generated using the PCR method with the following primers: L101P (forward primer, 5′-AGACAGTGCGCAGGGCACCTGTGATCAACATGCGAG-3′; reverse primer, 5′-CTCGCATGTTGATCACAGGTGCCCTGCGCACTGTCT-3′); N568D (forward primer, 5′-ATCACCGTCCTGCTGGGCCACGACGGTGCCGGGAAGAC-3′; reverse primer, 5′-GTCTTCCCGGCACCGTCGTGGCCCAGCAGGACGGTGAT-3′); and G1221S (forward primer, 5′-ATCTTCAACATCCTGTCAGCCATCGCCACCTTCCTG-3′; reverse primer, 5′-CAGGAAGGTGGCGAGGCCTGACAGGATGTTGAAGAT-3′), where the mutated nucleotides are underlined. The PCR fragments were constructed using QuikChange II XL site-directed mutagenesis Kit (Stratagene). Mammalian Cell Lines, Culture, and Transfection—Mammalian cell culture and transfection were performed as described previously (8Mulugeta S Gray J.M. Notarfrancesco K.L. Gonzales L.W. Koval M. Feinstein S.I. Ballard P.L. Fisher A.B. Shuman H. J. Biol. Chem. 2002; 277: 22147-22155Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). hABCA3-GFP/HEK293, GFP/HEK293, L101P-hABCA3-GFP/HEK-293, and G1221S hABCA3-GFP/HEK293 stable cell lines were selected with 500 μg/ml G418 and maintained with 200 μg/ml G418 in growth medium. 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. Immunoblot and Immunofluorescence Analysis—Crude membrane protein preparation and deglycosylation reactions were performed as described previously (9Nagata K. Yamamoto A. Ban N. Tanaka A.R. Matsuo M. Kioka N. Inagaki N. Ueda K. Biochim. Biophys. Res. Commun. 2004; 324: 262-268Crossref PubMed Scopus (68) Google Scholar, 14Hamada H. Tsuruo T. J. Biol. Chem. 1988; 263: 1454-1458Abstract Full Text PDF PubMed Google Scholar). Immunoblot and immunofluorescence analyses were performed as described previously (8Mulugeta S Gray J.M. Notarfrancesco K.L. Gonzales L.W. Koval M. Feinstein S.I. Ballard P.L. Fisher A.B. Shuman H. J. Biol. Chem. 2002; 277: 22147-22155Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Anti-GFP or antiactin antibodies were diluted 1:4000 or 1:1000 for immunoblot. LAMP-1, LAMP-2, SP-B, and DC-LAMP antibodies were diluted 1:100 for immunofluorescence analysis. After primary antibody binding and washing, Texas-Red-conjugated goat anti-mouse IgG (Sigma) or rhodamine-conjugated goat anti-rabbit IgG (Sigma) were diluted 1:250 for immunocytochemistry. Confocal imaging was performed using a BioRad Radiance 2000 imaging system equipped with a krypton/argon ion laser source. Human Type II Cell Culture and Transfection—Human fetal epithelial cells were prepared and cultured as described previously (15Gonzales L.W. Guttentag S.H. Wade K.C. Postle A.D. Ballard P.L. Am. J. Physiol. 2002; 283: L940-L951Crossref PubMed Scopus (115) Google Scholar). For ABCA3 or nonspecific siRNA transfection, predesigned hABCA3 siRNA (Ambion; sense sequence, 5′-GGGCACUUGUGAUCAACAUtt-3′; antisense sequence, 5′-AUGUUGAUCACAAGUCtt-3′; coding region 294-315 relative to the start codon) or nonspecific siRNA (Qiagen; sense sequence, 5′-UUCUCCGAACGUGUCACGUtt-3′; antisense sequence, 5′-ACGUGACACGUUCGGAGAAtt-3′) were transfected using Oligofectamine reagent (Invitrogen) following the manufacturer's instructions. 24 h post-transfection, the medium was changed to Waymouth's medium containing dexamethasone/8-bromine-cAMP/isobutylmethylxanthine (DCI), and incubation continued for 4 days (15Gonzales L.W. Guttentag S.H. Wade K.C. Postle A.D. Ballard P.L. Am. J. Physiol. 2002; 283: L940-L951Crossref PubMed Scopus (115) Google Scholar). The hABCA3-GFP plasmid was electroporated into human fetal lung epithelial cells according to the manufacturer's protocol (Nucleofector, Amaxa Biosystem GmbH, Cologne, Germany). Once nucleofected, the cells were transferred into fresh Waymouth's medium containing 10% fetal calf serum for attachment. 24 h post-nucleofection, medium was changed to serum-free Waymouth's medium containing DCI and cells cultured for an additional 4 days (15Gonzales L.W. Guttentag S.H. Wade K.C. Postle A.D. Ballard P.L. Am. J. Physiol. 2002; 283: L940-L951Crossref PubMed Scopus (115) Google Scholar). Real-time RT-PCR—Total RNAs were extracted from untransfected or siRNA-transfected hATII cells using the RNeasy Mini Kit (Qiagen) following the manufacturer's instruction, and on-column DNase digestion was performed using RNase-free DNase (Qiagen) to remove trace genomic DNA. The yield and purity of RNA was spectrophotometrically determined. Real-time RT-PCR was performed on a LightCycler (Roche Applied Science) using a one-step LightCycler-RNA Master SYBR Green I Kit (Roche). The cycling condition for RT-PCR was as follows: 48 °C for 30 s, 95 °C for 10 s, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. Quantification of the target gene mRNA level was obtained as a threshold PCR cycle number (CT) when the increase in the fluorescent signal of the PCR product showed exponential amplification. This value was then normalized to the threshold PCR cycle number obtained for actin mRNA from a parallel sample. Real-time RT-PCR was performed using the following primers; hABCA3 (forward. 5′-TTCTTCACCTACATCCCCTAC-3′; reverse, 5′-CCTTTCGCCTCAAA TTTCCC-3′); Actin (forward, 5′-CTCCTCCTGA GCGCAAGTACTC-3′; reverse, 5′-TCGTCATACTCCTGCTTGCTGAT-3′). Liposome Preparation and Lipid Uptake—Liposomes were prepared from l-αDPPC, C12-NBD-PC, egg PC, egg phosphatidylglycerol, and cholesterol in molar ratios 5:5:5:3:2. C12-NBD-SM- and C12-NBD-PE-containing liposomes were prepared with l-αDPPC, egg PC, egg phosphatidylglycerol, cholesterol, and C12-NBD-SM or C12-NBD-PE in molar ratios 10:5:3:2:2. NBD-cholesterol-containing liposomes were prepared from αDPPC, egg PC, egg phosphatidylglycerol, and NBD-cholesterol in molar ratios 10:5:3:2. Lipid uptake experiments were performed as described previously (16Muller W.J. Zen K. Fisher A.B. Shuman H. Am. J. Physiol. 1995; 269: L11-L19Crossref PubMed Google Scholar, 17Chinoy M.R. Fisher A.B. Shuman H. Am. J. Physiol. 1994; 266: L713-L721PubMed Google Scholar). Confocal Microscopy Imaging of Live Cells—Untransfected cells or cells transfected with hABCA3-GFP, hABCA3-DsRed, hABCA3 siRNA, or nonspecific siRNA were loaded with LysoTracker Red (0.01 μm), ERTracker Red (0.01 μm), Nile-Red (0.1 μg/ml), NBD-labeled phospholipids, or cholesterol (150 μg/ml) and washed twice with fresh medium. Coverslips were affixed to a chamber and mounted in a PDMI-2 open perfusion microincubator (Harvard apparatus, Holliston, MA) maintained at 37 °C on a Nikon TE300 inverted microscope. Confocal imaging was performed using a Bio-Rad Radiance 2000 imaging system equipped with a krypton/argon ion laser source. After staining the live cells, images were collected under confocal microscopy, and 10 different images were taken from each sample. Images were Kalmanaveraged three times. Lipid Uptake—NBD-lipid in membrane fractions was measured as previously described (18Madesh M. Hajnoczky G. J. Cell Biol. 2001; 155: 1003-1015Crossref PubMed Scopus (448) Google Scholar). Briefly, cells were incubated with NBD-lipid containing liposomes for 30 min and washed with ice-cold phosphatebuffered saline two times each for 5 min. Equal numbers of cells (1 × 107 cells) were permeabilized with 1 ml of intracellular medium composed of 120 mm KCl, 10 mm NaCl, 1 mm KH2PO4, 20 mm Tris-HEPES, and 1 μg/ml each aprotinin, leupeptin, and pepstatin, at pH 7.2, supplemented with 80 μg/ml digitonin (Sigma, 50% (w/w)). After 10 min of incubation, the intracellular medium was removed, and the cells were resuspended in 1 ml of phosphate-buffered saline. Fluorescence from the suspension was monitored in a multiwavelength excitation dual wavelength emission fluorometer (Delta RAM, Photon Technology International) using 460-nm excitation and 534-nm emission. Experiments were performed at 37 °C with constant stirring. Data are representative of four independent experiments. Electron Microscopy—For electron microscopy, cells cultured on glass slides were fixed with ice-cold 5% glutaraldehyde in 0.1 m cacodylate buffer (pH 7.4) for 2 h, followed by two steps of post-fixation: 1% OsO4 in cacodylate buffer and then 2% uranyl acetate in distilled water, 1 h for each step. The cells were then dehydrated in graded concentrations of ice-cold acetone, embedded on glass slides with electron microscopy bed 812 resin (EMS, Fort Washington, PA), and polymerized at 60 °C for 48 h. After the glass slides were removed with 48% hydrofluoric acid (Sigma) from the surface of the epon plastic blocks, the cells remaining in the plastic blocks were cut into ultrathin sections (70 nm), counter stained with uranyl acetate and lead citrate, and imaged with a JEOL 100 CX microscope. hABCA3 Localizes to Lamellar Bodies and Lysosomes—ABCA3 was previously shown to localize to lamellar body membranes in ATII cells and to intracellular vesicles in lung-derived cell lines (7Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (239) Google Scholar, 8Mulugeta S Gray J.M. Notarfrancesco K.L. Gonzales L.W. Koval M. Feinstein S.I. Ballard P.L. Fisher A.B. Shuman H. J. Biol. Chem. 2002; 277: 22147-22155Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 19Zen K. Notarfrancesco K. Oorschot V. Slot J.W. Fisher A.B. Shuman H. Am. J. Physiol. 1998; 275: L172-L183PubMed Google Scholar). Many secretory organelles appear to be derived from or consist of terminal lysosomes (20Dell'Angelica E.C Mullins C. Caplan S. Bonifacino J.S. FASEB J. 2000; 14: 1265-1278Crossref PubMed Google Scholar, 21Hook G.E.R. Gilmore L.B. J. Biol. Chem. 1982; 257: 9211-9220Abstract Full Text PDF PubMed Google Scholar, 22Gibson K.F. Windnell C.C. Am. J. Respir. Cell Mol. Biol. 1991; 4: 504-513Crossref PubMed Scopus (14) Google Scholar, 23Wasano K. Hirakawa Y. Histochemistry. 1994; 102: 329-335Crossref PubMed Scopus (30) Google Scholar, 24Weaver T.E. Na C.-L. Stahlman M. Semin. Cell Dev. Biol. 2002; 13: 263-270Crossref PubMed Scopus (180) Google Scholar). We therefore hypothesized that lamellar bodies are lysosome-derived secretory organelles. hABCA3 fused to green fluorescent protein (hABCA3-GFP) was transiently expressed in A549 and hATII cells. In hormone-induced, differentiated human fetal lung ATII cells, which produce and secrete surfactant comparable with adult ATII cells (15Gonzales L.W. Guttentag S.H. Wade K.C. Postle A.D. Ballard P.L. Am. J. Physiol. 2002; 283: L940-L951Crossref PubMed Scopus (115) Google Scholar), confocal microscopy showed hABCA3-GFP in the membranes of vesicles that were labeled with the lysosomal marker Lyso-Tracker Red and the lamellar body membrane marker dendritic cell-specific lysosome-associated membrane protein (DC-LAMP) (Fig. 1, a-f)) (25Akasaki K. Nakamura N. Tsukui N. Yokota S. Murata S. Katoh R. Michihara A. Tsuji H. Marques Jr., E.T. August J.T. Arch. Biochem. Biophys. 2004; 425: 147-157Crossref PubMed Scopus (13) Google Scholar). In A549 cells, a lung-derived epithelial tumor cell line that neither contains lamellar bodies nor expresses DC-LAMP and surfactant proteins (26Li F. Rosenberg E. Smith C.I. Notarfrancesco K. Reisher S.R. Shuman H. Feinstein S.I. Am. J. Physiol. 1995; 269: L241-L247PubMed Google Scholar), hABCA3-GFP, was found in membranes of vesicles that were labeled with LysoTracker Red and two lysosomal membrane proteins, LAMP-1 and LAMP-2 (Fig. 1, g-o). These results further support a close relationship between lysosomes and lamellar bodies in ATII cells. hABCA3 Mutations Associated with RDS Alter Trafficking and Processing of the Protein—Missense mutations of ABCA3 linked to fatal surfactant deficiency and abnormal lamellar body formation have been found in different functional domains of hABCA3 (6Shulenin S Nogee L.M. Annilo T. Wert S.E. Whitsett J.A. Dean M. N. Engl. J. Med. 2004; 350: 10-17Crossref Scopus (576) Google Scholar, 10Bullard J.E Wert S.E Whitsett J.A Dean M. Nogee L.M. Am. J. Respir. Crit. Care Med. 2005; 172: 1026-1031Crossref PubMed Scopus (273) Google Scholar). To determine whether these mutations lead to improper organelle targeting or loss of ATPase activity, constructs of hABCA3-GFP were engineered with representative missense mutations in residues conserved across species for ABCA3 (6Shulenin S Nogee L.M. Annilo T. Wert S.E. Whitsett J.A. Dean M. N. Engl. J. Med. 2004; 350: 10-17Crossref Scopus (576) Google Scholar). 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. These three mutant ABCA3 constructs (shown schematically in supplemental Fig. 1) were then expressed in A549 cells (supplemental Fig. 2). Fluorescence images of live cells stained with either LysoTracker Red or ERTracker Red indicated that the three constructs had graded trafficking defects. 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)). The construct containing mutation G1221S occasionally localized to lysosomes (Fig. 2A, j-l, arrows) and otherwise remained in the ER (Fig. 2B, j-l). 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). Thus, single missense mutations of hABCA3 can alter its localization, suggesting that RDS in some affected infants is likely associated with improper intracellular trafficking of hABCA3. Western blotting with GFP antibody revealed that mutant protein is expressed at a lower overall level than wild-type protein and that wildtype and mutant fusion proteins (L101P, N568D, and G1221S) are processed differently (Fig. 2C). hABCA3 protein was found as two molecular mass forms by SDS-PAGE, a 190-kDa form (GFP fusion protein = 220 kDa) and a 150-kDa form (GFP fusion protein = 180 kDa) (Fig. 2C) (7Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (239) Google Scholar). However, the relative amount of the lower molecular mass protein band was markedly reduced in all of the mutants compared with wild type. 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. The higher molecular mass (full-length) bands (220 kDa) for wild-type and mutant proteins were shifted by ∼10 kDa to lower molecular masses after treatment with PNGase F (Fig. 2C, 210 kDa), whereas the positions of the lower molecular mass bands (180 kDa) were not affected by glycosidase, as shown previously for the wild-type protein (9Nagata K. Yamamoto A. Ban N. Tanaka A.R. Matsuo M. Kioka N. Inagaki N. Ueda K. Biochim. Biophys. Res. Commun. 2004; 324: 262-268Crossref PubMed Scopus (68) Google Scholar). Effect of 4-PBA on Trafficking of hABCA3 Missense Mutations—Improper protein folding or trafficking is associated with a number of genetic diseases (27Nishibori Y. Liu L. Hosoyamada M. Endou H. Kudo A. Takenaka H. Higashihara E. Bessho F. Takahashi S. Kershaw D. Ruotsalainen V. Tryggvason K. Khoshnoodi J. Yan K. Kidney Int. 2004; 66: 1755-1765Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 28Sharma M. Pampinella F. Nemes C. Benharouga M. So J. Du K. Bache K.G. Papsin B. Zerangue N. Stenmark H. Lukacs G.L. J. Cell Biol. 2004; 164: 923-933Crossref PubMed Scopus (283) Google Scholar). Chemical or pharmacological chaperones can subsequently correct abnormal folding or trafficking of defective proteins (29Liu X.L. Done S.C. Yan K. Kilpelainen P. Pikkarainen T. Tryggvason K. J. Am. Soc. Nephrol. 2004; 15: 1731-1738Crossref PubMed Scopus (81) Google Scholar, 30Welch W.J. Semin. Cell Dev. Biol. 2004; 15: 31-38Crossref PubMed Scopus (99) Google Scholar). Abnormal trafficking of the most common mutation of the cystic fibrosis gene (ABCC7), ΔF508-CFTR (cystic fibrosis transmembrane regulator), can be rescued by application of the chemical chaperone 4-phenylbutyrate (4-PBA) (31Lim M. McKenzie K. Floyd A.D. Kwon E. Zeitlin P.L. Am. J. Respir. Cell Mol. Biol. 2004; 31: 351-357Crossref PubMed Scopus (68) Google Scholar). To 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. 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). 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. 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. ABCA3 Promotes Formation of Lamellar Vesicles—Mutations of hABCA3 protein are associated with abnormal lamellar body formation in hATII cells (6Shulenin S Nogee L.M. Annilo T. Wert S.E. Whitsett J.A. Dean M. N. Engl. J. Med. 2004; 350: 10-17Crossref Scopus (576) Google Scholar). To test whether ABCA3 is sufficient for biogenesis of vesicles containing lamellae, stable hABCA3-GFP expression was established in non-lung-derived HEK293 cells that normally express low levels of endogenous ABCA3. As with the other cells, hABCA3-GFP was localized to lysosomal membranes in HEK293 cells (supplemental Fig. 5). To examine the effects of ABCA3 expression on cell function and morphology, GFP/HEK293 or hABCA3-GFP/HEK293 cells were stained with Nile-Red, a lipophilic dye used to label lamellar bodies in hATII cells (15Gonzales L.W. Guttentag S.H. Wade K.C. Postle A.D. Ballard P.L. Am. J. Physiol. 2002; 283: L940-L951Crossref PubMed Scopus (115) Google Scholar). Cells expressing hABCA3-GFP exhibited more vesicles that were stained with Nile-Red compared with GFP/HEK293 cells (Fig. 4A), suggesting that ABCA3 expression may promote formation of lipid-containing vesicles. Ultrastructural visualization by electron microscopy of hABCA3-GFP/HEK293 cells stain
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