Cloning and the nucleotide sequence of rat glutathione S-transferase P cDNA.

A cDNA library prepared from poly(A)+ RNA of 2-acetylaminofluorene (AAF) induced rat hepatocellular carcinoma was screened by synthetic DNA probes deduced from a partial amino acid sequence of glutathione S-transferase P subunit that had been isolated from the tumor by two-dimensional gel electrophoresis. One of the four clones analyzed contained an mRNA region encoding the total amino acid sequence of this enzyme subunit and the complete 3'-noncoding region. The nucleotide sequence indicates that this enzyme subunit has 209 amino acids (calculated Mr=23,307) distinct from other glutathione S-transferase subunits such as Ya and Yc. Comparison of the amino acid sequences between these proteins indicates that glutathione S-transferase P subunit gene has been evolved from the ancestral gene at an earlier stage than the separation of Ya and Yc and that there are at least three domains having a considerable homology with each other in these enzymes. The very large increase of this mRNA in chemically induced hepatocellular carcinoma suggests a characteristic derepression of this gene during hepatocarcinogenesis.     

Multiple Ya subunits of glutathione S-transferase detected by monoclonal antibodies

Monoclonal antibodies to ligandin (YaYa) and glutathione (GSH) S-transferase B (YaYc) were produced by hybridomas derived from the fusion of mouse myeloma cells and spleen cells of mice immunized with the YaYa or YaYc proteins, respectively. Enzyme-linked immunosorbent assay was used to screen for antibody-producing clones. Immunoblotting of the subunits of transferase B, ligandin, and another GSH S-transferase containing Yb subunits showed that the monoclonal antibodies produced by two anti-YaYa subclones recognized the Ya subunits of both ligandin and transferase B, but they did not bind Yc or Yb subunits. It was also revealed that antibodies produced by several anti-YaYc subclones recognized the Yc subunit, but not the Ya subunit of the antigen which was used for the immunization of the mice. However, these monoclonal antibodies did bind the Ya subunit of ligandin. These results indicate that the Ya subunits of GSH S-transferase B and of ligandin do share at least one common determinant. However, these two Ya subunits are structurally distinct as evidenced by their differences in binding by monoclonal anti-YaYc antibodies.     

Ligandin heterogeneity : evidence that the two non-identical subunits are the monomers of two distinct proteins.

Purified ligandin (Y-protein) a 46000-dalton protein, has been shown to consist of two subunit species (mol. wts. 22 000 and 24 000) on discontinuous polyacrylamide gel electrophoresis in sodium dodecyl sulphate. This technique was used to define further the nature of these subunits. The Y sulphobromophthalein-binding fraction of rat hepatic cytosol was shown to contain three major subunit bands designated subunit Ya, subunit Yb and subunit Yc in ascending order of size. Purified ligandin was found to comprise Ya and Yc subunit species, and also gave two bands on isoelectric focusing. The two subunit species in purified ligandin were partially separated by an additional purification step. Antiserum to ligandin reacted mono-specifically with the purified protein, as well as hepatic, renal and small intestinal mucosa cytosol, but gave lines of identity and partial identity with cytosol from testis, ovary and adrenal gland. The Y fraction of testis was found to contain only Yb and Yc species, while all three major bands were found in liver, kidney and small intestinal mucosa. Phenobarbital treatment increased the concentration of Ya and Yb in the liver, but had little effect on Yc. These findings suggest that the Ya and Yc ligandin subunits are the monomers of two proteins: YaYa and YcYc.     

Identity of ligandin in rat testis and liver.

1. One of the main problems in the field of multifunctional proteins such as ligandin is the possibility that multiple forms and isoproteins may exist. Because liver ligandin [GSH (reduced glutathione) S-transferase B] consists of equal amounts of Ya (22 000 Da) and Yc (25 000 Da) subunits, and testis ligandin, prepared by the standard technique of anion-exchange and molecular-exclusion chromatography, contains more Yc subunit than Ya, it has been claimed that testis and liver ligandin are different entities. 2. We purified testis ligandin by immunoaffinity chromatography and have obtained a product identical with liver ligandin (Yc = Ya). This suggests that the differences previously described may be due to contamination of testis ligandin by a closely related species. In fact sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of testis GSH S-transferases separated by CM-cellulose chromatography showed that GSH S-transferase AA, present in large amounts, migrated in the same region as Yc subunit. 3. Testis ligandin prepared by the standard technique was similar to that reported [Bhargava, Ohmi, Listowsky & Arias (1980) J. Biol. Chem. 255, 724-727] and contained more Yc subunit than Ya. CM-cellulose chromatography of this 'pure' preparation revealed significant amounts of GSH S-transferase AA migrating as Yc subunit, in addition to ligandin consisting of equal amounts of Ya and Yc subunits. 4. Our studies show that testis ligandin is identical with liver ligandin. Previously described differences are due to a contaminant identified as GSH S-transferase AA.     

Isolation of two cholic acid-binding proteins from rat liver cytosol using affinity chromatography

Using an affinity matrix coupled with cholic acid, two proteins that recognise bile acids were isolated from rat liver cytosol. One protein of molecular weight 68 000 was immunologically identical to rat albumin. The other protein was of molecular weight 46 000. On discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis the 46 000 molecular weight protein dissociated to a single band with an RF value identical to the Yb subunit of the bromosulphophthalein-binding fraction (Y-fraction) of whole liver cytosol. The monomers of purified ligandin under these conditions resolved into two bands which corresponded to the Ya and Yc subunits of liver cytosol Y-fraction. Anti-serum to the purified ligandin reacted monospecifically with purified ligandin and whole liver cytosol, but did not cross-react with the Yb dimer eluted from the affinity column. The Yb dimer was shown to possess glutathione-S-transferase activity with a substrate specificity distinct from ligandin but similar to glutathione-S-transferase C. Cholic acid inhibited the catalytic activity of the transferase.     

Cloning and sequence analysis of a cDNA for a rat liver glutathione S-transferase Yb subunit.

We have isolated a Yb-subunit cDNA clone from a GSH S-transferase (GST) cDNA library made from rat liver polysomal poly(A) RNAs. Sequence analysis of one of these cDNA, pGTR200, revealed an open reading frame of 218 amino acids of Mr = 25,915. The deduced sequence is in agreement with the 19 NH2-terminal residues for GST-A. The sequence of pGTR200 differs from another Yb cDNA, pGTA/C44 by four nucleotides and two amino acids in the coding region, thus revealing sequence microheterogeneity. The cDNA insert in pGTR200 also contains 36 nucleotides in the 5' noncoding region and a complete 3' noncoding region. The Yb subunit cDNA shares very limited homology with those of the Ya or Yc cDNAs, but has relatively higher sequence homology to the placental subunit Yp clone pGP5. The mRNA of pGTR200 is not expressed abundantly in rat hearts and seminal vesicles. Therefore, the GST subunit sequence of pGTR200 probably represents a basic Yb subunit. Genomic DNA hybridization patterns showed a complexity consistent with having a multigene family for Yb subunits. Comparison of the amino acid sequences of the Ya, Yb, Yc, and Yp subunits revealed significant conservation of amino acids (approximately 29%) throughout the coding sequences. These results indicate that the rat GSTs are products of at least four different genes that may constitute a supergene family.     

Rat liver glutathione S-transferases. Complete nucleotide sequence of a glutathione S-transferase mRNA and the regulation of the Ya, Yb, and Yc mRNAs by 3-methylcholanthrene and phenobarbital

With the use of cDNA probes reverse transcribed from purified glutathione S-transferase mRNA templates, four cDNA clones complementary to transferase mRNAs have been identified and characterized. Two clones, pGTB38 and pGTB34, have cDNA inserts of approximately 950 and 900 base pairs, respectively, and hybridize to a mRNA(s) whose size is approximately 980 nucleotides. In hybrid-select translation experiments, pGTB38 and pGTB34 select mRNAs specific for the Ya and Yc subunits of rat liver glutathione S-transferases. Clone pGTB33, which harbors a truncated cDNA insert, hybrid-selects only the Ya mRNA. All of the clones, pGTB38, pGTB34, and pGTB33, hybrid-select another mRNA which is specific for a polypeptide with an electrophoretic mobility slightly greater than the Ya subunit. The entire nucleotide sequence of the full length clone, pGTB38, has been determined and the complete amino acid sequence of the corresponding polypeptide has been deduced. The mRNA codes for a protein comprising 222 amino acids with Mr = 25,547. We have also identified a cDNA clone complementary to a Yb mRNA of the rat liver glutathione S-transferases. This clone, pGTA/C36, hybrid-selects only Yb mRNA(s) and hybridizes to a mRNA(s) whose size is approximately 1200 nucleotides. Although the Ya, Yb, and Yc mRNAs are elevated coordinately by phenobarbital and 3-methylcholanthrene, the Ya-Yc mRNAs are induced to a much greater extent compared to the Yb mRNA(s). These data suggest that the mRNAs for each transferase isozyme are regulated independently.     

Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.     

The nucleotide sequence of a rat liver glutathione S-transferase subunit cDNA clone

We have determined the nucleotide sequence of a cloned cDNA derived from liver poly(A) RNA of pentobarbital-treated rats encoding a glutathione S-transferase subunit. This cDNA clone pGTR261 contains one open reading frame of 222 amino acids, a complete 3' noncoding region, and 63 nucleotides in the 5' noncoding region. The cloned DNA hybridizes to rat poly(A) RNA in a tissue-specific fashion, with strong signals to liver and kidney poly(A) RNA(s) of approximately 1100 and approximately 1400 nucleotides in size but little or no hybridization to poly(A) RNAs from heart, lung, seminal vesicles, spleen, or testis under stringent conditions. Our sequence covers the cDNA sequence of pGST94 which contains a partial coding sequence for a liver glutathione S-transferase subunit of Ya size. Comparison of sequences with our earlier clone pGTR112 suggests that there are at least two mRNA species coding for two different subunits of the Ya (Mr = 25,600) subunit family with very limited amino acid substitutions mainly of conserved polarity. The divergent 3' noncoding sequences should be useful molecular probes in differentiating these two different but otherwise very similar subunits in induction and genomic structure analyses. Our results suggest that tissue-specific expression of the glutathione S-transferase subunits represented by the sequences of pGTR261 and pGTR112 may occur at or prior to the level of RNA processing.     

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.     

Hepatic ligandin subunits and mRNAs during development

Eight-week-old rats had twofold higher hepatic ligandin concentration than 10-day-old animals as determined immunologically and by steroid isomerase and glutathione S-transferase assays. Increased ligandin content was accompanied by parallel increase in subunit synthesis as determined by [³H]leucine incorporation into each subunit relative to incorporation into total cytosolic proteins. The mRNA content for each ligandin subunit was twofold higher in older animals as determined by cell-free in vitro translation followed by immunoprecipitation and dot hybridization using a ligandin cDNA probe. When poly A mRNA from the postmitochondrial fraction of liver from young or old rats was subjected to agarose gel electrophoresis under denaturing conditions and hybridized to ligandin cDNA probe, a single 11 S band was obtained. With RNA from total liver, an additional 13 S band was obtained, suggesting the existence of a precursor form of ligandin mRNA. Since precursor polypeptides were not observed with RNA from total liver in cell-free in vitro translation systems, the precursor form requires processing to the 11 S form before the mRNA becomes functional.     

Human liver glutathione S-transferases: complete primary sequence of an Ha subunit cDNA

Multiple human liver GSH S-transferases (GST) with overlapping substrate specificities may be essential to their multiple roles in xenobiotics metabolism, drug biotransformation, and protection against peroxidative damage. Human liver GSTs are composed of at least two classes of subunits, Ha (Mr = 26,000) and Hb (Mr = 27,500). Immunological cross-reactivity and nucleic acid hybridization studies revealed a close relationship between the human Ha subunit and rat Ya, Yc subunits and their cDNAs. We have determined the nucleotide sequence of the Ha subunit 1 cDNA, pGTH1. The alignments of its coding sequence with the rat Ya and Yc cDNAs indicate that they are approximately 80% identical base-for-base without any deletion or insertion. Regions of sequence homology (greater than 50%) have also been found between pGTH1 and a corn GST cDNA and rat GST cDNAs of the Yb and Yp subunits. Among the 62 highly conserved amino acid residues of the rat GST supergene family, 56 of them are preserved in the Ha subunit 1 coding sequences. Comparison of amino-acid replacement mutations in these coding sequences revealed that the percentage divergence between the rat Ya and Yc genes is more than that between the Ha and Ya or Ha and Yc genes.     

A study of the structures of the YaYa and YaYc glutathione S-transferases from rat liver cytosol. Evidence that the Ya monomer is responsible for lithocholate-binding activity.

The two dimeric lithocholic acid-binding proteins previously identified as ligandin (YaYa) and glutathione S-transferase B (YaYc) were isolated from rat liver cytosol. These proteins have molecular weights of 44000 and 47000 respectively. The recovery of these two proteins from liver was not affected by the addition of the proteinase inhibitor Trasylol. No spontaneous interconversion between these two proteins was observed on storage. YaYa and YaYc proteins yielded peptides of identical molecular weight after limited digestion with Staphylococcus aureus V8 proteinase. Analytical and preparative tryptic-digest peptide 'maps' showed that all the soluble peptides obtained from YaYa protein were also recovered from YaYc protein. Approximately six extra soluble peptides, which were not recovered from YaYa protein, were obtained from the tryptic digest of YaYc protein. Subdigests of the insoluble tryptic-digest 'cores' also resulted in the recovery of identical peptides from both proteins. Evidence is presented that the Ya subunit possessed by both proteins is identical; glutathione S transferase B is a hybrid of ligandin and glutathione S-transferase AA. The Ya monomer is responsible for lithocholate binding.     

Studies on subunit structure and evidence that ligandin is a heterodimer

Several lines of evidence indicate that ligandin consists of two different subunits. The protein dissociates into two components that are detected by electrophoresis in a discontinuous sodium dodecyl sulfate system, or in acid-urea gels, and by isoelectric focusing in the presence of urea. The apparent molecular weights of the two polypeptides are 25,000 and 22,000. Alkylated or succinylated ligandins also exhibit subunit heterogeneity and resolved into two bands in these electrophoretic systems. Cross-linked ligandin showed only one band in sodium dodecyl sulfate-gel electrophoresis indicating that the two subunits are part of a heterodimeric protein rather than monomers of two different proteins. No dansylated terminal amino acids were detected suggesting that the NH2-terminal residues of both chains are blocked. One mole of arginine or phenylalanine was released per mole of ligandin after digestion with carboxypeptidase B or A, respectively. Tryptic maps of succinylated ligandin were consistent with identical disposition of arginine residues in both chains, but several additional tryptic peptides were obtained with native ligandin as compared to the predicted number if both subunits were identical. These observations are consistent with the possibility that both subunits contain common sequences and that a small peptide of about 25 to 30 amino acid residues is cleaved from the COOH-terminal of the larger subunit to produce the smaller subunit.     

Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney

cDNA complementary to mRNA coding for the beta subunit of dog renal (Na+ + K+)-ATPase has been cloned into lambda gt11 and the nucleotide sequence of the DNA has been determined. The amino acid sequence of the beta subunit polypeptide has also been deduced from the DNA. The mature form of the dog kidney beta subunit contains 302 amino acids with three potential asparagine-linked attachment sites for carbohydrate. The initiation methionine is removed during processing of the polypeptide to its mature form. Although the beta subunit is an integral membrane protein there is no signal sequence for the polypeptide, and hydropathy analysis predicts that the beta subunit polypeptide spans the cell membrane only once. Secondary structure predictions and a model for the structure of the beta subunit are proposed. DNA sequencing of the 5' non-coding region of the mRNA revealed a 200 bp inverted repeat from the coding region. Blot hybridization of a fragment of the beta subunit cDNA identified a single mRNA species of 2.7 kb in dog kidney and several rat tissues. RNA from rat liver was deficient in mRNA that hybridized to the dog kidney beta subunit cDNA, although mRNA that hybridized to an alpha subunit cDNA was detected. RNA from a human hepatoma cell line, HepG2, however, contained comparable levels of mRNA for both the alpha and the beta subunits.     

Amino acid sequence of the a subunit of human factor XIII

Factor XIII is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The complete amino acid sequence of the a subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gtll cDNA library prepared from human placenta mRNA was screened with an affinity-purified antibody against the a subunit of human factor XIII and then with a synthetic oligonucleotide probe that coded for a portion of the amino acid sequence present in the activation peptide of the a subunit. Six positive clones were identified and shown to code for the a subunit of factor XIII by DNA sequence analysis. A total of 3831 base pairs was determined by sequencing six overlapping cDNA clones. This DNA sequence contains a 5' noncoding region or a region coding for a portion of a pro-piece or leader sequence, the mature protein (731 amino acids), a stop codon (TGA), a 3' noncoding region (1535 nucleotides), and a poly(A) tail (10 nucleotides). When the a subunit of human factor XIII was digested with cyanogen bromide, 11 peptides were isolated by gel filtration and reverse-phase HPLC. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 363 amino acid residues were identified. These amino acid sequences were in complete agreement with those predicted from the cDNA. The a subunit of factor XIII contained the active site sequence of Tyr-Gly-Gln-Cys-Trp, which is identical with that of tissue transglutaminase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction and characterization of a plasmid containing complementary DNA to mRNA encoding the N-terminal amino acid sequence of the rat glutathione transferase Ya subunit.

Free polyribosomal poly(A)-containing RNA isolated from normal rat liver was used to prepare a complementary DNA plasmid library in the Pst1 site of the plasmid pAT153 . A plasmid pGSTr155 complementary to mRNA coding for a glutathione transferase Ya subunit was selected by differential hybridization in situ and preliminary characterization was performed by hybrid-selected mRNA translation, immunoprecipitation and polyacrylamide-gel electrophoresis of the product synthesized in vitro. The nucleotide sequence of the complementary DNA contained within pGSTr155 was determined and shown to contain a single open reading frame corresponding to the first 129 amino acids of the N-terminus of the Ya subunit and a further 63 nucleotides upstream of the initiating methionine codon.     

Cloning of a putative γ-aminobutyric acid (GABA) receptor subunit ϱ3 cDNA

Cloned cDNA encoding a putative member of GABA receptor ϱ-subunit class was isolated from rat-retina-mRNA-derived libraries. The cDNA encodes a signal peptide of 21 amino acids followed by the mature ϱ3 subunit sequence of 443 amino acids. The proposed amino acid sequence exhibits 63 and 61% homology to the previously-reported human ϱ1 and rat ϱ2 sequences, respectively. Northern blot analysis demonstrated the expression of mRNA for ϱ3 subunit in retina.     

Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.