THEJOURNAL OF BIOLOGICAL CHEMISTRY ( 0
Vol. 266, No. 22, Issue of August 5, pp. 14686-14691, 1991 Printed in U.S . A.
1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Organization and Chromosomal Localization of the Gene for the El@Subunit ofHuman Branched Chaina-Keto Acid Dehydrogenase* (Received for publication, January 11, 1991)
Hiroshi Mitsubuchi, Yoshitaka Nobukuni, Fumio Endo, and IchiroMatsudaS From the Department of Pediatrics, Kumamoto University Medical School, Honjo1-1-1, Kumamoto 860, Japan
A defect in the E& subunit of the branched chaina- leucine, and isoleucine (Reaction 1) keto aciddehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an TPP,Mg2’ attempt to elucidate themolecular basis of MSUD, we R-COCOOH + COA-SH + NAD’ (1) isolated and characterized the cDNA of the EIBsubunit R-CO S-COA + CO, + NADH + H+ of BCKDH.Using thecDNA as a probe, a chromosomal gene related to E,B subunit of human BCKDH was The BCKDHcomplex consists of three catalytic components: isolated from human gene libraries. The gene of EIB dihydrolipoyl subunit is over 100 kilobases long and is split into 10 branched chain a-ketoacid decarboxylase (E1), exons. All of the splice donor and acceptor sites con- transacylase ( E2),and dihydrolipoamide dehydrogenase ( E 3 ) . form to the GT/AG rule. The transcription initiation El is further composed of two subunits, Ela and
[email protected] and site wasdetermined bynuclease S1 mapping and E2 components are specific to BCKDH. On the other hand, acid primer extension and was located47 bases upstream the E3 componentis common among the three keto from the initiation codon. A “CAAT” box and its re- dehydrogenase complexes, BCKDH, pyruvate dehydrogenase verse complement sequences were present at 39 bases (PDH),anda-ketoglutarate dehydrogenase. TheBCKDH and 76 bases upstream from the cap site, but there was complex alsocontains two specificregulatoryenzymes, a no “TATA” box-like sequence. There were three sets kinase and a phosphatase, responsible for regulation of the of sequences resembling the transcription factor Spl- catalytic activity through phosphorylation and dephosphorylbinding sites and twosets of sequences resemblingthe ation (1-3). enhancer core sequence. We also analyzed the chroEla isthecatalyticsubunitphosphorylated at 2 serine mosomal localization of the gene for the EIB subunit of residues responsible for regulation of the catalytic activityby BCKDH. Thegenewas mappedtochromosome 6. Knowledge of the gene structure of human BCKDH El@ covalent modification. Ez catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A and forms the subunit will facilitate further studies on the expression and regulation of this gene and provide necessary in- structured coreof the enzyme complex.T o this, El,E3,kinase, formation for analyses of mutations in patients with and phosphatase are bound through noncovalent interactions. MSUD. The functionof El@is unknown (1-3). Impaired BCKDH activity leads t o maple syrup urine disease (MSUD), an autosomalrecessive inborn error of metabolism (1,2). Several different phenotypes of MSUD have been Mammalianbranchedchaina-keto aciddehydrogenase elucidated on the basis of clinical features asfollows: classical, (BCKDH)’ (EC1.2.4.4) is a mitochondrial multienzyme com- intermittent, intermediate, and thiamine responsive type (1, plex catalyzing the oxidative decarboxylation of branched 2). Etiology of MSUDis heterogeneous, asmutationsin chain a-keto acids derived from amino acids such as valine, different regions of any of the BCKDH proteinscould lead to * This work was supported by aGrant in aidfor Scientific Research decreased functions of the entire complex (4-7).’ We reported that a defect in the BCKDH-EIP subunit is 04180553 from the Ministry of Education, Science and Culture of Japan, Grant 2-6 from the National Center of Neurology and Psy- one causeof MSUD (8,9). The isolation and characterization chiatry of the Ministry of Health and Welfare, Japan, and Grant of cDNAs encoding all or a part of the human BCKDH-Ela 63A-01 for Pediatric Research from Ministry of Health and Welfare. (10, ll),BCKDH-E,@ component(12, 13), BCKDH-E, comThe costs of publication of this article were defrayed in part by the payment of pagecharges. Thisarticlemusttherefore be hereby ponent (14-17), and E3 component (18,19), but not the gene marked “advertisement” in accordance with 18 U.S.C. Section 1734 structures of these subunits have beenreported. solely to indicate thisfact. To better understand the structure-function relationships, The nucleotide sequence(s) reportedin this paper has been submitted gene organization, and biosynthetic regulatory mechanisms, to theGenBankTM/EMBLDataBankwith accession number(s) 090382-090386. we isolatedandcharacterizedthe gene for thehuman $ To whom correspondence should be addressed. BCKDH-E1@ subunit. This gene which is over 100 kilobases I The abbreviations used are: BCKDH, branched chain a-keto acid was mapped tochromosome dehydrogenasecomplex; &a, decarboxylase a-subunit; E,& decar- (kb) long consists of 10 exons and 6. boxylase P-subunit; E P ,dihydrolipoyl transacylase; E3, dihydrolipo-
-
amide dehydrogenase; MSUD, maple syrup urine disease; PDH, pyruvate dehydrogenase;kb, kilobase(s);bp,basepair;SDS,sodium dodecyl sulfate; PIPES, piperazine-N,N’-bis(2-ethanesulfonic acid).
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I. Matsuda, Y. Nobukuni,H.Mitsubuchi, R. Heidenreich, Y. Indo, F. Endo, and S. Segal, manuscript in preparation.
14686
El/3Subunit of BCKDH
14687
TABLEI Exon-intron organization of the gene for the human BCKDH-E,P subunit Exon sequence are shown in capital letters, and intron sequences are in small letters. T h e numbers shown at the exon-intron junctions indicate ofpositions the corresponding amino acids in BCKDH-E,O the gene, deduced as from the BCKDH-E,Bc D N A sequence (13). Exon
Size
Intron
1 2
243
78
1 2
3
69
3
4
134
4
5
156
5
6
109
6
7
8 9 10
98 111 87 298
Size
Exon
Exon
Intron
kb
bp
7 8 9 10
17
0.8 125 0.7 2.6 >20 2.1 25 10
TAC
G
Tyr
Gly-16
GCA
G
Ala
Val-42
TAT
G
Tyr
Gly-65
GAT
CAG
ASP
Gln-109
ATC
AAG
Ile
LYS-161
GCA
G
Ala
Ala-198
ACT Thr TGT
CAG
CYS
LYS-267
GTT
CAG
Val
Gln-296
TAT
TGA
Tyr-342
Stop
Gln-230
AAG
gtgagccctgggact . . . . . . . . . .ttttgattttcacag
GG
CAA
Gly-16
Gln
gtaaccctgatatgt . . . . . . . . . .aatactgtttttcag
TA
ATA
Val-42
Ile
gtaagtaaataccta . . . . . . . . . .cttttctattttaag
GA
AAA
Gly-65
gtaagtgaatgaaca . . . . . . . . . .ctttctgaccctcag
ATT
L Ys
Ile-110
Val
gtatgttcatttatg . . . . . . . . . .aatctgtttttgcag
GTG
GTT
GTT
Val-162
Val
gtaaagattttcttt . . . . . . . . . .ctgttctgtatttag
CG
GAA
Ala-198
Glu
gtgagtagcattgat . . . . . . . . . . ttctctttatttcag
GTT
CAT
Val-231
His
gtatgaatataatgg . . . . . . . . . . tcttttctctttcag
TCT
GTG
Ser-268
Val
gtagagtaatttttg . . . . . . . . . . tgactctgtctgcag
GAG
GAA
Glu-297
Glu
probe, was also detected by primer extension analysis. From theseresults, the 5’ end of the BCKDH-Elp mRNAwas assigned to a position 47 bases upstream from the first nuRESULTS cleotide of the initiation triplet. The assigned 5‘ end was the Isolation and Characterization of the BCKDH-EIP Generesidue A, the generally preferred cap site (31, 32). The Two phage libraries constructed from human placenta and sequence CCAAT, resembling the canonical “CAAT”box, and human leukocytes were screened for the BCKDH-EIP gene. its reverse complement (ATTGG) arelocated at -39 and -75. Approximately 53 independent clones were isolated and anaThe latter sequence is situated at the usual location, that is lyzed by restriction enzyme digestion and partial sequencing (Fig. 1).These clones overlapped, except for two regions, and around 70-80 bases upstream from the cap site (32) (Fig. 2 A ) . spanned over 100 kb. Since a part of intron 3 and a part of A sequence resembling the canonical TATA boxwas not intron 6 were not cloned completely, the gene must be larger observed. Three setsof potential binding sites for the cellular than indicated in Fig. 1. To define positions and boundaries transcription factor Spl, CCGCCC (33), an inverted form of of the exon blocks, the restriction fragments identified by GGGCGG were present at positions -98 to -93, -56 to -51 Southern hybridization were subcloned and their sequences and -13 to -8. Two sets of Enhancer core sequence TGGAAA determined (Fig. 1, Table I). The gene was divided into 10 (34) were present at positions -532 to -527 and -311 to -306 exons which ranged from 69 bases (exon 3) to 298 (exon lo), (Fig. 2 A ) . The 3”untranslated region of the BCKDH-E,P and the nine introns ranged in size from 0.7 kb (intron 4) to gene contains 160 nucleotides (Fig. 2B).The site of the 20 kb longer (intron 3 and intron 6). Since the exons total polyadenylation signal was inferred from the cDNA (13). A typical poly(A)+ addition signal sequence AATAAA was not about 1.4 kb, 98.6% of the gene is occupied by introns. Allof the splice donor and acceptor sites conform to the found but another poly(A)+ addition signal sequence AAGT/AG rule (31) for nucleotides immediately flanking exon TAAT was detected 13 bases upstream from the poly(A)+tail. Chromosomal Localization of BCKDH-EIP Gene-The huborders (Table I). We found one nucleotide substitution between the cDNA sequence (13) and genomic sequence, A to man BCKDH-EIP cDNA probe detected 1.7-, 2.5-, 5.5-, 6.8-, G at nucleotide position 1347, which was present in the 3‘- 7.4-, and 11-kb hybridizing bands in the EcoRI-digested human DNAs, and detected 5.5- and l l - k b hybridizing bands in untranslated region. Characterization of the 5‘ and 3’ Ends of the BCKDH El@ the EcoRI-digested mouse DNAs. The 6.8-kb band was well Gene-The nucleotide sequence around the 5’ and 3’ ends of resolved from cross-hybridizing mouse sequences. The 1.7-, the BCKDH-EIP gene is shown in Fig. 2. The 5’ end of the 2.5-, and 7.4-kb bands could not be resolved from the mouse mRNA was determined by nuclease S1 mapping and primer sequences since the bands were too weak. The 6.8-kb band extension analysis (Fig. 3) and is numbered +l. The DNA was found in five hybrid cell lines (Fig. 4). The presence of fragment labeled at position 181 downstream from the first the 6.8-kb band correlated only with the presence of human nucleotide of the initiation triplet was used as aprobe for the chromosome 6 (Fig. 5), thereby indicating that the human S1 mapping. We noted a protectedgene fragment of 228 bases BCKDH-EIP gene is located on that chromosome. for kidney poly(A)+ RNA. The 228-base long fragment, startDISCUSSION ing from the primer labeled at the same position as the S1 We determined the structural organization of the human ’’ Portions of the paper (including “Experimental Procedures” and chromosomal gene for the BCKDH-E1@subunit by analyzing Figs. 1,3,and 7) are presented in miniprint end at of the this paper. the overlapping genomic clones obtained from two different Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the human gene libraries. This 100-kb long gene is one of the Journal that is available from Waverly Press. largest thus far determined (35). Since the sum of the length EXPERIMENTALPROCEDURES3
EIP Subunit of BCKDH
14688 A -511
-541 -581 -571 -561 -551 -591 T T C T ATTCAGTGGA TTTGCAGGAT GAAATCTAAG TTTACCTCTT TCATCTCTCA
-521
-531 ."
-501
T T T A A A T ~ T GGAAF~ACGAA
-441 -431 -421
-491
ATCAATG ATCTATATAT GCTTATTAAG DR1 - -411
-481
7
7
1
""K
-391 DR2d
-181
CAGTGACTACAGCAGTTTTTCGAAAGTCAAAACAATAACCAATGCCAATCAGTAGCTCTCTTCACCTCTCACACAATAAT
-351
-141
-331
T Y,\U -321 -271
-461
-451
CATTTTAAGG AGGGTGTGAA T ~ G T G A AAGCTTTGAG
DR2 -371
.."
na7-
-161 AGAGACAGGA
I
-281 -111 -291
-301
CAGGATAAAA AAGGGTTACA TTTAAA~AAA AGTATGGATG T T A A A C C A * T Z Z ~ G G C A
EC
-221 -231 -241 -251 -261
CTGTAAATGA CACCGGRRAAGATCGCCTCC
-2RR4"
-201
-181 -191
FIG. 2. Nucleotide sequence of 5' AGGCACTGTG T G G M C T T T G CAAACTCGGGTCCTCACCCG AGGACCGCAC GGCCGACCTC GGGGAGGCTC CACCCCAGAAACTATTTCAT ( A ) and 3' ( B )termini. The sequence DR4-l;1 -161 -151 -141 -131 -121 -111-91 -101 on both strands was determined. A , the TGGCGCCACCTCTAGCCCACACTTCCCCTCTTCCGCATGC GCACAAGGAG CAGAAGCCGA CCAGCTCGTCACGTCGCCTT c c B T sequence underlined (+1 to +243) despl -1 -81 -71 -51 -61 -41 -31 -21 -11 notes the first exon. The initiation codon AAATTTCCAG T T C C TCTGTTTCAT ~ ~ GGGCTCTGGCCCG -TGG AAGCCTCCTC G G G T G C X ~ ~ ~ C C E "CAAT' SP1 R"CAAT" is in triangles. The boxed area with the 50 60 70 80 90 1 10 20 30 0 4 ' " RGGCGGCGTGCGGCTGCATAGCCTGAGAATCCCGGTGGTG AGCGGGGATG GCGGTTGTAGCGGCGGCTGCCGGCTGGCTACTCAGGCTCA symbols resembles the following seAAA quences: CAAT, CAAT box (32); S P l , 130 120 DS2 100 140 150 160 170 180 110 GGGCTGCAGGGGCTGAGGGGCACTGGCGTCGGCTTCCTGGCGCGGGGCTGGCGCGGGGCTTTTTGCACCCCGCCGCGACT GTCGAGGATG Spl-binding site (33);EC, enhancer core sequence (34). The bracket with 220DR5 the in-210 200 190 231) DR5
