FEBS 25447 FEBS Letters 508 (2001) 221^225 CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher ConnectorABCA3 is a lamellar body membrane protein in human lung alveolar type II cells1 Gen Yamanoa, Hisayuki Funahashib, Oichi Kawanamic, Li-Xia Zhaoa, Nobuhiro Bana, Yoshiyuki Uchidad, Toshio Morohoshie, Junichi Ogawaf , Seiji Shiodab, Nobuya Inagakia;* aDepartment of Physiology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan bDepartment of Anatomy, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-Ku, Tokyo 142-8555, Japan cInstitute of Gerontology, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-Ku, Kawasaki 211-8533, Japan dDepartment of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8375, Japan eDepartment of Pathology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-Ku, Tokyo 142-8555, Japan f Department of Surgery, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan Received 18 September 2001; revised 10 October 2001; accepted 16 October 2001 First published online 30 October 2001 Edited by Veli-Pekka Lehto Stargardt's macular dystrophy [5] in human, respectively. In Abstract The ABCA3 gene, of the ABCA subclass of ATP- binding cassette (ABC) transporters, is expressed exclusively in addition to ABCA1 and ABCA4, ¢ve other members, lung. We report here the cloning, molecular characterization, and ABCA2/ABC2 [6], ABCA3/ABC3 (ABC-C) [7,8], ABCA6 distribution of human ABCA3 in the lung. Immunoblot analysis [9], ABCA7 [10], and ABCA8 [9], have been shown to belong using the specific antibody reveals a 150-kDa protein in the crude to the ABCA subclass to date. ABCA1 and ABCA4 are pro- membrane fraction of human lung. Immunohistochemical anal- posed to be transmembrane transporters for intracellular cho- yses of alveoli show that ABCA3 is expressed only in the type II lesterol/phospholipids [3,4,11] and protonated N-retinylidene- cells expressing surfactant protein A. At the ultrastructural level, phosphatidylethanolamine [12], respectively. ABCA2, ABCA3 immunoreactivity was detected mostly at the limiting ABCA3, and ABCA7 mRNA levels have been reported to membrane of the lamellar bodies. Since members of the ABCA be upregulated by the sustained cholesterol in£ux mediated transporter family are known to be involved in transmembrane by modi¢ed low density lipoprotein [13,14], suggesting that transport of endogenous lipids, our findings suggest that ABCA3 plays an important role in the formation of pulmonary surfactant ABCA transporters including ABCA3 are involved in trans- in type II cells. ß 2001 Federation of European Biochemical membrane transport of endogenous lipids [4]. Societies. Published by Elsevier Science B.V. All rights re- ABCA3, which was originally cloned from a cDNA library served. constructed from human medullary thyroid carcinoma cell line [7] or from human genomic DNA by exon-trapping [8], Key words: ATP-binding cassette transporter; is expressed predominantly in the human lung [7,8]. However, Alveolar type II cell; Lipid transport; Surfactant; the distribution and function of ABCA3 in the lung remain Lamellar body; Surfactant protein A unknown, and it has not been cloned from lung tissues. In the present study, we cloned ABCA3 from human lung and generated a speci¢c antibody against it, and found that 1. Introduction the 150-kDa ABCA3 protein is expressed predominantly at the limiting membrane of the lamellar bodies in the type II The ATP-binding cassette (ABC) transporter superfamily is cells in human lung alveolar structures. The data suggest that one of the largest gene families, encoding highly conserved ABCA3 may play an important role in the formation of pul- proteins involved in energy-dependent transport of a variety monary surfactant, which is composed mainly of phospholip- of substrates across membrane, including ions, amino acids, ids and speci¢c surfactant proteins [15]. peptides, carbohydrates, and lipids [1]. Among the several subclasses of ABC transporters, the ABCA subclass [2] has received considerable attention because mutation of the 2. Materials and methods ABCA1/ABC1 or the ABCA4/ABCR gene causes Tangier dis- 2.1. Construction of human ABCA3 expression vector ease accompanying high density lipoprotein de¢ciency [3,4] or 7.2U105 plaques of a human lung cDNA library (Stratagene) were screened under the standard hybridization conditions [16] using DNA fragments of mouse EST clones AW900257 and AA638829, which contain the regions corresponding to the 5P-untranslated region to *Corresponding author. Fax: (81)-18-884 6442. nucleotide +1020 (relative to the putative translation start site) and E-mail address: [email protected] (N. Inagaki). nucleotide +4820 to the 3P-untranslated region of human ABCA3 cDNA, respectively, as probes. Three of the positive 71 clones, VhAB- 1 The nucleotide sequence for human ABCA3 has been deposited in CA3-5-1, VhABCA3-5-4, and VhABCA3-3-17, were subcloned and the DDBJ/EMBL/GenBank nucleotide sequence database under sequenced. In addition, to obtain the DNA fragment covering the accession number AB070929. middle region of human ABCA3, the DNA fragment from +2475 to +4313 was ampli¢ed by polymerase chain reaction (PCR) using Abbreviations: ABC, ATP-binding cassette; PCR, polymerase chain human lung cDNA (Clontech), subcloned, and sequenced. Using reaction; SP-A, surfactant protein A; PBS, phosphate-bu¡ered saline; these DNA fragments, the expression vector (pCMVhABCA3) carry- DAB, 3,3P-diaminobenzidine-4HCl ing the full-length cDNA of human ABCA3 was constructed. 0014-5793 / 01 / $20.00 ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0014-5793(01)03056-3 FEBS 25447 12-11-01 Cyaan Magenta Geel Zwart 222 G. Yamano et al./FEBS Letters 508 (2001) 221^225 2.2. Tissue preparation, cell culture, and transfection Human lung tissue was obtained from adult patients undergoing surgical removal for various reasons. Culture and transfection of HEK 293 cells were carried out as previously described [17]. 2 Wgof pCMVhABCA3 or pCMV vector was transfected into cells with Lipo- fectamine and Opti-MEM I (Life Technologies, Inc.), according to the manufacturer's instructions. 2.3. Crude membrane preparation Crude membrane of transfected HEK 293 cells or human lung tissue was prepared as previously described [6,17]. Brie£y, for trans- fected HEK 293 cells, 3 days after transfection with pCMVhABCA3 or pCMV vector alone, the cells were washed, suspended in bu¡er A consisting of 50 mM Tris (pH 7.5) and 1 mM EDTA containing protease inhibitor cocktail (10 Wl/ml) (Sigma), homogenized, and then ultracentrifuged at 100 000Ug at 4³C for 1 h. The pellets were resuspended in 500 Wl of bu¡er A and stored at 380³C until immu- noblot analysis. For lung tissue, the tissue in bu¡er A (10% volume) containing protease inhibitor cocktail (10 Wl/ml) was homogenized in a Te£on pestle-glass homogenizer on ice and centrifuged at 800Ug for 7 min at 4³C. The supernatant was then subjected to ultracentrifuga- tion, and the pellet was resuspended and stored as described above. Protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA). 2.4. Primary antibodies The speci¢c antibody for ABCA3 was raised in a rabbit against the synthetic peptide corresponding to 13 C-terminal amino acid residues (FAHLQPPTAEEGR) of human ABCA3, which di¡ers construc- Fig. 2. Immunohistochemical labeling of ABCA3 by DAB reaction of human lung tissue cryosections. A: Immunoreactive round or cu- boid cells (arrow) are scattered throughout the alveoli. B: A repre- sentative immunoreactive cell; vesicle-like structures are strongly stained on the surface (arrowhead). Scale bar, 10 Wm. tively from other ABCA transporters and any other protein from the sequence databases. The antibody was puri¢ed using a¤nity chro- matography (HiTrap Protein G, Amersham Pharmacia Biotech). For double-immuno£uorescence staining, a mouse monoclonal IgG anti- body against human surfactant protein A (SP-A, PE-10, Dako, Japan) or a mouse monoclonal antibody against Hsp-60 of mitochondria (MBL, Nagoya, Japan) was used. 2.5. Immunoblot analysis The crude membrane proteins prepared from transfected HEK 293 cells (10 Wg) and human lung tissue (20 Wg) were boiled in SDS reducing sample bu¡er, electrophoresed on 7% SDS^polyacrylamide gel, and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech) as previously described [6]. After blocking and washing [6], the membrane was incubated with 1:1000 diluted anti-ABCA3 antibody for 2 h at room temperature. After washing, the membrane was incubated with 1:5000 diluted horserad- ish peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia Bio- tech) for 1 h, followed by washing [6]. Proteins were detected using an enhanced chemiluminescence system (ECL, Amersham Pharmacia Bi- otech). 2.6. Immunohistochemistry Portions of human lung tissue were ¢xed by immersion with 4% paraformaldehyde containing 0.1% glutaraldehyde in 0.1 M phos- phate bu¡er (PB, pH 7.2) for 1 day at 4³C. After washing with 0.1 Fig. 1. Immunoblot analysis of ABC3 protein. Membrane proteins M PB, the ¢xed tissues were trimmed to smaller pieces, incubated for prepared from human lung tissue (20 Wg) (lane 1) and HEK 293 two nights in 30% sucrose in 0.1 M phosphate-bu¡ered saline (PBS), cells transfected with pCMVhABCA3 (10 Wg) (lane 2) and empty and cut into 8 Wm thick sections on a cryostat (MICROM HM 500; vector (pCMV; mock) (10 Wg) (lane 3) were electrophoresed on a MICROM, Heidelberg, Germany). The sections were rinsed with 7% SDS^polyacrylamide gel. For immunoblot analysis, an anti- PBS, preincubated with 10% normal goat serum in PBS, and then ABCA3 rabbit antibody and a horseradish peroxidase-conjugated incubated with 1:10 000 diluted anti-ABCA3 antibody.