Molecular cloning of cDNA for proteasomes from rat liver: primary structure of component C3 with a possible tyrosine phosphorylation site

Proteasomes are multicatalytic proteinase complexes consisting of multiple components. Previously, we reported the cloning and sequencing of cDNA for the largest component, C2, of rat liver proteasomes [Fujiwara, T., Tanaka, K., Kumatori, A., Shin, S., Yoshimura, T., Ichihara, A., Tokunaga, F., Aruga, R., Iwanaga, S., Kakizuka, A., & Nakanishi, S. (1989) Biochemistry 28, 7332-7340]. In the present study, the nucleotide sequence of another component (C3) of proteasomes has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The deduced sequence of component C3 consists of 234 amino acid residues with a calculated molecular weight of 25,925. These values are consistent with those obtained by protein chemical analyses. A single mRNA species hybridizing to the C3 cDNA of rat liver was expressed in all rat tissues examined and in a variety of other eukaryotic organisms, its distribution being similar to that of C2 mRNA. The wide distribution of the gene product, possibly C3, suggests that this protein functions similarly in most eukaryotes. C3 is an unmodified protein of a single gene and differs from any other known protein, but its overall amino acid sequence resembles those of other proteasomal components, including C2, suggesting that these components belong to a single family of proteins with the same evolutionary origin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver arginase

Arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis in the liver of ureotelic animals. The nucleotide sequence of rat liver arginase cDNA, which was isolated previously (Kawamoto, S., Amaya, Y., Oda, T., Kuzumi, T., Saheki, T., Kimura, S., and Mori, M. (1986) Biochem. Biophys. Res. Commun. 136, 955-961) was determined. An open reading frame was identified and was found to encode a polypeptide of 323 amino acid residues with a predicted molecular weight of 34,925. The cDNA included 26 base pairs of 5'-untranslated sequence and 403 base pairs of 3'-untranslated sequence, including 12 base pairs of poly(A) tract. The NH2-terminal amino acid sequence, and the sequences of two internal peptide fragments, determined by amino acid sequencing, were identical to the sequences predicted from the cDNA. Comparison of the deduced amino acid sequence of the rat liver arginase with that of the yeast enzyme revealed a 40% homology.     

cDNA cloning of a neural visinin-like Ca(2+)-binding protein.

A 21,000-dalton Ca(2+)-binding protein (Walsh, M.P., Valentine, K.A., Ngai, P.K., Carruthers, C.A., and Hollengerg, M.D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca(2+)-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca(2+)-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain.     

Mammalian protein serine/threonine phosphatase 2C: cDNA cloning and comparative analysis of amino acid sequences.

Complementary DNA encoding rat protein phosphatase 2C alpha was obtained from a liver library and used to isolate the homologous cDNAs from rabbit liver and human teratocarcinoma libraries. The amino acid sequences of the three enzymes deduced from the cDNA (382 amino acids) were extremely similar (greater than 99% identity), the maximum number of differences (between rat and human) being four. Amino acid sequences of peptides corresponding to 238 residues (61%) of the protein phosphatase 2C beta isoform from rabbit skeletal muscle were determined and showed 12 differences from the recently published sequence of the rat liver enzyme deduced from the cDNA (95% identity).     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

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.     

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.     

Amino acid sequence of rat liver cathepsin L

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their Mr values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N-terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176-177 (Asp-Ser) were not present at the C-terminus and alanine was replaced by proline at residue 125. Asn-108 in the heavy chain is modified with carbohydrate.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.     

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Expression of rat liver S-adenosylhomocysteinase cDNA in Escherichia coli and mutagenesis at the putative NAD binding site

The cDNA for rat liver S-adenosylhomocysteinase has been cloned, and the nucleic acid sequence has been determined. By comparison of the deduced amino acid sequence for S-adenosylhomocysteinase with that of the dinucleotide binding region for other proteins, the sequence from amino acids 213 to 244 in rat liver S-adenosylhomocysteinase was proposed to be part of the NAD binding site (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 719-723). A vector has been constructed that expresses S-adenosylhomocysteinase in Escherichia coli in the presence of isopropyl beta-D-thiogalactopyranoside by inserting the coding sequence of rat liver S-adenosylhomocysteinase cDNA downstream from the lac promoter of plasmid pUC118. The enzyme that is produced comprises as much as 10% of the soluble cellular proteins. The purified enzyme is a tetramer, contains 4 mol of tightly bound NAD, and has kinetic properties indistinguishable from those of the liver enzyme. Tryptic peptide mapping and NH2-terminal sequence analysis indicate that the recombinant enzyme is structurally identical to the liver enzyme except for the absence of the NH2-terminal blocking group. The rat liver enzyme has a blocked NH2-terminal alanine residue (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 719-723). By oligonucleotide-directed mutagenesis mutant vectors have been generated that express proteins in which each of the glycines in the Gly-Xaa-Gly-Xaa-Xaa-Gly sequence of the putative NAD binding site (residues 219-224) was changed to valine. Immunoblot analysis of extracts of the cells transformed with these vectors reveals the presence of immunoreactive proteins with the subunit molecular weight of S-adenosylhomocysteinase. The mutant proteins have no catalytic activity, contain no bound NAD, and do not form the same quaternary structure as the recombinant S-adenosylhomocysteinase.     

Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.     

Molecular cloning and sequence analysis of cDNA coding for rat liver hemoprotein H-450

cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity.     

cDNA cloning and sequencing for the import precursor of coupling factor 6 in H(+)-ATP synthase from rat liver mitochondria

The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 458 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions of both the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame consisted of 108 and 76 amino acid residues with a molecular weight of 12,494 and 8,927, respectively. The presequence of 32 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-gamma-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis

L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.     

Isolation and sequence analysis of a cDNA encoding rat liver alpha-L-fucosidase.

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].     

cDNA cloning and inducible expression during pregnancy of the mRNA for rabbit pulmonary prostaglandin omega-hydroxylase (cytochrome P-450p-2)

We have isolated cDNA clones of the mRNA for prostaglandin omega-hydroxylase (cytochrome P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. (Tokyo) 96, 593-603) in rabbit lung by using synthetic oligonucleotides as probes. The cDNA sequence contains an open reading frame of 1,470 nucleotides, the first 9 amino acids of which correspond to the residues 17-25 of cytochrome P-450p-2 determined from protein analysis. The predicted primary structure contains amino acid sequences of 23 tryptic fragments of cytochrome P-450p-2 and the deduced amino acid composition is in agreement with that determined from the purified protein. The complete polypeptide, including residues 1-16, contains 506 amino acids with a calculated molecular weight of 58,515. Cytochrome P-450p-2 shared 74% amino acid similarity with rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega) (Hardwick, J.P., Song, B.-J., Huberman, E., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 801-810), whereas it showed less than 25% similarity to other forms of cytochrome P-450, indicating that the two cytochrome P-450s constitute a unique cytochrome P-450 gene family. DNA blot analysis of the total genomic DNA of rabbits suggest the presence of several genes or gene-like DNA sequences which cross-hybridized with the cloned cDNA. RNA blot analysis showed that progesterone treatment increased the amount of mRNA hybridizable to the cDNA by about 100-fold in the lung of rabbits as compared with the basal level without the treatment. This high level of the mRNA was also observed in the lung of pregnant rabbits.     

Deduced primary structure of rat tryptophan-2,3-dioxygenase

The complete amino acid sequence of the tryptophan 2,3-dioxygenase (TO) of rat liver was determined from the nucleotide sequence of a full length TO cDNA isolated from a rat liver cDNA library and determined its primary structure. TO was encoded in a mRNA of about 1.7 kb containing an open reading frame of 1218 bp. According to the deduced amino acid sequence, the monomeric polypeptide of TO consisted of 406 amino acid residues with a calculated molecular weight of 47,796 daltons. It has twelve histidine residues around its hydrophobic region, which has homology with some heme proteins and oxygenase, suggesting that this hydrophobic region might to be the core of TO for the activity.