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Cloning of Human Very-Long-Chain Acyl-Coenzyme a Dehydrogenase

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Cloning of Human Very-Long-Chain Acyl-Coenzyme a Dehydrogenase Am. J. Hum. Genet. 57:273-283, 1995 Cloning of Human Very-Long-Chain Acyl-Coenzyme A Dehydrogenase and Molecular Characterization of Its Deficiency in Two Patients Toshifumi Aoyama,' Masayoshi Souri,' Ichiro Ueno,2 Takehiko Kamijo,2 Seiji Yamaguchi,4 William J. Rhead,5 Kay Tanaka,6 and Takashi Hashimoto' Departments of 'Biochemistry and 2Pediatrics, Shinshu University School of Medicine, and 3Central Clinical Laboratories, Shinshu University Hospital, Matsumoto; 4Department of Pediatrics, Shimane Medical University, Izumo; 'Department of Pediatrics, University of Iowa, Iowa City; and 'Department of Genetics, Yale University School of Medicine, New Haven Summary zyme A (CoA) dehydrogenase and acyl-CoA oxidase Two overlapping cDNA clones (1,991 bp and 736 bp, (AOX), respectively. Four distinct acyl-CoA dehydroge- respectively) encoding the precursor of human mito- nases, i.e., short-chain acyl-CoA dehydrogenase (SCAD), chondrial very-long-chain acyl-coenzyme A dehydroge- medium-chain acyl-CoA dehydrogenase (MCAD), long- nase (VLCAD) were cloned and sequenced. The cDNA chain acyl-CoA dehydrogenase (LCAD), and very-long- inserts of these clones together encompass a region of chain acyl-CoA dehydrogenase (VLCAD), have been 2,177 bases, encoding the entire protein of 655 amino identified in the mitochondrial P-oxidation system acids, including a 40-amino acid leader peptide and (Rhead 1991; Coates and Tanaka 1992a, 1992b; Engel a 615-amino acid mature polypeptide. PCR-amplified 1992; Hale and Bennett 1992; Izai et al. 1992). cDNAs VLCAD cDNAs were sequenced in cultured fibroblasts encoding human SCAD and MCAD have been cloned, from two VLCAD-deficient patients. In both patients, and mutation analyses of the two deficiencies, particu- a 105-bp deletion encompassing bases 1078-1182 in larly of the latter, have been extensively performed VLCAD cDNA was identified. The deletion seems to (Coates et al. 1988; Naito et al. 1990; Yokota et al. occur due to exon skipping during processing ofVLCAD 1991; Tanaka et al. 1992; Andresen et al. 1994). On pre-mRNA. This is the first demonstration of a mutation the other hand, mutations of the LCAD and VLCAD causing VLCAD deficiency. Quantitative cDNA expres- genes underlying their deficiencies have not yet been sion of normal human VLCAD was performed in the identified for two reasons. First, VLCAD was identified patients' fibroblasts, using vaccinia viral system, which only 2 years ago (Izai et al. 1992), and the primary demonstrated that the deficiency of the normal VLCAD sequence of human VLCAD was hitherto unknown. Sec- protein causes impaired long-chain fatty acid P-oxida- ond, there has been clinical and biochemical uncertainty tion activity in the patients' fibroblasts. In patient fibro- concerning the identity of the LCAD and VLCAD defi- blasts, raising VLCAD activity to -20% of normal con- ciencies, because of the overlap of the substrate specific- trol fibroblast activity raised palmitic acid P-oxidation ity of the two enzymes (Hale et al. 1985, 1990a, 1990b; flux to the level found in control fibroblasts, which may Indo et al. 1991a). offer important information for the rational design of In the last two years, several patients with inherited future somatic gene therapy for VLCAD deficiency. VLCAD deficiency have been identified by this and other laboratories (Aoyama et al. 1993; Bertrand et al. 1993). Also, in three patients previously diagnosed as having LCAD deficiency on the basis of activity assays, the Introduction LCAD sequence was found to be perfectly normal, while In mammals, fatty acids are oxidized by two major sys- immunoblot analysis indicated that these patients were tems, namely the mitochondrial and peroxisomal P-oxi- in fact deficient in VLCAD rather than in LCAD (Yama- dation systems. The first step in the mitochondrial and guchi et al. 1993). Recently, we reported detailed bio- peroxisomal P-oxidation cycle is catalyzed by acyl-coen- chemical and immunochemical studies of seven patients having VLCAD deficiency (Aoyama et al., in press). Clinically notable features of these patients were also Received March 14, 1995; accepted for publication May 3, 1995. summarized: all patients were found to have hepatocel- Address for correspondence and reprints: Dr. Toshifumi Aoyama, lular dysfunction, severe lipid storage in organs, and Department of Biochemistry, Shinshu University School of Medicine, cardiac at least four with Matsumoto, Nagano 390, Japan. disease; patients presented hy- © 1995 by The American Society of Human Genetics. All rights reserved. pertrophic cardiomyopathy. Additionally, the cDNA- 0002-9297/95/5702-0009$02.00 encoding rat VLCAD has been cloned, and the im- 273 274 Am. J. Hum. Genet. 57:273-283, 1995 portant role of VLCAD as a rate-limiting enzyme in an EcoRI digest of the phage DNA was subcloned into long-chain fatty acid 5-oxidation system has been dem- pTZ1 8U plasmid vector (TOYOBO). Plasmid DNA was onstrated (Aoyama et al. 1994c). isolated in large scale, using DNA preparation kits We report here the isolation and sequencing ofcDNAs (Qiagen). encoding the entire precursor of human VLCAD, the first identification of mutations in the VLCAD gene in DNA Sequencing two patients with VLCAD deficiency, and the restora- Serial sequence deletions of the clone were carried tion of long-chain fatty acid P-oxidation activity in the out via exonuclease III digestion, using Kilo-sequence patients' fibroblasts by means of cDNA-expression of deletion kits (Takara), on the basis of the method de- normal human VLCAD. scribed by Henikoff (1984). DNA sequencing was per- formed directly on the cDNA insert in the plasmid by the dideoxy-sequencing method (Sanger et al. 1977) us- Material and Methods ing Dye primer cycle sequencing kits (Applied Biosys- Material tems). An antibody that reacts with human VLCAD was RNA Preparation and Northern Blot Analysis prepared using purified human VLCAD protein (Ao- Total RNA was extracted from fibroblasts, using the yama et al., in press). cDNAs encoding rat VLCAD guanidinium isothiocyanate procedure (Chomczynski (Aoyama et al. 1994c) and human AOX (Aoyama et al. and Sacchi 1987). Twenty micrograms each of total 1994b) were prepared as described, respectively. Human RNA were denatured in formaldehyde/formamide and liver cDNA libraries prepared in the Xgt 11 expression were electrophoresed in 1% agarose gel containing vector were obtained from Dr. Frank J. Gonzalez of the formaldehyde, and RNA was transferred to a nylon National Institutes of Health (Aoyama et al. 1989c). membrane (Hybond-N+; Amersham) by capillary blot- Reagents used for both DNA sequencing and amino acid ting (Sambrook et al. 1989). Prehybridization and hy- sequencing of proteolytic peptides were purchased from bridization were carried out according to Amersham Applied Biosystems. Vaccinia virus strain WR, pSC 11, protocol. and human TK- 143 cells were obtained from Dr. Ber- nard Moss of the National Institutes of Health. CV-1 Amplification of cDNA by PCR and hepatoma H4IIEC3 cells were from the American Two micrograms of total RNA were reverse-tran- Type Culture Collection. Electron transfer flavoprotein scribed with Murine leukemia virus reverse transcriptase (ETF) was prepared from rat liver mitochondria (Furuta and random hexanucleotide primers according to manu- et al. 1981). [1-14C]Palmitic acid was obtained from facturer's instructions. One twentieth of the cDNA American Radiolabeled Chemicals. Sodium 2-[5-(4- product was annealed to 1 pmol each of the sense and chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) was antisense primers, and 40 cycles of PCR reaction were a generous gift from Byk Gulden Pharmazeutika. performed using a DNA Thermal Cycler (Idaho Tech- nology). Each cycle consisted of 5-s denaturation at Source of the Cells and Culture Method 94°C, 2-s annealing at 52°C, and 1-min extension at The case histories of the two VLCAD-deficient cells, 750C. The final extension was for 10 min. The PCR patient 2 and 3, together with five others (Aoyama et products were purified by electrophoresis using 1% al., in press), have been reported elsewhere (Aoyama et agarose gel. The purified fragments were directly used al. 1993; Yamaguchi et al. 1993). Skin fibroblasts were for DNA sequencing. cultured in the medium containing Dulbecco's modified Amino Acid Sequence Analsis Eagle's medium, 10% (v/v) FCS, 0.1 mM nonessential Native human VLCAD protein (Aoyama et al., in amino acids, 1 x antibiotic-antimycotic solution press) and its proteolytic polypeptides prepared by endo- (GIBCO/BRL Oriental) and 4.5 mg D-glucose/ml. proteinase Glu-C or endoproteinase Lys-C (Boehringer Mannheim) treatment were electrophoresed on 10% cDNA Cloning Method polyacrylamide gel containing sodium dodecyl sulfate Four-hundred thousand independent plaques from and were transferred to a polyvinylidene difluoride the human liver cDNA library were screened using the membrane (Bio-Rad). Amino-terminal sequence was de- rat VLCAD cDNA (RVL-57) (Aoyama et al. 1994c) as termined using a model 477A gas-phase sequencer (Ap- a probe (Maniatis et al. 1982). After positive clones plied Biosystems) and an in-line model 120A phenylthio- were obtained, another human liver cDNA library was hydantoin analyzer. screened using the longest cDNA insert in previously identified positive clones as a probe. Phage DNA of posi- Vaccinia Viral Expression of VLCAD cDNA tive clones was then prepared using the plate-lysate The vaccinia virus system was used to express VLCAD method (Maniatis et al. 1982). The
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