Cloning and sequence analysis of mRNA for mouse aspartate aminotransferase isoenzymes

The nucleotide sequences of mRNAs for the mouse mitochondrial and cytosolic aspartate aminotransferase isoenzymes (mAspAT and cAspAT) (EC 2.6.1.1) were determined from complementary DNAs. The mAspAT mRNA comprises minimally 2460 nucleotides and codes for a polypeptide of 430 amino acid residues corresponding to the precursor form of the mAspAT (pre-mAspAT). The cAspAT mRNA comprises minimally 2086 nucleotides and codes for a polypeptide of 413 amino acid residues. The region coding for the mature mAspAT and that for the cAspAT show about 53% overall homology. The former shares 49% and the latter 48% of homology, respectively, with that of the Escherichia coli aspC gene, which has been shown to code for the E. coli AspAT (Kuramitsu, S., Okuno, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 1259-1262). When the deduced amino acid sequence of the mouse pre-mAspAT was compared with that of the pig pre-mAspAT polypeptide, we found that they share a 94% homology and that the mouse pre-mAspAT yields a presequence consisting of 29 amino acid residues and a mature mAspAT, consisting of 401 amino acid residues. These numbers and the amino acid residues present at the putative cleavage site are all in complete agreement in these two species. The deduced amino acid sequence of the mouse cAspAT shares 91% homology with that of the pig cAspAT. Comparisons of the nucleotide and deduced amino acid sequences between the mouse and E. coli AspATs suggest that the mammalian mAspAT gene is more closely related to the E. coli aspC gene than is the mammalian cAspAT gene.     

cDNA cloning and expression of pig cytosolic aspartate aminotransferase in Escherichia coli: amino-terminal heterogeneity of expressed products and lack of its correlation with enzyme function

A full-length cDNA encoding the pig cytosolic aspartate aminotransferase (EC 2.6.1.1) (cAspAT) was constructed from two overlapping cDNA clones. One clone (Lm pcAAT-8) isolated from a lambda gt10 pig heart cDNA library contained a 3' untranslated sequence, a poly(A) segment, and a part of the coding region for amino acid positions 127-412. Another clone (Lm pcAAT-107) isolated from a lambda gt10 primer extension library contained the coding region for amino acid positions 1-148 and a 5' untranslated sequence. Rejoining of the cDNA inserts of the two clones and recloning into pUC18 gave rise to a cDNA covering an entire coding sequence for pig cAspAT mRNA. Insertion into pKK223-3 yielded an expression plasmid, ppcAAT200. Escherichia coli JM105 cells transfected with ppcAAT200 overproduced pig cAspAT to an extent of about 3% of the total cellular soluble proteins. The expressed product was indistinguishable from the alpha subform of cAspAT isolated from pig heart in terms of specific activity, absorption spectra, molecular size, crystalline form, and immunological reactivity with anti pig cAspAT antibody. Compared with the amino-terminal sequence (Ala-Pro-Pro-) reported for pig heart cAspAT, the recombinant pig cAspAT showed heterogeneity in the amino-terminal sequence: Ala 1 (26%), Pro2 (54%), and Pro3 (19%). Construction of a mutant cAspAT with deletion of residues 1-3 and its comparison with the wild-type enzyme revealed that loss of the three amino-terminal residues does not affect the catalytic activity and structural integrity of the enzyme.     

Branched-chain alpha-ketoacid dehydrogenase kinase. Molecular cloning, expression, and sequence similarity with histidine protein kinases.

A cDNA for branched-chain alpha-ketoacid dehydrogenase kinase was cloned from a rat heart cDNA library. The cDNA had an open reading frame encoding a protein of 382 amino acid residues with a calculated molecular weight of 43,280. The clone codes for the branched-chain alpha-ketoacid dehydrogenase kinase based on the following: 1) the deduced amino acid sequence contained the partial sequence of the kinase determined by direct sequencing; 2) expression of the cDNA in Escherichia coli resulted in synthesis of a 43,000-Da protein that was recognized specifically by kinase antibodies; and 3) enzyme activity that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex was found in extracts of E. coli expressing the protein. Northern blot analysis indicated the mRNA for the branched-chain alpha-ketoacid dehydrogenase kinase was more abundant in rat heart than in rat liver, as expected from the relative amounts of kinase activity expressed in these tissues. The deduced sequence of the kinase aligned with a high degree of similarity within subdomains characteristic of procaryotic histidine protein kinases. This first mitochondrial protein kinase to be cloned appears more closely related in sequence to procaryotic histidine protein kinases than to eucaryotic serine/threonine protein kinases.     

Rat cytosolic aspartate aminotransferase: regulation of its mRNA and contribution to gluconeogenesis

Induction of cytosolic aspartate aminotransferase (cAspAT) was observed in rat liver on administration of a high-protein diet, or glucagon and during fasting. The enzyme activity in the liver of rats given 80% protein diet or glucagon injection during starvation increased to 2- to 2.4-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of hybridizable cAspAT mRNA, measured by blot analysis using the cloned rat cAspAT cDNA as a probe. No increase in the enzyme was detected in kidney, heart, brain, or skeletal muscle. The activity of mitochondrial aspartate aminotransferase (mAspAT) did not increase. Induction of cAspAT was observed when glucose metabolism tended toward gluconeogenesis. The physiological function of the induction of cAspAT is considered to be to increase the supply of oxaloacetate as a substrate for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) [EC 4.1.1.32] for gluconeogenesis.     

Isolation of a cDNA encoding the rat liver S-adenosylmethionine synthetase

We have isolated cDNA clones encoding the rat liver S-adenosylmethionine synthetase by means of immunological screening from a phage lambda gt 11 expression library containing cDNA synthesized from adult rat liver poly(A)-RNA. The amino acid sequence deduced from the cDNA indicates that the rat liver enzyme for this protein contains 397 amino acid residues and has a molecular mass of 43697 Da. The deduced amino acid sequence of rat liver S-adenosylmethionine synthetase was 68% similar to those of yeast S-adenosylmethionine synthetases encoded by two unlinked genes SAM1 and SAM2. The rat liver S-adenosylmethionine synthetase also shows 52% similarity with the deduced amino acid sequence of the MetK gene encoding the S-adenosylmethionine synthetase in Escherichia coli.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.     

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.     

Molecular cloning and in vivo expression of a precursor to rat mitochondrial aspartate aminotransferase

A 2.4 kilobase cDNA for rat mitochondrial aspartate aminotransferase (E.C. 2.6.1.1.) was isolated and sequenced. The predicted presequence is 93% homologous to the presequences of the enzyme from pig and mouse. The predicted amino acid sequence of the mature enzyme differs from that determined directly by amino acid sequencing (Huynh, Q.K., Sakakibara, R., Watanabe, T., and Wada, H. (1981) J. Biochem. (Tokyo) 90, 863-875) at 13 amino acids residues. The most important difference is at position 140 where the cDNA encodes a tryptophanyl residue rather than the previously reported glycine. This critical residue is now seen to be conserved in all aspartate aminotransferases. The coding region of this cDNA was inserted into the plasmid cloning vector pKK233-2 and used to stably express an unfused precursor in Escherichia coli JM105.     

Protein structure of pig liver 4-aminobutyrate aminotransferase and comparison with a cDNA-deduced sequence.

The amino acid sequence of pig liver 4-aminobutyrate aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments. The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work [Kwon, O., Park, J. & Churchich, J. E. (1992) J. Biol. Chem. 267, 7215-7216]. Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107-142. Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work. The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli.     

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.     

cDNA cloning of rat prolyl oligopeptidase and its expression in the ovary during the estrous cycle

A cDNA for rat prolyl oligopeptidase was cloned which contained an open reading frame of 2,130 nucleotides encoding a protein of 710 amino acids. The deduced amino acid sequence is around 95% homologous to other mammalian prolyl oligopeptidases and about 40% to bacterial prolyl oligopeptidases. The recombinant prolyl oligopeptidase generated in E. coli was purified and its properties were examined. The substrate specificity and the susceptibility to proteinase inhibitors were similar to those of the native enzyme. Northern blot analysis showed wide expression of the prolyl oligopeptidase gene. Using ovaries from hormone-treated rats, it was found that both the mRNA expression and enzyme activity increased in the luteal phase. These findings suggest the involvement of prolyl oligopeptidase in events associated with corpus luteum formation and/or luteal regression.     

Molecular cloning of cDNA for rat liver general acyl CoA dehydrogenase and homology between the rat liver and pig kidney enzymes

cDNA clone for general acyl CoA dehydrogenase (GAD) was isolated from a rat liver cDNA expression library in lambda gt11 using anti-pig kidney GAD antibody. Size of the isolated cDNA was estimated to be 1.5-1.6 kb. By immunological analysis of fusion protein and epitope selection, the cDNA clone was identified as that containing the GAD gene. Partial amino acid sequence deduced from nucleotide sequence of the cDNA coincided with that of the pig kidney enzyme. The antibody cross-reacted with rat liver enzyme and molecular weights of these enzyme proteins were shown to be almost the same. All these results indicate that rat liver GAD shares a common structure with pig kidney enzyme.     

Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells.

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.     

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis

Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from partial amino acid sequencing of the purified protein were consistent with the amino acid sequence deduced from the cDNA. However, the amino terminus of the protein was blocked, and therefore, direct protein sequence information confirming the cDNA reading frame of this region could not be obtained by Edman degradation. We have now employed an integrated approach using fast atom bombardment mass spectrometry, tandem mass spectrometry, and conventional sequencing methodologies to establish the amino-terminal sequence of the protein and to identify an amino acid sequence (35 residues) present in the purified protein but missing from the amino acid sequence deduced from cDNA clones for this protein. The missing piece of cDNA corresponds to an exon found in mouse genomic clones for a protein similar to rat SBP. Therefore, the cDNA clones for rat SBP may represent splicing variants that lack the sequence information of one exon. The blocked amino terminus of the protein was identified as 5-oxopyrrolidine-2-carboxylic acid. Mass spectrometry also provided evidence regarding glycosylation of the protein. The first of two potential glycosylation sites clearly carries carbohydrate; the second site is, at most, only partially glycosylated.     

Rat Brain Glutamic Acid Decarboxylase Sequence Deduced from a Cloned cDNA

A cDNA clone complementary to the rat brain glutamic acid decarboxylase mRNA was isolated from a rat brain cDNA expression library using an antibody specific to the enzyme. The cDNA insert has been shown to direct the synthesis of an active protein in Escherichia coli. In this study, the nucleotide sequence of this clone, which includes the complete coding region, is presented. The predicted protein is 593 amino acids in length. The first 557 residues display a 95% identity when compared with the corresponding cat sequence. However, the deduced amino acid sequence of the carboxy-terminal end of the rat protein, downstream of residue 557, is totally different from the cat, whereas it agrees with a published partial peptidic sequence of the rat protein.     

cDNA and deduced amino acid sequence of a mouse DNA repair enzyme (APEX nuclease) with significant homology to Escherichia coli exonuclease III

We purified a mouse DNA repair enzyme having apurinic/apyrimidinic endonuclease, DNA 3'-phosphatase, 3'-5'-exonuclease and DNA 3' repair diesterase activities, and designated the enzyme as APEX nuclease. A cDNA clone for the enzyme was isolated from a mouse spleen cDNA library using probes of degenerate oligonucleotides deduced from the N-terminal amino acid sequence of the enzyme. The complete nucleotide sequence of the cDNA (1.3 kilobases) was determined. Northern hybridization using this cDNA showed that the size of its mRNA is about 1.5 kilobases. The complete amino acid sequence for the enzyme predicted from the nucleotide sequence of the cDNA (APEX nuclease cDNA) indicates that the enzyme consists of 316 amino acids with a calculated molecular weight of 35,400. The predicted sequence contains the partial amino acid sequences determined by a protein sequencer from the purified enzyme. The coding sequence of APEX nuclease was cloned into pUC18 SmaI and HindIII sites in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain cells of Escherichia coli. The transformed cells expressed a 36.4-kDa polypeptide (the 316 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide of beta-galactosidase) and were less sensitive to methyl methanesulfonate than the parent cells. The fusion product showed priming activity for DNA polymerase on bleomycin-damaged DNA and acid-depurinated DNA. The deduced amino acid sequence of mouse APEX nuclease exhibits a significant homology to those of exonuclease III of E. coli and ExoA protein of Streptococcus pneumoniae and an intensive homology with that of bovine AP endonuclease 1.     

Nucleotide sequence of the cDNA encoding the precursor for mitochondrial serine:pyruvate aminotransferase of rat liver

The nucleotide sequence of the mRNA coding for the precursor of mitochondrial serine:pyruvate aminotransferase of rat liver was determined from those of cDNA clones. The mRNA comprises at least 1533 nucleotides, except the poly(A) tail, and encodes a polypeptide consisting of 414 amino acid residues with a molecular mass of 45,834 Da. Comparison of the N-terminal amino acid sequence of mitochondrial serine:pyruvate aminotransferase with the nucleotide sequence of the mRNA showed that the mature form of the mitochondrial enzyme consisted of 390 amino acid residues of 43,210 Da. The amino acid composition of mitochondrial serine:pyruvate aminotransferase deduced from the nucleotide sequence of the cDNA showed good agreement with the composition determined on acid hydrolysis of the purified protein. The extra 24 amino acid residues correspond to the N-terminal extension peptide (pre-sequence) that is indispensable for the specific import of the precursor protein into mitochondria. In the extension peptide there are four basic amino acids distributed among hydrophobic amino acids and, as revealed on helical wheel analysis, the putative alpha-helical structure of the peptide was amphiphilic in nature. The secondary structures of the mature serine:pyruvate aminotransferase and three other aminotransferases of rat liver were predicted from their amino acid sequences. Their secondary structures exhibited a common feature and so we propose the specific lysine residue which binds pyridoxal phosphate as the active site of serine:pyruvate aminotransferase.     

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.