- Email: [email protected]
Isolation and characterisation of a cDNA encoding the precursor for a novel member of the Acyl-CoA dehydrogenase gene family
Biochimica et Biophysica Acta 1446 (1999) 371^376 www.elsevier.com/locate/bba
Short sequence-paper
Isolation and characterisation of a cDNA encoding the precursor for a novel member of the Acyl-CoA dehydrogenase gene family Elizabeth A.R. Telford a
a;
*, Leanne M. Moynihan a;b , Alexander F. Markham a , Nicholas J. Lench a
Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, UK b Cancer Research Laboratories, University of Nottingham, University Park, Nottingham NG7 2RD, UK Received 25 March 1999; received in revised form 3 June 1999; accepted 8 June 1999
Abstract A gene encoding the precursor for a novel member of the human acyl-CoA dehydrogenase (ACD) gene family has been isolated which maps to human chromosome 11q25. The cDNA contains an open reading frame of 1248 nucleotides encoding a predicted 415-amino-acid peptide, and shares considerable sequence similarity with other members of the ACD family. ß 1999 Elsevier Science B.V. All rights reserved.
We have recently identi¢ed a locus on human chromosome 11q25 linked to histiocytosis with associated features of sensorineural deafness and joint contractures, by autozygosity mapping in a large consanguineous Pakistani family [1]. The minimal critical region of homozygosity in this family was mapped to an approximately 1cM interval encompassed by the DNA markers D11S1309 and D11S968. We are currently constructing a detailed transcript map of this region so that a positional candidate gene approach can be used to identify and characterise gene sequences at this locus. Expressed sequence tag (EST) database searches show that approximately 30 transcripts have already been mapped within this small region of interest. Initially we have concentrated on transcripts expressed in the appropriate target tissues including the myeloid cell
* Corresponding author. Fax: +44-113-244-4475; E-mail: [email protected]
lines HL60, U387, KG-1 and/or the cochlea as suitable candidates for mutation screening. Here we describe the isolation, characterisation and pattern of tissue expression of a novel gene (ACAD-8) encoding a protein similar to members of the acyl coenzyme A dehydrogenase family of enzymes and its exclusion as a candidate gene for this novel form of familial histiocytosis. Acyl-CoA dehydrogenases (ACDs) are a family of mitochondrial enzymes that catalyse the ¢rst dehydrogenation step in the L-oxidation of fatty acylCoA derivatives. The mitochondrial L-oxidation pathway is a cycle of four sequential reactions in which the fatty acid substrate is shortened by two carbon atoms with each cycle, releasing an acetylCoA molecule that can then be used in the tricarboxylic acid cycle or for ketogenesis [2]. Seven human ACDs have already been described, including four involved in the initial step of mitochondrial L-oxidation of straight chain fatty acids (short- (SCAD), medium- (MCAD), long- (LCAD) and very long chain (VLCAD) ACDs) and three (isovaleryl-
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 0 2 - 5
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(IVD), isobutyryl- (SBCAD) and glutaryl-CoA (GCD) dehydrogenases) involved in the degradation of amino acids. All ACDs catalyse the same initial dehydrogenation of the substrate at the L-carbon atom and require electron transfer £avoprotein (ETF) as an electron acceptor [3]. However, they di¡er distinctly from each other with regard to the length and con¢guration of the hydrocarbon chain of their respective substrates and have accordingly received appropriate names. The enzymes are similar when compared at the nucleotide and amino acid levels, and each speci¢c enzyme is conserved across species. Their close structural and functional similarities suggest that these genes may have evolved from a single ancestral gene and diverged in the course of evolution [4]. ACDs are nuclear encoded and are synthesised as precursor proteins in the cytosol with an amino terminal leader peptide, which is cleaved o¡ on import to the mitochondria, producing a mature monomer [4]. All, except VLCAD, are found as soluble proteins in the inner mitochondrial matrix, where monomers are assembled into the functional homotetrameric enzyme with one molecule of £avin adenine dinucleotide (FAD) noncovalently bound to each monomer [5]. VLCAD is a membrane-associated homodimer with monomers of larger size than the other enzymes of this class [6,7]. Fatty acids provide important respiratory fuel for many tissues including heart, skeletal muscle, brown adipose tissue, kidney and liver [8,9], as is evident in individuals with defects in any one of the ACDs. The phenotypes of these patients have some similar characteristics including hypoglycaemia, hypoketonaemia, muscle fatigue, hepatic lipidosis, and an organic aciduria that is often diagnostic of the enzyme de¢ciency [2]. MCAD is the best-studied member of the extended family as it has a very high incidence of genetic defects in humans [10]. Overlapping sequences homologous to the EST stSG297 were assembled from the Unigene public web server (http://www.ncbi.nlm.nih.gov/Schuler/ UniGene) into a 2238 bp contiguous fragment of DNA. Using reverse transcription^polymerase chain reaction (RT^PCR) on RNA from human adult brain and skin ¢broblasts, a 1610 bp fragment was ampli¢ed using the primers L2 and 40327-2 (dTGGGCTGTCACGTCTTG; dCCTAGACCAC-
AGGCGTCTTAAC; nt 382^398 and 1971^1992, respectively). DNA was sequenced on both strands using a combination of the Thermosequenase cycle sequencing kit (Amersham) and AmpliTaq cycle sequencing kit (Perkin Elmer) and the ABI Prism 377 Genescanner. The remaining 381 bp extending beyond the translation start signal was veri¢ed by sequencing clone 92A24 from the RPCII PAC human genomic library (UK HGMP Resource Centre) and 250 bp from the 3P end to the poly(A) tail was sequenced from I.M.A.G.E. clone 40327 [11]. The sequence obtained is characterised by a single open reading frame of 1248 bp encoding a predicted protein of 415 amino acids (Fig. 1). Two putative polyadenylation signals are located downstream of the translation termination codon (Fig. 1; nt 2172^ 2177 and 2199^2104). A search of the GenBank database using the BLAST algorithm [12] identi¢ed a high degree of similarity to members of the ACD family. As this is the eighth human ACD to be isolated, we have named this sequence ACAD-8. The cDNA sequence has been submitted to GenBank and assigned accession number AF126245. Alignment of the novel protein sequence with those of other human ACDs (Fig. 2) showed that it shares 28%, 32%, 32%, 33%, 34%, 36% and 38% identity with GCD, LCAD, VLCAD, IVD, MCAD, SBCAD and SCAD, respectively. Established members of this family share a similar degree of homology and the conserved invariant sequences in this study are similar to those reported previously [4,13]. There is no homology shared between family members in the amino terminal leader peptide (24 to 40 amino acids). Glutamic acid residue 401 of MCAD (Glu-376 of the mature monomer) is located at the active site of the enzyme and initiates catalysis by abstracting the substrate K-hydrogen as H [14,15]. This residue is conserved in all human ACDs except LCAD and IVD and corresponds to Glu-398 of the novel sequence. Several other amino acids with a known role in MCAD function are conserved in this novel protein including threonine-161 (position 162 in MCAD) and serine-167 (position 168 in MCAD) which form hydrogen bonds to the £avin moiety of MCAD [15]. A tryptophan residue at position 192 in MCAD, which is believed to have a role in electron transfer from the FAD bound to MCAD to ETF, is replaced by another aromatic
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Fig. 1. The complete human ACAD-8 cDNA sequence and its putative translation product. The termination signal is marked with an asterisk and the poly(A) addition signals are underlined.
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Fig. 2. Comparison of seven human ACD enzymes and human ACAD-8. Alignment was achieved using the Pileup program of the University of Wisconsin Genetics Computer Group with ¢nal manual re¢nements. The mature amino termini of the enzymes are indicated by boldface italic print. Residues conserved between ACAD-8 and any of the mature monomers are shown in bold and residues that are the same in all eight enzymes are listed as a consensus (CON). The active site glutamate in SBCAD, SCAD, MCAD, ACAD-8, VLCAD and GCD is marked with an asterisk. Positions marked by a number are described in detail in the text: 1, threonine-162 in MCAD; 2, serine-168 in MCAD; 3, tryptophan-192 in MCAD; 4, arginine-306 in MCAD; 5, glutamine-374 in MCAD; 6, glycine-378 in MCAD; 7, glutamate-325 in MCAD; 8, arginine-408 in MCAD.
6
residue, phenylalanine-191 in the novel protein and in LCAD. It is known from the crystal structure of MCAD that the tetrameric enzyme may be regarded as a dimer of two dimers [15]. Several residues crucial for the interaction of the two subunits within the dimer and some important for dimer^dimer interaction have been identi¢ed on the basis of the crystal structure [15] and functional studies [16]. Arginine306 and glutamine-374 form hydrogen bonds to the pyrophosphate moiety of the FAD of the neighbouring subunit of the MCAD dimer while glycine-378 forms hydrogen bonds to the ribose moiety of FAD of the neighbouring subunit. These residues are conserved in the novel sequence (arginine-302, glutamine-371 and glycine-375), indicating that the region involved in FAD binding and dimerisation in the novel protein is very similar to that in MCAD. Glutamate-325 and arginine-408 form an important salt bridge between the two pairs of dimers of the tetrameric MCAD and this salt bridge is also likely to form between the corresponding residues, aspartate321 and arginine-405, of the novel protein, suggesting that, like most other ACDs, it is a tetrameric enzyme. In contrast, although arginine at position 469 is conserved in VLCAD, glutamate is replaced by an arginine residue at position 385, excluding salt bridge formation, which is in agreement with the fact that VLCAD is a dimer rather than a tetramer [6,7]. The tissue distribution of the novel mRNA was determined by overnight hybridisation of a multi-tissue Northern blot (Clontech) with a 32 P-labelled 1127 bp PCR product (nt 382^1508, Fig. 1) in a bu¡er consisting of 0.5 M sodium phosphate bu¡er (pH 7.2), 1% BSA, 7% SDS, 1 mM EDTA and 50 Wg/ml denatured calf thymus DNA (Fig. 3). A band approximately 2.1 kb in size was detected at comparable levels in all tissues examined, except in liver and kidney, where it was only weakly expressed. This band correlates well with the size of transcript ex-
pected from the cDNA sequence. Two additional bands approximately 4.0 and 2.4 kb in size appeared clearly in this Northern blot even after stringent wash conditions. It is possible that these transcripts represent cross-reacting ACDs. Transcripts for previously characterised human ACDs range in size from 2^2.4kb [6,7,17^21] and larger products of 4.6 and 3.8 kb have been described for IVD [20]. The gene encoding this novel ACD has previously
Fig. 3. Northern blot analysis of ACAD-8 expression in di¡erent human tissues. The Northern blot contains approximately 2 Wg of poly A RNA from eight di¡erent adult human tissues : heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7) and pancreas (lane 8). RNA size marker bands are indicated on the right-hand side. The 2.1 kb transcript is marked with an asterisk.
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been localised by radiation hybrid mapping to the interval of human chromosome 11q25 in which the gene(s) encoding a novel familial form of histiocytosis with associated sensorineural deafness and joint contractures has been mapped (human transcript map: http://www.ncbi.nlm.nih.gov/science96). Localisation of the gene to this region was con¢rmed by PCR screening of CEPH YAC clones contained within an integrated chromosome 11 contig map that spans the interval de¢ned by D11S1320-11qter (http://mcdermott.swmed.edu). The novel cDNA was shown by RT^PCR to be expressed in the myeloid cell line KG-1 and in a human cochlear cDNA library. It was therefore considered a candidate gene for the 11q25 disease, although none of the a¡ected individuals show any features typical of ACD de¢ciency [2]. Mutation analysis was carried out by sequencing RT^PCR products from RNA extracted from EBV-transformed lymphoblast cell lines obtained from an a¡ected patient and her una¡ected father as well as control RNA. No nucleotide variations were found in the coding region of the a¡ected or una¡ected individuals or in a normal control. Therefore, this gene can now be excluded as a possible candidate for the disease locus. In this study we have isolated a cDNA encoding a novel member of the acyl-CoA dehydrogenase family, as de¢ned by considerable homology with other ACDs, and have described its pattern of tissue expression. Further studies are required to characterise the substrate speci¢city of this new member of the ACD family, as this is the primary characteristic used to de¢ne related enzymes [22]. Until such studies are carried out we propose that this eighth member of the human acyl-coA dehydrogenase gene family to be described should be referred to as ACAD-8. This research is supported by the Wellcome Trust, MRC, Birth Defects Foundation, NYRHA and YCRC.
[3] [4]
[5] [6] [7]
[8]
[9] [10]
[11] [12] [13]
[14] [15] [16]
[17]
[18]
[19] [20]
References [1] L.M. Moynihan, S.E. Bundey, D. Heath, D.P. McHale, R.F. Mueller, A.F. Markham, N.J. Lench, Am. J. Hum. Genet. 62 (1998) 1123^1128. [2] C.R. Roe, P.M. Coates, in: C.R. Scriver, A.L. Beaudet,
[21] [22]
W.S. Sly, D. Valle (Eds.), The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 1995, pp. 1501^1533. Y. Ikeda, K. Okamura-Ikeda, K. Tanaka, J. Biol. Chem. 260 (1985) 1311^1325. Y. Matsubara, Y. Indo, E. Naito, H. Osaza, R. Glassberg, J. Vockley, Y. Ikeda, J. Kraus, K. Tanaka, J. Biol. Chem. 264 (1989) 16321^16331. C.L. Hall, H. Kamin, J. Biol. Chem. 250 (1975) 3476^3486. K. Izai, Y. Uchida, T. Orii, S. Yamamoto, T. Hashimoto, J. Biol. Chem. 267 (1992) 1027^1033. T. Aoyama, M. Souri, S. Ushikibo, T. Kamijo, S. Yamaguchi, R.I. Kelley, W.J. Rhead, K. Uetake, K. Tanaka, T. Hashimoto, J. Clin. Invest. 95 (1995) 2465^2473. J. Bremer, H. Osmundsen, in: S. Numa (Ed.), Fatty Acid Metabolism and its Regulation, Elsevier Science, New York, 1984, pp. 113^154. B.E. Hainline, D.J. Kahlenbeck, J. Grant, A.W. Strauss, Biochim. Biophys. Acta 1216 (1993) 460^468. N. Gregerson, V. Winter, D. Curtis, T. Deufel, M. Mach, J. Hendrickx, P.J. Willems, A. Ponzone, T. Parrella, R. Ponzone, J.H. Ding, W. Zhang, Y.T. Chen, S. Kahler, C.R. Roe, S. Kolvraa, K. Schneiderman, B.S. Andresen, P. Bross, L. Bolund, Hum. Hered. 43 (1993) 342^350. G. Lennon, C. Au¡ray, M. Polymeropoulos, M.B. Soares, Genomics 33 (1996) 151^152. S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, J. Mol. Biol. 215 (1990) 403^410. B.S. Andresen, P. Bross, C. Viany-saban, P. Divry, M.-T. Zabot, C.R. Roe, M.A. Nada, A. Byskov, T.A. Kruse, S. Neve, K. Kristiansen, I. Knudsen, M.J. Coryden, N. Gregersen, Hum. Mol. Genet. 5 (1996) 461^472. P. Bross, S. Engst, A.W. Strauss, D.P. Kelly, I. Rasched, S. Ghisla, J. Biol. Chem. 265 (1990) 7116^7119. J.J.P. Kim, M. Wang, R. Paschke, Proc. Natl. Acad. Sci. USA 90 (1993) 7523^7527. P. Bross, C. Jesperson, T.G. Jensen, B.S. Andresen, M.J. Kristensen, V. Winter, A. Nandy, S. Ghisla, I. Rasched, L. Bolund, J.J.P. Kim, N. Gregerson, J. Biol. Chem. 270 (1995) 10284^10290. R. Rozen, J. Vockley, L. Zhou, R. Milos, J. Willard, K. Fu, C. Vicanek, L. Low-Nang, E. Torban, B. Fournier, Genomics 24 (1994) 280^287. D.P. Kelly, J.J.P. Kim, J.J. Billadello, B.E. Hainline, T.W. Chu, A.W. Strauss, Proc. Natl. Acad. Sci. USA 84 (1987) 4068^4072. E. Naito, H. Ozasa, Y. Ikeda, K. Tanaka, J. Clin. Invest. 83 (1989) 1605^1613. Y. Matsubara, M. Ito, R. Glassberg, S. Satyabhama, Y. Ikeda, K. Tanaka, J. Clin. Invest. 85 (1990) 1058^1064. Y. Indo, T. Yang-Feng, R. Glassberg, K. Tanaka, Genomics 11 (1991) 609^620. Y. Ikeda, C. Dabrowski, K. Tanaka, J. Biol. Chem. 258 (1983) 1066^1076.
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Short sequence-paper
Isolation and characterisation of a cDNA encoding the precursor for a novel member of the Acyl-CoA dehydrogenase gene family Elizabeth A.R. Telford a
a;
*, Leanne M. Moynihan a;b , Alexander F. Markham a , Nicholas J. Lench a
Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, UK b Cancer Research Laboratories, University of Nottingham, University Park, Nottingham NG7 2RD, UK Received 25 March 1999; received in revised form 3 June 1999; accepted 8 June 1999
Abstract A gene encoding the precursor for a novel member of the human acyl-CoA dehydrogenase (ACD) gene family has been isolated which maps to human chromosome 11q25. The cDNA contains an open reading frame of 1248 nucleotides encoding a predicted 415-amino-acid peptide, and shares considerable sequence similarity with other members of the ACD family. ß 1999 Elsevier Science B.V. All rights reserved.
We have recently identi¢ed a locus on human chromosome 11q25 linked to histiocytosis with associated features of sensorineural deafness and joint contractures, by autozygosity mapping in a large consanguineous Pakistani family [1]. The minimal critical region of homozygosity in this family was mapped to an approximately 1cM interval encompassed by the DNA markers D11S1309 and D11S968. We are currently constructing a detailed transcript map of this region so that a positional candidate gene approach can be used to identify and characterise gene sequences at this locus. Expressed sequence tag (EST) database searches show that approximately 30 transcripts have already been mapped within this small region of interest. Initially we have concentrated on transcripts expressed in the appropriate target tissues including the myeloid cell
* Corresponding author. Fax: +44-113-244-4475; E-mail: [email protected]
lines HL60, U387, KG-1 and/or the cochlea as suitable candidates for mutation screening. Here we describe the isolation, characterisation and pattern of tissue expression of a novel gene (ACAD-8) encoding a protein similar to members of the acyl coenzyme A dehydrogenase family of enzymes and its exclusion as a candidate gene for this novel form of familial histiocytosis. Acyl-CoA dehydrogenases (ACDs) are a family of mitochondrial enzymes that catalyse the ¢rst dehydrogenation step in the L-oxidation of fatty acylCoA derivatives. The mitochondrial L-oxidation pathway is a cycle of four sequential reactions in which the fatty acid substrate is shortened by two carbon atoms with each cycle, releasing an acetylCoA molecule that can then be used in the tricarboxylic acid cycle or for ketogenesis [2]. Seven human ACDs have already been described, including four involved in the initial step of mitochondrial L-oxidation of straight chain fatty acids (short- (SCAD), medium- (MCAD), long- (LCAD) and very long chain (VLCAD) ACDs) and three (isovaleryl-
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 0 2 - 5
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(IVD), isobutyryl- (SBCAD) and glutaryl-CoA (GCD) dehydrogenases) involved in the degradation of amino acids. All ACDs catalyse the same initial dehydrogenation of the substrate at the L-carbon atom and require electron transfer £avoprotein (ETF) as an electron acceptor [3]. However, they di¡er distinctly from each other with regard to the length and con¢guration of the hydrocarbon chain of their respective substrates and have accordingly received appropriate names. The enzymes are similar when compared at the nucleotide and amino acid levels, and each speci¢c enzyme is conserved across species. Their close structural and functional similarities suggest that these genes may have evolved from a single ancestral gene and diverged in the course of evolution [4]. ACDs are nuclear encoded and are synthesised as precursor proteins in the cytosol with an amino terminal leader peptide, which is cleaved o¡ on import to the mitochondria, producing a mature monomer [4]. All, except VLCAD, are found as soluble proteins in the inner mitochondrial matrix, where monomers are assembled into the functional homotetrameric enzyme with one molecule of £avin adenine dinucleotide (FAD) noncovalently bound to each monomer [5]. VLCAD is a membrane-associated homodimer with monomers of larger size than the other enzymes of this class [6,7]. Fatty acids provide important respiratory fuel for many tissues including heart, skeletal muscle, brown adipose tissue, kidney and liver [8,9], as is evident in individuals with defects in any one of the ACDs. The phenotypes of these patients have some similar characteristics including hypoglycaemia, hypoketonaemia, muscle fatigue, hepatic lipidosis, and an organic aciduria that is often diagnostic of the enzyme de¢ciency [2]. MCAD is the best-studied member of the extended family as it has a very high incidence of genetic defects in humans [10]. Overlapping sequences homologous to the EST stSG297 were assembled from the Unigene public web server (http://www.ncbi.nlm.nih.gov/Schuler/ UniGene) into a 2238 bp contiguous fragment of DNA. Using reverse transcription^polymerase chain reaction (RT^PCR) on RNA from human adult brain and skin ¢broblasts, a 1610 bp fragment was ampli¢ed using the primers L2 and 40327-2 (dTGGGCTGTCACGTCTTG; dCCTAGACCAC-
AGGCGTCTTAAC; nt 382^398 and 1971^1992, respectively). DNA was sequenced on both strands using a combination of the Thermosequenase cycle sequencing kit (Amersham) and AmpliTaq cycle sequencing kit (Perkin Elmer) and the ABI Prism 377 Genescanner. The remaining 381 bp extending beyond the translation start signal was veri¢ed by sequencing clone 92A24 from the RPCII PAC human genomic library (UK HGMP Resource Centre) and 250 bp from the 3P end to the poly(A) tail was sequenced from I.M.A.G.E. clone 40327 [11]. The sequence obtained is characterised by a single open reading frame of 1248 bp encoding a predicted protein of 415 amino acids (Fig. 1). Two putative polyadenylation signals are located downstream of the translation termination codon (Fig. 1; nt 2172^ 2177 and 2199^2104). A search of the GenBank database using the BLAST algorithm [12] identi¢ed a high degree of similarity to members of the ACD family. As this is the eighth human ACD to be isolated, we have named this sequence ACAD-8. The cDNA sequence has been submitted to GenBank and assigned accession number AF126245. Alignment of the novel protein sequence with those of other human ACDs (Fig. 2) showed that it shares 28%, 32%, 32%, 33%, 34%, 36% and 38% identity with GCD, LCAD, VLCAD, IVD, MCAD, SBCAD and SCAD, respectively. Established members of this family share a similar degree of homology and the conserved invariant sequences in this study are similar to those reported previously [4,13]. There is no homology shared between family members in the amino terminal leader peptide (24 to 40 amino acids). Glutamic acid residue 401 of MCAD (Glu-376 of the mature monomer) is located at the active site of the enzyme and initiates catalysis by abstracting the substrate K-hydrogen as H [14,15]. This residue is conserved in all human ACDs except LCAD and IVD and corresponds to Glu-398 of the novel sequence. Several other amino acids with a known role in MCAD function are conserved in this novel protein including threonine-161 (position 162 in MCAD) and serine-167 (position 168 in MCAD) which form hydrogen bonds to the £avin moiety of MCAD [15]. A tryptophan residue at position 192 in MCAD, which is believed to have a role in electron transfer from the FAD bound to MCAD to ETF, is replaced by another aromatic
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Fig. 1. The complete human ACAD-8 cDNA sequence and its putative translation product. The termination signal is marked with an asterisk and the poly(A) addition signals are underlined.
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375
Fig. 2. Comparison of seven human ACD enzymes and human ACAD-8. Alignment was achieved using the Pileup program of the University of Wisconsin Genetics Computer Group with ¢nal manual re¢nements. The mature amino termini of the enzymes are indicated by boldface italic print. Residues conserved between ACAD-8 and any of the mature monomers are shown in bold and residues that are the same in all eight enzymes are listed as a consensus (CON). The active site glutamate in SBCAD, SCAD, MCAD, ACAD-8, VLCAD and GCD is marked with an asterisk. Positions marked by a number are described in detail in the text: 1, threonine-162 in MCAD; 2, serine-168 in MCAD; 3, tryptophan-192 in MCAD; 4, arginine-306 in MCAD; 5, glutamine-374 in MCAD; 6, glycine-378 in MCAD; 7, glutamate-325 in MCAD; 8, arginine-408 in MCAD.
6
residue, phenylalanine-191 in the novel protein and in LCAD. It is known from the crystal structure of MCAD that the tetrameric enzyme may be regarded as a dimer of two dimers [15]. Several residues crucial for the interaction of the two subunits within the dimer and some important for dimer^dimer interaction have been identi¢ed on the basis of the crystal structure [15] and functional studies [16]. Arginine306 and glutamine-374 form hydrogen bonds to the pyrophosphate moiety of the FAD of the neighbouring subunit of the MCAD dimer while glycine-378 forms hydrogen bonds to the ribose moiety of FAD of the neighbouring subunit. These residues are conserved in the novel sequence (arginine-302, glutamine-371 and glycine-375), indicating that the region involved in FAD binding and dimerisation in the novel protein is very similar to that in MCAD. Glutamate-325 and arginine-408 form an important salt bridge between the two pairs of dimers of the tetrameric MCAD and this salt bridge is also likely to form between the corresponding residues, aspartate321 and arginine-405, of the novel protein, suggesting that, like most other ACDs, it is a tetrameric enzyme. In contrast, although arginine at position 469 is conserved in VLCAD, glutamate is replaced by an arginine residue at position 385, excluding salt bridge formation, which is in agreement with the fact that VLCAD is a dimer rather than a tetramer [6,7]. The tissue distribution of the novel mRNA was determined by overnight hybridisation of a multi-tissue Northern blot (Clontech) with a 32 P-labelled 1127 bp PCR product (nt 382^1508, Fig. 1) in a bu¡er consisting of 0.5 M sodium phosphate bu¡er (pH 7.2), 1% BSA, 7% SDS, 1 mM EDTA and 50 Wg/ml denatured calf thymus DNA (Fig. 3). A band approximately 2.1 kb in size was detected at comparable levels in all tissues examined, except in liver and kidney, where it was only weakly expressed. This band correlates well with the size of transcript ex-
pected from the cDNA sequence. Two additional bands approximately 4.0 and 2.4 kb in size appeared clearly in this Northern blot even after stringent wash conditions. It is possible that these transcripts represent cross-reacting ACDs. Transcripts for previously characterised human ACDs range in size from 2^2.4kb [6,7,17^21] and larger products of 4.6 and 3.8 kb have been described for IVD [20]. The gene encoding this novel ACD has previously
Fig. 3. Northern blot analysis of ACAD-8 expression in di¡erent human tissues. The Northern blot contains approximately 2 Wg of poly A RNA from eight di¡erent adult human tissues : heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7) and pancreas (lane 8). RNA size marker bands are indicated on the right-hand side. The 2.1 kb transcript is marked with an asterisk.
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been localised by radiation hybrid mapping to the interval of human chromosome 11q25 in which the gene(s) encoding a novel familial form of histiocytosis with associated sensorineural deafness and joint contractures has been mapped (human transcript map: http://www.ncbi.nlm.nih.gov/science96). Localisation of the gene to this region was con¢rmed by PCR screening of CEPH YAC clones contained within an integrated chromosome 11 contig map that spans the interval de¢ned by D11S1320-11qter (http://mcdermott.swmed.edu). The novel cDNA was shown by RT^PCR to be expressed in the myeloid cell line KG-1 and in a human cochlear cDNA library. It was therefore considered a candidate gene for the 11q25 disease, although none of the a¡ected individuals show any features typical of ACD de¢ciency [2]. Mutation analysis was carried out by sequencing RT^PCR products from RNA extracted from EBV-transformed lymphoblast cell lines obtained from an a¡ected patient and her una¡ected father as well as control RNA. No nucleotide variations were found in the coding region of the a¡ected or una¡ected individuals or in a normal control. Therefore, this gene can now be excluded as a possible candidate for the disease locus. In this study we have isolated a cDNA encoding a novel member of the acyl-CoA dehydrogenase family, as de¢ned by considerable homology with other ACDs, and have described its pattern of tissue expression. Further studies are required to characterise the substrate speci¢city of this new member of the ACD family, as this is the primary characteristic used to de¢ne related enzymes [22]. Until such studies are carried out we propose that this eighth member of the human acyl-coA dehydrogenase gene family to be described should be referred to as ACAD-8. This research is supported by the Wellcome Trust, MRC, Birth Defects Foundation, NYRHA and YCRC.
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BBAEXP 91284 18-8-99