PII: 0014-5793(90)80215-5 Volume 262, number 2, 305-309 J?EBS 08265 March 1990 Molecular cloning of the mature Elba/I subunit of human branched-chain a-keto acid dehydrogenase complex Jacinta L. Chuang, Rody P. Cox and David T. Chuang ~e~ar~~ts of Bio~~~istry and Intern~i Me~~&~e, U~~~E~s~ty of Texas Southwestern Med~ol Center, basil, TX 75235, USA Received 29 January 1990 We have isolated a cDNA encoding the El b-b subunit of the human branchedchain cl-keto acid dehydrogenase complex. The human El b-p cDNA is 1401 base pairs in length. It encodes the entire mature Elb-B subunit consisting of 342 amino acid residues, and a mitochondrial targeting prese- quence of 31 residues. The calculated molecular mass of the mature &man Elb-/I subunit is 37851 Da, and the calculated isoelectric point is pH 5.18. A hydro~thy plot shows that the human Etb-B subunit is highly hydrophobic. Northern blot analysis shows that the human Elb-/I mRNA is approximately 1.4 kb in size. It is present at the normal level in fibroblasts from two unrelated maple syrup urine disease patients. cDNA; Branched-chain Elb-8; Nucleotide sequence; Amino acid sequence; (Human liver) 1. INTRODUCTION [4]. The isolation and expression of the bovine Elb-fl cDNA will be described elsewhere’. The mammalian branched-chain cu-keto acid dehydrogenase complex (the branched-chain complex) catalyzes the oxidative dec~boxylation of the a-keto acids derived from the transamination of the branched- chain amino acids leucine, isoleucine and valine. The multienzyme complex is associated with the inner- membrane of the mitochondrial matrix, and is both structurally and mech~istic~ly analogous to pyruvate and cy-ketoglutarate dehydrogenase complexes [ 1,2]. The mammalian branched-chain complex has 3 catalytic components: a branched-chain cu-keto acid decarboxylase (Elb), a dihydrolipoyl transacylase (E2b) and a dihydrolipoyl dehydrogenase (E3). The enzyme complex also contains two regulatory enzymes, a specific kinase and a specific phosphat~e that regulate the activity of the complex via a phosphorylation- dephosphorylation cycle [3]. Our laboratory has recent- ly isolated cDNAs encoding the entire Elb-fl subunits of both bovine and human branched-chain complexes. In this paper, we report for the first time the isolation and sequencing of the human Elb-fl cDNA. The availability of this cDNA will facilitate investigations into the molecular basis of inborn errors involving the branched-chain complex, i.e. maple syrup urine disease 2. MATERIALS AND METHODS 2.1. Screening of a Agt-fl library A bovine Elba cDNA isolated with the anti-Elb-fl antibody’ was radiolabeled by the random-priming method [5]. It was utilized as a probe to screen a Agt-11 library of human fetal liver cDNA (Clontech). Positive clones were plaque-purified and subcloned into the Bluescript SK- vector as described previously [a]. 2.2. Nucleotide sequencing The nucleotide sequencing of cDNA inserts was carried out by the dideoxynucleotide chain termination method 171 using T7 DNA polymerase. The templates used were double-stranded Bluescripts (pBSSK_) containing the cDNA inserts. These double-stranded templates were sequenced in both directions. The primers used for se- quencing included SK, KS, T3, T7, M13-20, the Mlf-reverse primer, and four 20-bp synthetic oligonucleotides specific for internal se- quences of the cDNA insert. 2.3. Peptide sequencing and CNBr digestion The sequences of the amino-terminal region and CNBr fragments of the bovine Elb-fl subunit were determined by gas-phase microse- quencing as described previously f8]. For CNBr digestion, the bovine Elb-fl subunit (20 pg) isolated by electroelution from SDS- polyacryiamide gel was mixed with 50 pI of CNBr solution (5 mg in 1 ml of 70% formic acid). The mixture was incubated at 25°C in the dark for 48 h. The digest was electrophoresed in 15% SDS- polyacrylamide gel and transblotted onto a polyvinylidene difluoride membrane for peptide sequencing [S]. Correspondence address: D.T. Chuang, apartment of Bio- chemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 752359038, USA 2.4. Cell culture and Nortber~ blot ~natys~ Abbreviations: Elb, branched-chain cr-keto acid decarboxylase; Elp, pyruvate dehydrogenase; E2b, dihydrolipoyl transacylase; E3, dihydrolipoyl dehydrogenase; SDS, sodium dodecyl sulfate; MSUD, maple syrup urine disease; bp, base pairs; kb, kilobase pairs Fibroblasts derived from MSUD patients (PK and Lo) [6] were cuhured as described previously [6]. Total or poly(A)+ RNA was prepared from normal fibroblasts, and Northern blot analysis was carried out using random-primed cDNA probes 161. ’ J.L. Chuang, C.W. Fisher, M.A. Hale, R.P. Cox and D.T. Chuang, submitted for publication. Published by Elsevier Science Publishers B. K (Biomedical Division) 00145793/90/$3.50 0 1990 Federation of European Biochemical Societies 305 Volume 262, number 2 FEBS LETTERS March 1990 -30 -20 -LO ArpLruProProAr~ArgLeuProClyAlaClyLeuAlaAc~ClyPheLeull~sProAlaAlaTt,rVnlCl,rAspALa CGGCTCCCTCCACCTCCGCTCCT~CGCGCGCCTGCCCCCCACCATGCG I 10 20 AlaGlnAr8ArgClnValAlaHisPheThl-PheClnProAspPraGluP~oArqGluT~d:lyClnThrGlnLysHet GCCCAGAGGCCGCAGGn;GCTCA~ACCCAC 30 40 AsnLeuPheClnSerValThrSerAlaLauAEpAsnSerLsp MTCTTTCCCAGTCTGTAACAAG'IlXCTTGGATMCTCATTGGCCAAAGATCCTACTGCAGTAATA~TGAAGAT 50 60 70 ValAlaPheGlyClyValPheArgCysThrVolGly~~~~spLysT~lyLysAspAr8ValPh~sn~rP~o GTTGCC GCGAGACAAATATGCAMAGATAGAGTlTlT.UTACCCCA 130 140 150 AsnCysGlySarLeuThrIleArgSe~ProTrpClyCysValGly~~ly~~euTyrHisSe~~e~Pr~lu AACTGTGGAAGCCTCACTAlXCGGTCCC-?XTG TTGGTCA-ATCATTCTCAGAGTCCIWA 180 190 200 CysIleGluAspLysArnProCysIle~ePheGl~oLysIleLeuTyrArgAlaAlalrlaGluGluValProIle TGCATAGAGGATAAAAUCC ~CC~~G~~CCTATA 210 220 GluPr~TyrAsnIleProLe~~~GluValIl~~Gl~l~e~~pVal~rLeuVa~~T~~l~~ GAACCATACMCATCCCACTGTCCCAGGCCGMGTCATACAGGAAGGGAGTGATGTTACTCTAG-CT 230 240 250 GlnVal~isValIleA~8GluValAlaSerHeUl~VSGluLysLeUGlVValSe~CysGluValIl~p~~rg CAGGlTCATGTGATCCGAGAGGTAGCTTCCAlWXAMAGAAU GCTTGGAG~CTTGTGACTGAGG 260 270 280 Tl~rileIleProTrpAspValAspThrIleCysLysSerValIleLysSerGlyA~8LeuLeuIleSerHisGluAla ACTATAATACC~~A~T~ACAC~~MGTCTGTGATC~TCA~CGA~CT~TCAGTCACGA~CT 290 300 ProLeuThrGlyClyPh~laSercluIleSe~Se~ThrValGlnGl~Gl~CysPheLeuAsnLeuCle CCCTIY;ACACGCCGCmCC~~TCACCTCTACAG~CA~AGGM~~C~MCCTAGA~CTC~ATA 310 320 330 SerArgValCysGlyTyrAsp~~ProPheProBi.IlePheGluP~oPhe~~IleProAspLysT~pLysCysTy~ TCAliGAGTATGTGGTPATGACCA~C~~~MC~~~ACATCC~GAC~~~GT~AT 340 342 AspAlaLeuArgLysMetIl&~Ty~*** GATGCCCTTCGACAACTATCGACCATATAGAAAAGCGGAAGATTATGACTAGATATGGAAATAT=I=l=C WCCC 78 156 234 312 468 546 624 702 780 850 936 1014 1092 1170 1248 1326 1393 1401 Fig. 1. Nucleotide and deduced amino acid sequences of the human Elb-fl cDNA (hEl,&l). The numbers on the far right correspond to ordinates of the last nucleotide in each row. The numbers above the sequence refer to the positions of amino acids relative to the amino-terminal Val residue (position 1) of the mature human Elb-/3 polypeptide. Amino acid sequences corresponding to the amino-terminal regions of the bovine Elb-fl subunit (residues 1 to 28) and a CNBr fragment (residues 240 to 248) determined by peptide sequencing are underlined. The putative polyadenyla- tion signal AATAAA (ordinates 1393 to 1398) is also underlined. 306 Volume 262, number 2 FEBSLETTERS March 1990 3. RESULTS AND DISCUSSION hybridized with the 1393 bp bovine Elb-fl cDNA’. Nucleotide sequencing disclosed that the 3 human The screening of the Xgt-1 1 library (4QOOOO pfu) pro- cDNA clones were identicaf. As shown in fig.1, the duced 3 human cDNA clones (hEl&1, -2 and -4) that d b -30 -20 -10 I H I. I’ I’ H H I, P G A G I. A H C P 1. II I’ A A 1’ ” E 0 A A Q R H 9 V A II F ‘r F E ________---__-----__------_--__-----~~~~,, -30 -20 -10 -L HAAVSGLVAETPSEVSGl,LKRRFllUTAPAA------ a b a b a b a b P b a b c 320. _ - 330 - ?-s3*orl a b c human E 1 b-,8 cDNA (hE l/3- 1) is 1401 bp in length. It Rg.2. Comparison of the amino acid sequences of three Elfi polypeptides. The deduced primary structures of human El b-p (sequence a) (the pre- sent study), Ps~do~o~~ Etb-8 (sequence b) 11 l] and the related Elp subunit (sequence c) of the human pyruvate dehy~ogenase complex [12] are aligned for maximal identity. The boxed residues are those which are identical between any two of the protein subunits. 307 Volume 262, number 2 FEBS LETTERS March 1990 encodes a mature peptide of 342 amino acids and a mitochondrial presequence of 31 residues. The insert contains a 282 bp 3 ‘-untranslated region with no po- ly(A)+ tail, although a potential polyadenylation signal AATAAA is present (underlined bases 1393 to 1398). The fidelity of the human Elb-fl cDNA is established by the matching of the deduced human amino acid se- quence with the determined bovine El\>-P sequence in the amino-te~inal region (underlined residues 1 to 28) and in a CNBr fragment (underlined residues 240 to 248) (fig.1). The only substitutions are in positions 13, 26 and 242 which are Val, Ala and Gln respectively in the bovine Elb-fl subunit. The calculated molecular mass is 37 851 Da and the isoelectric point is 5.18 for the human Elba subunit. A hydropathy plot according to Kyte and Doolittle 191 shows that the entire human Elb-,& chain is highly hydrophobic. Hydrophobicity (> 1.3) is invariably associated with predicted a-helices or P-pleated sheets based on Chou and Fasman [lo] (data not shown). It is tempting to suggest that the Elb-fi subunit is buried in- side the hydrophilic Elb-a subunit to form an Q&Z structure. The relatively inaccessible topography may explain the poor antigenicity of Elb-,@ when the purified bovine Elb component is used as an antigen 181. To assess possible sequence conservation, the deduc- ed primary structures of human EIb-8, Pseudomonas Elb-b Ill] and the human Elp-fl of the pyruvate dehydrogenase complex [12] are aligned for maximal identity as shown in fig.2. There is significant sequence conservation throughout the entire stretches of the 3 pol~eptides. The identity over a span of 342 amino acid residues is 46% between human Elb-,& and Pseudomonas E 1 b-B, and 3 1% between human E 1 b-P and human Elp-, subunits. The lengths of the Eli3 subunits are also similar with 342, 329 and 352 residues for human Elb-,&, pseudomonas Elb-,i? and human Elp-,&, respectively. The significant sequence conserva- tion suggests that these functionally related El@ subunits will have similar secondary, tertiary, and quaternary structures. The results also support the view that the genes for the Elfl subunits were evolved from a common ancestor. MSUD is genetically heterogeneous [ 131 as the branched-chain complex is encoded by at least 6 struc- tural genes. A mutation in any of these genes could result in the dysfunction of the branched-chain complex and consequently the MSUD phenotype. We have shown previously [6] that the Elb-cu mRNA is present at the normal level in fibroblasts derived from a Men- nonite MSUD patient (P.K.); however, both Elb-a and Elb-fl subunits are markedly reduced as observed by Western blotting. In fibroblasts from a second unrelated MSUD patient Lo, the Elb-lr mRNA was signific~tly reduced, and both the Elb-cu and Elb-p subunits were nearly absent. Fig.3 shows that the Elb-P 308 285 - IBS- Elb,8 - - Actin -Elbp Actin - Fig.3. Northern blot analysis of the Elb-/3 mRNA in normal and MSUD fibroblasts. Total (indicated) or poly(A)+ RNA prepared from normal or MSUD ~brobl~ts was subjected to Northern blotting with the hEbY- cDNA as a probe. P.K. is a Mennonite classical patient. Lo is a varient MSUD patient. The actin mRNA served as internal standards was probed either separately (panel A) or along with the Elb-fl mRNA using a “‘P-labeled cDNA mixture (panel B). The lower level of normal Elb-b mRNA in panel B resulted from the lower amount of poly(A)+ RNA applied. The size of human Elb-fi mRNA (1.4 kb) was estimated using 28 S and 16 S rRNAs as standards. mRNA (1.4 kb) is present at normal levels in fibroblasts from P.K. (panel A) and Lo (panel B). With respect to PK., the MSUD mutation might involve either the Elb-cr or Elb-fl gene. However, in Lo the mutation af- fects the Elb-cu gene as demonstrated by a marked reduction in its level of mRNA. The presence of a nor- mal level of Elb-,0 mRNA in Lo thus strengthens the previous suggestion that the failure to assemble into a stable a& structure results in degradation of the un- paired Elb-fi subunits in the cell [6,14]. Acknowledgements: This work was supported by Grants DK26758 and DK37373 from the National Institutes of Health and Basic Research Grant l-1 149 from the March of Dimes Birth Defects Foun- dation. REFERENCES [II PI 131 141 PI 161 Reed, L.J., Pettit, F.M., Yeaman, S.J., Teague, W.M. and Bleile, D.M. (1980) Proc. FEBS Meet. 60, 47-56. Yeaman, S.J. (1989) Biochem. J. 257, 625-632. Randle, P. J., Fatania, H.R. and Lau, KS. (1984) in: Enzyme Regulation by Reversible Phosphoryiation-Advances (Cohen, P. ed.) pp. l-26, Elsevier, Amsterdam. Dancis, J., Jansen, V., Hutzler, J. and Levitz, M. (1963) Biochim. Biophys. Acta. 77, 523-524. Reinberg, A.P. and Volgelstein. B. (1983) Anal. Biochem. 132, 6-13. Fisher, C.W., Chuang, J.L., Griffin, T.A., Lau, KS., Cox, R.P. and Chuang, D.T. (1989) J. Biol. Chem. 264, 3448-3453. Volume 262, number 2 FEBS LETTERS March 1990 [7] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 181 Hu, C.-W.C., Lau, K.S., Griffin, T.A., Chuang, J.L., Fisher, C.W., Cox, R.P. and Chuang, D.T. (1988) J. Biol. Chem. 263, 90079014. [9] Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132. [lo] Chou, P.Y. and Fasman, G.D. (1978) Adv. Enzymol. 47, 45-148. [Ill Burns, G., Brown, T., Hatter, K., Idriss, J. and Sokatch, J.R. (1988) Eur. J. B&hem. 176, 311-317. 1121 Koike, K., Ohta, S., Urata, Y., Kagawa, Y. and Koike, M. (1988) Proc. Natl. Acad. Sci. USA 85, 41-45. [13] Lyons, L.B., Cox, R.P. and Dancis, J. (1973) Nature 243, 533-535. [14] Ho, L., Hu, C.-W.C., Packman, S. and Patel, MS. (1986) J. Clin. Invest. 78, 844-847. 309