Cloning and cDNA sequence of the β-subunit component of human pyruvate dehydrogenase complex

Cloning and cDNA sequence of the β-subunit component of human pyruvate dehydrogenase complex

Gene. 86 (1990) 297-302 Elsevier 297 GENE 03354 Cloning and e D N A sequence of the p-subunit component of human pyruvate dehydrogenase complex (Re...

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Gene. 86 (1990) 297-302 Elsevier

297

GENE 03354

Cloning and e D N A sequence of the p-subunit component of human pyruvate dehydrogenase complex (Recombinant DNA; deduced primary amino acid sequence; inborn errors in pyruvate metabolism) Lap Ho and Mulehand S. Patel Department of Biochemistr), and Pew Centerfor Molecular Nutrition, Case Western Reserve UniversitySchool of Medicine. Cleveland, OH 44106

(e.s.a.) Received by D.M. Skinner: 11 July 1989 Accepted: 28 August 1989

SUMMARY

Two eDNA clones (ZEIpl, 1469 bp; ~Et/~2, 1437 bp) encoding the p-subunit of the pyruvate dehydrogenase (Et) component of the human pyruvate dehydrogenase complex were isolated from a human liver ~,gtll cDNA library. The composite human liver Etp eDNA encoded the entire mature EtP [329 amino acids (aa)] as well as a portion (26 aa) of the E~p leader peptide. Significant discrepancies were identified between the nucleotide and deduced aa sequences of human liver Em~cDNAs and the corresponding sequences of a previously reported cultured human foreskin fibroblast E~/~cDNA [Koike et al., Proc. Natl. Acad. Sci. USA 85 (1988) 41-45]. The composite human liver Etp cDNA generated in this study provides a reference sequence for investigating the structure-function relationship of human E~/~and for characterizing genetic mutations in patients with Et deficiency.

INTRODUCTION

The mammalian PDC is comprised of multiple copies of three catalytic component enzymes, namely pyruvate dehydrogenase (Et), dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase (Reed, 1974). El catalyzes the initial irreversible decarboxylation of pyruvate (Reed, 1974). It is a tetrameric enzyme composed of two copies each of an ~ and/~ subunit (Reed, 1974). It has been proposed that the Et • subunit catalyzes the pyruvate decarCorrespondence to: Dr. M.S. Patel, Department of Biochemistry, Case Western Reserve University School of Medicine, 2119 Abington Road, Cleveland, OH 44106 (U.S.A.) Tel. (216)368-3354; Fax (216)368-4544. Abbreviations: aa, amino acid(s); bp, base pair(s); ~Gal, p.gaiactosidase; eDNA, DNA complementary to RNA; Et, pyruvate dehydrogenase; Et, gene encoding Et; kb, kilobase(s) or 1000bp; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PDC, pyruvate dehydrogenase complex; SDS, sodium dodecyl sulfate; ss, single strand(ed). 0378-11191901503.50 © 1990ElsevierSciencePublishers B.V.(BiomedicalDivision)

boxylation reaction while the subsequent reductive acetylation of E2 iipoyl moieties is catalyzed by the EIP subunit (Roche and Reed, 1972; Hubner et al., 1978). There have been more than 100 reported cases of PDC deficiency. Analyses of PDC component enzymatic activities have established that the majority of PDC-deficient patients have low levels of Et activity only (Robinson et aft., 1987; Wexler et al., 1988). Since Et is a heterotetrameric enzyme and it is activated by a specific phospho-Et phosphatase (Reed, 1974), findings of low levels of Et activity in these patients could be due to defects of either the Et~ subunit, the Et~ subunit or the phospho-Et phosphatase. In isolated cases of Et deficiency, the diagnosis of specific E ~ defect has been based on finding of low levels of Et~ mRNA in the presence of normal levels of E ~ (Wexler et al., 1988). In the majority of cases of Et defects, however, it is not possible to identify mutations specific to either the Et~eor the Etp peptide by simple analysis of enzymatic activities, protein or mRNA profdes. The human Et~ gene has recently been identified on the X-chromosome (Brown

298 (b) Sequence analysis of E~p cDNAs

etal., 1989), indicating that Et-deficiency among female patients is due likely to specific Et~ mutations. In fight of recent advances in the application of the PCR technique (Wong et al., 1987), direct analysis of specific E~ mRNAs is expected to be the method ofchoice for analysis ofhuman Et mutations. This experimental approach for diagnosis of genetic defects, however, requires the availability of nt sequences which accurately represent normal human Et and ~mRNAs. Here, we present the characterization oftwo human liver E~/~cDNA clones. Analysis of these two clones revealed significant differences between the sequence of human liver Et~ cDNA and that of the previously reported cultured human foreskin fibroblast Et~cDNA (Koike etal., 1988).

The ;tEmpi eDNA contained 1469 bp, with the 5' terminus located at nt 65 of the composite human liver E+B cDNA (Figs. 1 and 2). The Y-tmtranslated region of the ZE~/tl eDNA contained two polyadenylation signals: a variant signal (ATI'AAA) and a consensus signal (AATAAA) that were located at nt 1417 and 1463, respectively, of the composite human liver El/~ eDNA (Fig. 2). A poly(A) tail of 50 nt was located 16 bp downstream from the consensus polyadenylation signal (Fig. 2). The translation start codon (AUG) was not present in the 5' end of the eDNA, indicating the ZE~/I1 cDNA did not encode the entire Elp prepeptide. The second EIP cDNA clone (~,E1/~2)contained 1437 bp. The 5' terminus of the ~E~82 cDNA was located at nt 12 and the eDNA extended up to nt 1435 of the composite human EI/~eDNA (Fig. 2), followed by a short poly(A) tail of 13 nt (not shown). The ZEtp2 cDNA contained only the variant polyadenylation signal which was located 14 bp upstream from the ZEta2 polyadenylation site. Thus, both the variant and the consensus polyadenylation signals are utilized during in vivo processing of the E~p mRNA. The codon for translation initiation, methionine, was not present in the ~ E t ~ eDNA. Re-screening the same human liver Zgtl I library using a 32P-labeled ZEI~2 eDNA probe generated by the random priming method (Feinberg and Vogelstein, 1983) was not successful in identifying clones which would further extend the 5' of the ~.E,p2 eDNA.

EXPERIMENTAL AND DISCUSSION

(a) Isolation of E ~ eDNA clones We have reported the isolation and partial characterization of an E,/~ eDNA clone (~E,/~I) from a human liver Zgtl I cDNA library (Ho etal., 1988). Its identity was confirmed based on partial nt sequence analysis and our aa sequence analysis ofthe N terminus of purified bovine heart Et/~(Ho et al., 1988). The ,~E,/~I eDNA clone was not large enough to encode the entire Et/~ prepeptide (Ho etal., 1988). To isolate larger Et~ eDNA clones, ~Gal fusion proteins from six additional putative £ ~ eDNA clones from the same ~gtl I libr~xy were generated using a plate lysate system (Dong etal., 1986). The sizes of these fusion proteins were compared in an immunoblot using mouse anti-~Gal antibodies (not shown). A phage ~. recombinant Q.E~/~2) producing the largest fusion protein was selected for sequencing.

(e) Amino acid sequence of human liver EIP The composite nt sequence of human liver E,~ eDNA as well as the deduced aa sequence of the human liver E,/~ prepeptide are presented in Fig.2. The N-terminal aa ofthe

~ql3s

(A)+ s N

s'

kb

P

~r"

O

I"" 0.2

Pv

E

P

X

I

I

I

I

I 0,4

I 0,6

" I O.O

I 1.0

s' "'1 1.2

I 1,4

I 1,6

Fig, I. Sequencing strategies and a partial restriction endonuclease map of human liver EI~ eDNA clones (~Ei~l and ~El~2). Both ~,El~l and ~El~2 cDNAs were subcloned into Ml3mpJ9. Overlapping Ml3mpl9 deletion clones were generated using ass directional deletion method (Dale et el., 1985). Sequencing of ss recombinant Ml3mpl9 DNA was performed by the dideoxy chain-termination method employing the MI3 universal primer and Sequenase® (U.S. Biochemical Co.; Sanger et el., 1977). Regions on the ZEsg2 DNA that were not conveniently covered by the directional deletion procedure were sequenced using ol;go primers synthesized based on the sequence of our E,# cDNAs. Blackened boxes, coding regions for the mature Eggpept/de; hatched boxes, coding regions for portions of the Etg leader peptide; thin fines, untranslated regions; horizontal arrows, starting position, direction and extent of sequence determinations. Asterisks mark the sequences analysed with oligo primers. E, EcoRI; N, Nael; P, Psll; Pv, PVull; X, Xbal.

299 TABLE I Discrepancies between the nt and the deduced aa sequences from human liver Etp cDNA and the corresponding sequences from a previously reported cultured human foreskin fibroblast E ~ El~ cDNA (Koike et al., 1988) aa No." (nt No.)

nt and aa sequences b

Remarks c

leader sequence *

- 24 to - 17

*

*

*

*

L V R R P L R E TTG GTG C G G AGA CCC CTT CGG GAG TTG G T T G C G GAG ACC CCT T C G GAG L V A E T P S E

insertion (1") and deletion ((3); frame shi~

- 2 to +2 (85 to 96)

A A L Q GCT GCG CTG CAG GCT G C C GTG CAG A A V Q

G~Cand C-, G

115 to 117 (433 to 441)

R G P AGG GGA CCC AGG GGG CCC

(19 to 42)

Mature polypepflde

R

129 to 131 (475 to 483)

G

A-.G

P

S Q C TCA CAG TGC TCA CAG TGC S

G

C

*

*

mistake in deducing aa

*

*

*

*

*

*

182 to 192 (634 to 666)

F P P E A Q S K D F L TTT CCT CCG GAA GCT CAG TCA AAA GAT TTT CTG TTT CTC CGG AAG CTC AGT CAA AAG ATT TTG CTG F L R K L S Q K I L L

deletion (C) and insertion (G); frame shi~

279 to 283 (925 to 939)

1 M E O P ATC A T G GAA GGTCCT ATC AAT GGA AGTCCT

insertion (A) and deletion (G); frame shi~

I

N

G

S

P

3'-Untranslated region A-*C; deletion (G); C-~ T

(! 168 to 1182)

ATA AAG GAA AAC GAT ATA ACG AA AAT GAT

(1234 to 1242)

GTA TGG AAA GTA TG AAA

deletion (G)

(1318 to 1326)

TAT ATC ACA TAT ATA ACA

C-* A

(1428 to 1440)

AAT ACA CAT TrA AAT ATA CGT TrA

C--, T A --, G

(1477 to 1483)

TAA CAT G TAT AGC TAG

a stretch of 4 nt changed to 6

a The numbering of aa and nt (in parentheses) is based on the composite human Esp eDNA in Fig. 2. b The upper two sequences of each four-line entry are derived from human liver EIP cDNAs (Fig. 2) and the lower two sequences from foreskin E,~ cDNA (Koike et al., 1988). The resulting changes in the deduced liver EIp prepeptide are identified by asterisks. c Relative to Fig. 2, these are the discrepancies present in foreskin EiP eDNA.

300 IAT~ A CCG A GCG A G ~ A TCT S GGC G TTG L GTG GV CGG R A RG A C CPC _ CTT L CGG R GAG E CTC V TCC S GGG C CTG L CTG L

K

AGG CGC TTT CAC TGG ACC GCG CCC GCT GCG CTG CAG GTG ACA GTT CGT CAT GCT A T A A A T R R F H W T A P A A L Q V T V R D A I N

-1 G

M

D

E

E

L

E

R

D

E

K

V

F

L

L

C

CCC CAG TAT OAT GGG GCA TAC AAG GTT ACT CGA GGG CTG TGG A A G A A A T A T A Q Y D G A Y K V S R G L N K K Y

E

E

V

GGA GAC AAG G D K

ACG ATT ATT CAC ACT CCC ATA TCA GAG ATG GGC TTT GCT GGA ATT GCT GTA GGT GCA GCT R

I

I

D

T

P

I

S

E

M

C

F

A

G

I

A

V

G

A

A

ATG GCT GGG TTG CGG CCC ATT TGT GAA TTT ATG ACC TTC AAT TTC TCC ATG CAA GCC ATT M

A

G

L

R

P

I

C

E

F

120 (10)

+1

CAG GGT ATG OAT GAG GAG CTG G A A A G A GAT GAG /tAG GTA TTT CTG CTT GGA GAA GAA GTT

Q

60

(-11)

M

T

F

N

F

S

M

Q

A

I

180 (30) 240

(so) 300 (70) 360

(90)

420

GAC CAG GTT A T A A A C TCA GCT GCC AAG ACC TAC TAC ATG TCT GGT GGC CTT CAG CCT GTG D Q V I N S A A K T Y Y M S G G L Q P V

(11o)

CCT ATA GTC TTC ACG GGA CCC AAT GGT GCC TCA GCA GGT GTA GCT GCC CAG CAC TCA CAG P I ~ F R G P N G A S A G V A A Q H S Q

(13o)

TGC TTT OCT GCC TGG TAT GGG CAC TGC CCA GGC TTA A~G GTG GTC ACT CCC TGG AAT TCA C F A A N Y G H C P G L K V V S P N N S

s4o (150)

GAG CAT OCT AAA GGA CTT ATT A A A T C A GCC ATT CGG OAT AAC AAT CCA GTG GTG GTG CTA

6OO (170)

E

D

A

K

G

GAG AAT G A A T T C A T G E

N

E

L

M

L

I

K

S

A

I

R

D

N

N

P

V

V

V

L

TAT GGG GTT CCT TTT G A A T T T CCT CCG G A A G C T CAG T C A A A A OAT Y

G

V

P

F

E

F

P

P

E

A

0

S

K

D

480

660 (190)

ATA G A A A G G C A A G G A ACA CAT ATA ACT GTG GTT I E R Q O T H I T V V

720 (21o)

TCC CAT TCA ACA CCT GTG GGC CAC TGC TTA GAA OCT GCA GCA GTG CTA TCT AAA CAA GGA S H S R P V G H C L E A A A V L S K E G

780 (230)

GTT GAA TOT OAO OTG ATA AAT ATG COT ACC ATT AGA CCA ATG OAC ATG OAA ACC ATA GAA V E C E V I N M R T l R P M D H E T I E

84o (250)

OCC AOT GTC ATG AAO ACA AAT CAT CTT GTA ACT GTG GAA OOA OGC TOO CCA CAO TTT COA A S V M K T N H L V T V E G G W P Q F G

900 (270)

GTA GGA GCT GAA ATC TGT GCC AGG ATC ATG GAA GGT CCT GCG TTC AAT TTC CTO GAT OCT V O A E I C A R I H E O P A F N F I, D A

(290)

CCT OCT OTT COT GTC ACT GOT OCT OAT GTC CCT ATO COT TAT COA AAO ATT CTA OAO GAC P A V R V T O A D V P H P Y A g I L E D

1020 (310)

AAC TCT ATA CCT CAO GTC AAA GAC ATC ATA TTT GCA ATA AAG AAA ACA TTA AAT ATT TAG s s z e Q V g D I I F A I g g T L N I

1080

TTT GOA CTT GAATAT C A A G T C OTT OAA ATT TAT TTG A A A T A C TTO CTO OCA CTO CAC CTG

1140

TTT CTG ATT CCT ATT G G A A ~ F L I P I G K

GCC ~ A K

960

(329)

CAT TTG TAC TGC AAO ACC TGA CTA TTC A T A A A G G A A A A C GAT TTC T A A A G C AAC AGC AGG

1200

TAT TTT TOT ACA OGG AAG TTT AAA TGT GTT TGT GTA TGG A A A A C T CTC CAC TCT CCT CCC

1260

CTA GAT GCC ATG CTT CCT TTT GTC TGT TAC CGT TGC CAT GTT CTT TGA ATA A C A A A T TAT

1320

ATC ACA TTT TAT CCT CTC TCA CCA C A A G G A C A A A G T A T G

GAT GTG GCA GAG TCC TGA TGA

1380

AA0 ATG TAT CCAAAC AAG &TA ACT TAT ATG TAT A A A A T T A A A G C A TAT /tAT ACA CAT TTA

1440

CTO TTA OTT TGT TTT GAT AAG G A A T A A A G G AAAAAAAAAAA~A~

AAT TTC T A A C A T G A A A A A A ~ A A A A A A A A A

AAAAAAAAAAAAAAAAAA

1500 1533

Fig, 2. Nucleotide and deduced aa sequences of human liver £1BcDNA. The nt sequence is a composite from both ~lEl#l and #lEip2 cDNAs. The nt and deduced aa (in single-letter codes; number in parentheses) are numbered on the right. For the purpose of compadnB the entire El~leader sequence, the first I ! nt (boxed) are taken from a previously published nt sequence for a cultured human foreskin fibroblast Era# cDNA (Koike et al., 1988). The 329 aa mature Etp peptide starts at Leu( + !). Consensus polyadenylation signal (AATAAA) and variant polyadenylation signal (ATrAAA) at nt 1463 and 1417, respectively, are underlined.

mature mitochondrial EJ3 peptide is Leu( + I) (Ho et al., 1988). Starting from Leu(+ I), the deduced mature E~/~ peptide consisted of 329 aa with a calculated Mr of 35 863. This is consistent with the estimated 36 kDa for bovine

kidney EJ3 (Reed, 1974). Upstream from Leu(+ 1), the composite human liver E,p eDNA encodes an adLlitional 26 aa that represent part of the Ed~ leader peptide. In comparison with a published E~p eDNA from cultured

301 human foreskin fibroblasts (Koike et al., 1988), the ORF of the human liver eDNA (~Et/F2) lacked 11 bp, including the. AUG. These 11 bp, as well as the corresponding deduced aa, are included in Fig. 2. The stretch of 30 aa beginning with Met (-30) to Ala(-l) represents the leader peptide of the Et/I prepeptide. The deduced Es/~ prepeptide (359 aa) has a calculated Mr of 39188, which is consistent with the molecular mass of 39 kDa for bovine kidney precursor Et/~ based on migration pattern on S D S - P A G E (DeMarcucci et al., 1988). (d) Sequence comparison with a cultured human foreskin fibroblast EtP cDNA The composite sequence of the human liver El/3 eDNA (Fig. 2) conf'mms the absence of a G at nt 210 that was reported in the partial nt sequence of ;LEt/~I (Ho et al., 1988). Although the present Et/~cDNA sequence (Fig. 2) is in general agreement with a recently published sequence for a cultured human foreskin fibroblast Et/~ eDNA (Koike et al., 1988), the two sequences differ from each other at 20 positions distributed over the entire EJ~cDNA. The nature of these differences and their locations on the Et/~ eDNA are detailed in Table I. One set of a single nt insertion and a deletion is identified in the coding region for the leader peptide of the Et/~ prepeptide at nt 24 and 38, respectively (Table I). This modified 5 out ofthe 30 aa ofthe human foreskin fibroblast Et/~ leader sequence (Table l) (Koike eta]., 1988). A consequence of altering these 5 aa is an increase in the net positive charge of the leader peptide by 4, due to addition of two Arg residues and replacement of one Glu residue with another Arg (Table I). Secondary structure prediction, nucleation-site prediction and distribution of charged and hydroxylated aa residues in the human liver Et/I leader sequence indicate that it has the features of a mitochondrial matrix-targeting signal peptide (Hurt and Van Loon, 1986). In the discrepancies documented between the liver and foreskin eDNA sequences, the possibility of sequencing error was minimized in the present study by repeated ss DNA sequencing of at least one of the two ,~gtl1-Et/] cDNAs in both the forward and reverse directions (Fig. 1). All nt changes reported in Table I that are located within the overlapping 137 l-bp region of the ~Et/~l and the ,~Et/~2 cDNAs are present in both cDNAs. The observation that these nt changes were found in two independent eDNA clones provides further support that our findings accurately reflect the human liver Et/~mRNA and are not the result of a cloning or sequencing artifact. Differences in the sequence that were present in the cultured foreskin fibroblast eDNA may represent tissue specificity, peculiarity of the fibroblast cell line used in the construction of the eDNA library, and cloning or sequencing artifacts.

(e) Identification and characterization of human E t defects

Most ofthe defects in human PDC deficiency involve the Et component (Robinson et al., 1987; Wexler et al., 1988). The availability of nt sequences which accurately represent normal human E~ mRNAs and the application of the PCR technique will facilitate the localization and characterization of E~ defects. Additionally, primary structure information on the E~/~and the El0Cpeptides derived from these nt sequences is integral to the understanding of the structure-function relationship of Es with respect to its catalysis and regulation as well as the interaction of these two subunits with each other and with other PDC components. We recently generated an unambiguous sequence for human Et~ eDNA (Ho et al., 1989). The composite nt sequence for human Etp eDNA presented in this study (Fig. 2) will serve as a reference standard for future analysis of human Es/? in both the normal and the disease states.

ACKNOWLEDGEMENTS We thank Drs. T. Chandra and S.L.C. Woo (Baylor College of Medicine, Houston) for the ~gtl I human eDNA library, Dr. Thomas Thekkumkara for technical advice and help, Beth Kleinhenz and Kevin Bailey for excellent technical assistance, and Drs. D. Samols, W. Merrick, D.S. Kerr and I.D. Wexler for their critical reading of the manuscript. This work was supported by NIH Grant DK 20478 and Metabolism Training Grant AM 07319 (L.H.).

REFERENCES

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