Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

Cloning and expression in Escherichia coli of mature E1 beta subunit of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex. Mapping of the E1 beta-binding region on E2.

A cDNA encoding the mature E1 beta subunit of the bovine branched-chain alpha-keto acid dehydrogenase complex was isolated from a lambda ZAP expression library. The bovine E1 beta cDNA is 1,393 base pairs in length. It encodes the entire mature E1 beta subunit consisting of 342 amino acid residues and a partial mitochondrial targeting presequence of 26 residues. The calculated molecular mass of the mature bovine E1 beta subunit is 37,776 daltons, and the calculated isoelectric point is pI 5.04. The mature bovine E1 beta subunit was expressed in Escherichia coli via the pKK233-2 vector in the presence of isopropyl beta-D-thiogalactopyranoside (IPTG). When expression was induced by IPTG at 37 degrees C, the soluble recombinant E1 beta subunit existed as a single high molecular weight form (Mr congruent to 3.5 x 10(5)), which sedimented during sucrose gradient ultracentrifugation at 2 x 10(5) x g. However, lowering the induction temperature to 25 degrees C resulted in the occurrence of both high and low molecular weight forms of the recombinant E1 beta protein. The low molecular weight form (Mr congruent to 9.1 x 10(4)) remained soluble after sucrose gradient centrifugation and was utilized in binding studies with a series of truncated recombinant E2 proteins. The results showed that the E1 beta subunit bound to the region between Ala-115 and Lys-150 of the E2 chain, which lay within the putative E3-binding domain. In contrast, the recombinant E1 alpha subunit did not bind the E2 component. The data suggest an apparent binding order of E2-E1 beta-E1 alpha, which supports and extends the model of E2 inner core deduced previously from the data of scanning transmission electron microscopy (Hackert, M.L., Xu, W.-X., Oliver, R.M., Wall, J.S., Hainfeld, J.F., Mullinax, T.R., and Reed, L.J. (1989) Biochemistry 28, 6816-6821). The relatively inaccessible topology of E1 beta may explain the lack of antigenicity and resistance to limited proteolysis of this subunit as it exists in the complex.     

Nucleotide and deduced amino acid sequence of the E1 alpha subunit of human liver branched-chain alpha-ketoacid dehydrogenase

A 1552-bp cDNA for the E1 alpha subunit of branched-chain alpha-ketoacid dehydrogenase (BCKDH) was isolated from a human liver cDNA library. The cDNA contained a 1134-bp open reading frame that encoded 378 amino acid (aa) residues of the enzyme and 418 bp of 3'-untranslated sequence. The deduced amino acid sequence of the human protein shows 96% identity with that of the rat enzyme subunit. Those 117-aa residues surrounding the phosphorylation sites are completely conserved between man and rat. BCKDH E1 alpha showed considerable amino acid sequence similarity with pyruvate dehydrogenase E1 alpha, particularly in the region of the two principal phosphorylation sites of these proteins. Northern blots of human liver and skin fibroblasts demonstrated a single 1.8-kb mRNA band, with a higher level of E1 alpha mRNA in liver than in normal fibroblasts. Fibroblasts from a patient with thiamine-responsive maple syrup urine disease (MSUD) contained an mRNA of the same size and abundance as that of normal fibroblasts. Genomic DNA from normal and MSUD fibroblasts gave the same restriction maps on Southern blots, and the gene was approximately 10-kb in size.     

Human F1-ATPase: molecular cloning of cDNA for the beta subunit

F1-ATPase is the major enzyme for ATP synthesis, and its beta subunit is the catalytic site. To date, no full-length cDNA for the eukaryotic F1 gene has been reported. Human F1 was studied because of its importance in medicine and cell biology. Here we report molecular cloning of a full-length cDNA for the human F1 beta subunit and purification of the human F1 beta subunit. The HeLa cell cDNA library constructed in an expression vector gamma gt11 was screened with antiserum against the yeast F1 beta subunit. One of the positive phage DNAs containing the human F1 beta gene and its flanking regions (1.8 kilobase pairs) was sequenced by the dideoxy chain termination method. The open reading frame started from a putative signal presequence, which was rich in both serine and arginine. There was a homologous segment in the signal presequence of human ornithine transcarbamoylase and that of F1 beta. The precursor of F1 beta was expressed in E. coli harboring a plasmid which had been constructed with T5 promotor and the F1 beta cDNA. Both the precursor and mature form of F1 beta were detected in HeLa cells in a pulse-chase experiment. The amino acid sequence of 480 residues (51,568.3 daltons) following the presequence was highly homologous with that of mature beef heart F1 beta (97.5%) and E. coli F1 beta (71.7%), but the codon usage in the human gene was very different from those of reported genes coding for F1 beta of other species.     

Isolation of a full-length complementary DNA coding for human E1 alpha subunit of the pyruvate dehydrogenase complex

A 1.5-kilobase cDNA clone for human pyruvate dehydrogenase E1 was isolated from a lambda gt11 expression library by screening with polyclonal antiserum to the E1 alpha subunit of the porcine pyruvate dehydrogenase complex, a polyclonal antibody against bovine pyruvate dehydrogenase complex and a synthetic oligonucleotide based on the known amino acid sequence of the amino-terminal of the bovine pyruvate dehydrogenase-E1 alpha subunit. Nucleotide sequence analysis of the cDNA revealed a 5'-untranslated sequence of 72 nucleotides, a translated sequence of 1170 nucleotides, and a 3'-untranslated sequence of 223 nucleotides with a poly(A) tail. The cDNA structure predicts a leader sequence of 29 amino acids and a mature protein of 362 amino acids comprising an amino-terminal peptide identical to that of the bovine E1 alpha subunit and three serine phosphorylation sites whose sequence was also identical to those in the bovine E1 alpha subunit. The translated sequence for the mature protein differs substantially from that described by Dahl et al. (Dahl, H. H., Hunt, S. M., Hutchison, W. M., and Brown, G. K. (1987) J. Biol. Chem. 262, 7398-7403) by virtue of a frameslip between bases 390 and 594. This amended sequence is confirmed by the presence of additional restriction sites for the enzymes NaeI and HaeII at the beginning and end, respectively, of this section. The leader sequence is typical for mitochondrial enzymes being composed of a combination of neutral and basic residues. The amino acid composition is strikingly similar to that of the bovine protein. This cDNA clone hybridizes with a 1.8-kilobase mRNA on a Northern blot analysis of human fibroblasts, and a second minor band of 4.4 kilobases is also detected.     

Cloning and sequencing of a cDNA encoding the precursor to the 24 kDa iron-sulfur protein of human mitochondrial NADH dehydrogenase

1. We have isolated a cDNA encoding the 24 kDa subunit, an iron-sulfur protein, of mitochondrial NADH dehydrogenase from a human fibroblast cDNA library by colony hybridization using a rat 24 kDa subunit cDNA as a probe. 2. The presequence predicted from the human cDNA sequence is typical of precursors to mitochondrial proteins in a high content of basic residues and in the absence of acidic ones. 3. The mature form of the human 24 kDa subunit shows 95% homology with its rat counterpart. Five cysteine residues are conserved among human, rat and bovine; four of these are expected to be involved in the binding of a binuclear iron-sulfur cluster.     

Isolation and characterization of a complementary DNA clone coding for the E1 beta subunit of the bovine branched-chain alpha-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing

A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human pyruvate dehydrogenase complex. Isolation of cDNA clones for the E1 alpha subunit, sequence analysis, and characterization of the mRNA

cDNA clones corresponding to the entire length of mRNA for the alpha subunit of human pyruvate dehydrogenase (EC 1.2.4.1), the E1 component of the pyruvate dehydrogenase complex, have been isolated from liver cDNA libraries. Two classes of cDNA clones were obtained and these correspond to two forms of pyruvate dehydrogenase E1 alpha mRNA. Both mRNA species have been demonstrated in a variety of human tissues and cultured fibroblasts. The cDNA sequence has been determined and, from it, the protein sequence of the human E1 alpha subunit was deduced. The protein is synthesized with a typical mitochondrial import leader sequence and the peptide bond at which this sequence is cleaved after transport into the mitochondrion has been determined by direct amino acid sequencing of the mature E1 alpha subunit. The human pyruvate dehydrogenase E1 alpha subunit contains identical phosphorylation sites to those found in the corresponding porcine protein. Preliminary studies of pyruvate dehydrogenase E1 alpha mRNA in cultured fibroblasts from patients with severe pyruvate dehydrogenase deficiency have revealed considerable heterogeneity as would be expected from protein studies.     

A T-to-A substitution in the E1 alpha subunit gene of the branched-chain alpha-ketoacid dehydrogenase complex in two cell lines derived from Menonite maple syrup urine disease patients

We cloned and sequenced cDNAs of the E1 alpha and E1 beta subunits of the branched chain alpha-ketoacid dehydrogenase complex (BCKDH) in two cell lines derived from two different Menonite MSUD patients (GM 1655, GM 1099). A T-to-A substitution which generates an asparagine in place of a tyrosine at amino acid 394 of the mature E1 alpha subunit was present in both alleles in these two cell lines, whereas cDNAs of the E1 beta subunit in these cell lines were identical to that of normal human lymphoid cell line and that of the clone from a human placenta cDNA library. It is suggested that the Menonite MSUD is caused by the missense mutation of the E1 alpha subunit of the BCKDH complex.     

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.     

Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family.

A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full-length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full-length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk- cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti-plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk- cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre-PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine-linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1-proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of two cDNA clones for pyruvate dehydrogenase E1 beta subunit and its regulation in tricarboxylic acid cycle-deficient fibroblast

Two distinct types of cDNA clones encoding for the pyruvate dehydrogenase (PDH) E1 beta subunit were isolated from a human liver lambda gt11 cDNA library and characterized. These cDNA clones have identical nucleotide sequences for PDH E1 beta protein coding region but differ in their lengths and in the sequences of their 3'-untranslated regions. The smaller cDNA had an unusual polyadenylation signal within its protein coding region. The cDNA-deduced protein of PDH E1 beta subunit revealed a precursor protein of 359 amino acid residues (Mr 39,223) and a mature protein of 329 residues (Mr 35,894), respectively. Both cDNAs shared high amino acid sequence similarity with that isolated from human foreskin (Koike, K.K., Ohta, S., Urata, Y., Kagawa, Y., and Koike, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 41-45) except for three regions of frameshift mutation. These changes led to dramatic alterations in the local net charges and predicted protein conformation. One of the different sequences in the protein coding region of liver cDNA (nucleotide position 452-752) reported here was confirmed by sequencing the region after amplification of cDNA prepared from human skin fibroblasts by the polymerase chain reaction. Southern blot analysis verified simple patterns of hybridization with E1 beta cDNA, indicating that the PDH E1 beta subunit gene is not a member of a multigene family. The mechanisms of differential expression of the PDH E1 alpha and E1 beta subunits were also studied in established fibroblast cell lines obtained from patients with Leigh's syndrome and other forms of congenital lactic acidosis. In Northern blot analyses for PDH E1 alpha and E1 beta subunits, no apparent differences were observed between two Leigh's syndrome and the control fibroblasts studied: one species of PDH E1 alpha mRNA and three species of E1 beta mRNA were observed in all the cell lines examined. However, in one tricarboxylic acid cycle deficient fibroblast cell line, which has one-tenth of the normal enzyme activity, the levels of immunoreactive PDH E1 alpha and E1 beta subunits were markedly decreased as assessed by immunoblot analyses. These data indicated a regulatory mutation caused by either inefficient translation of E1 alpha and E1 beta mRNAs into protein or rapid degradation of both subunits upon translation. In contrast, the PDH E1 alpha and E1 beta subunits in two fibroblast cell lines from Leigh's syndrome patients appeared to be normal as judged by 1) enzyme activity, 2) mRNA Northern blot, 3) genomic DNA Southern blot, and 4) immunoblot analyses indicating that the lactic acidosis seen in these patients did not result from a single defect in either of these E1 alpha and E1 beta subunits of the PDH complex.     

Cloning, characterization, and expression of the beta subunit of pig heart succinyl-CoA synthetase.

The form of succinyl-CoA synthetase found in mammalian mitochondria is known to be an alpha beta dimer. Both GTP- and ATP-specific isozymes are present in various tissues. We have isolated essentially identical complementary DNA clones encoding the beta subunit of pig heart succinyl-CoA synthetase from both newborn and adult tissues. These cDNAs include a 1.4-kb sequence encoding the cytoplasmic precursor to the beta subunit comprised of 417 amino acid residues including a 22-residue mitochondrial targeting sequence. The cDNA encoding the 395-amino acid, 42,502-Da mature protein was confirmed to be the succinyl-CoA synthetase beta subunit by agreement with the N-terminal protein sequence and by high homology to prokaryotic forms of the beta subunit that were previously cloned (about 45% identical to beta from Escherichia coli). In contrast to a previous report (Nishimura, J.S., Ybarra, J., Mitchell, T., & Horowitz, P.M., 1988, Biochem. J. 250, 429-434), we found no tryptophan residue to be encoded in the sequence for the mature beta subunit, and this finding is corroborated by the fact that highly purified pig heart succinyl-CoA synthetase shows no tryptophan fluorescence or tryptophan content in amino acid compositional analysis. The cDNA clones encoding the mature pig heart beta subunit and its counterpart alpha subunit were coexpressed in a deletion mutant strain of E. coli. Recovery of succinyl-CoA synthetase activity demonstrated that this combination of subunits forms a productive enzymatic complex having GTP specificity.     

Characterization of rainbow trout branched-chain alpha-keto acid dehydrogenase complex: inter-domain segments of the E2 component affect the overall activity

Branched-chain alpha-keto acid dehydrogenase complex (BCKADH) contains decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3) as catalytic components. BCKADH purified from rainbow trout (Oncorhynchus mykiss) liver was comparable with mammalian BCKADH in various enzymatic characteristics, but less efficient in catalyzing the overall reaction. The trout E2 subunit was larger than the mammalian subunit and rather similar to the chicken one in relative molecular mass on SDS-PAGE, whereas the E1 component was similar between trout and mammalian both in relative molecular mass of its alpha and beta subunits and in the catalytic activity. Trout E2 cDNA cloning and nucleotide sequencing revealed that the mature trout E2 subunit consists of 435 residues, and possesses 14 additional residues compared with mammalian E2. Eleven of these are localized in two interdomain segments as two sequences with two and nine residues, respectively. Trout E2 was inferior to rat E2 in the capacity for binding the E1 component, similar to chicken E2. Thus, it appears that non-mammalian BCKADH E2 is distinct from that in mammals in the structure of interdomain segments, resulting in reduction of overall activity of the enzyme complex.     

Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments

Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII.     

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.