Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae).

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.     

Nucleotide sequence of a class mu glutathione S-transferase from chicken liver

A clone coding for glutathione S-transferase (GST) CL2 was isolated from a chicken liver cDNA library. This clone (819 bp) encodes a polypeptide comprising 219 amino acids with a molecular weight of 25,717, excluding the initiator methionine. The primary amino acid sequence of the enzyme has 47% identical sequence with other class mu GSTs.     

Cloning and partial characterization of a Boophilus microplus (Acari: Ixodidae) glutathione S-transferase

A cDNA of glutathione S-transferase (GST) was isolated from a cDNA library of salivary glands of Boophilus microplus. The recombinant protein was purified by glutathione affinity chromatography and assayed upon the chromogenic substrate CDNB. The 864 bp cloned fragment was sequenced and showed an open reading frame coding for a protein of 220 amino acids. Expression of the GST gene was tested by RT-PCR in tick tissues and larvae mRNA. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian class mu GSTs.     

Differential expression of alpha, mu and pi classes of isozymes of glutathione S-transferase in bovine lens, cornea, and retina

Isozyme characterization of glutathione S-transferase (GST) isolated from bovine ocular tissue was undertaken. Two isozymes of lens, GST 7.4 and GST 5.6, were isolated and found to be homodimers of a Mr 23,500 subunit. Amino acid sequence analysis of a 20-residue region of the amino terminus was identical for both isozymes and was identical to GST psi and GST mu of human liver. Antibodies raised against GST psi cross-reacted with both lens isozymes. Although lens GST 5.6 and GST 7.4 demonstrated chemical and immunological relatedness, they were distinctly different as evidenced by their pI and comparative peptide fingerprint. A corneal isozyme, GST 7.2, was also isolated and established to be a homodimer of Mr 24,500 subunits. Sequence analysis of the amino-terminal region indicated it to be about 67% identical with the GST pi isozyme of human placenta. Antibodies raised against GST pi cross-reacted with cornea GST 7.2. Another corneal isozyme, GST 8.7, was found to be homodimer of Mr 27,000 subunits. Sequence analysis revealed it to have a blocked amino-terminus. GST 8.7 immunologically cross-reacted with the antibodies raised against cationic isozymes of human liver indicating it to be of the alpha class. Two isozymes of retina, GST 6.8 and GST 6.3, were isolated and identified to be heterodimers of subunits of Mr 23,500 and 24,500. Amino-terminal sequence analysis gave identical results for both retina GST 6.8 and GST 6.3. The sequence analysis of the Mr 23,500 subunit was identical to that obtained for lens GSTs. Similarly, sequence analysis of the Mr 24,500 subunit was identical to that obtained for the cornea GST 7.2 isozyme. Both the retina isozymes cross-reacted with antibodies raised against human GST psi as well as GST pi. The results of these studies indicated that all three major classes of GST isozymes were expressed in bovine eye but the GST genes were differentially expressed in lens, cornea, and retina. In lens only the mu class of GST was expressed, whereas cornea expressed alpha and pi classes and retina expressed mu and pi classes of GST isozymes.     

Primary and secondary structural analyses of glutathione S-transferase pi from human placenta

The primary structure of glutathione S-transferase (GST) pi from a single human placenta was determined. The structure was established by chemical characterization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact enzyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide sequence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47, 5626-5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleotide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one individual placenta studied at least two GST pi genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation identified a hydrophobic region in GST pi (residues 155-181) that was predicted to be either a buried transmembrane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme especially in regard to the potential involvement of a histidine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of pi, one of mu, two of alpha, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form.     

Primary sequence heterogeneity and tissue expression of glutathione S-transferases of Fasciola hepatica.

Glutathione S-transferases (GSTs) from Fasciola hepatica have been purified by glutathione affinity chromatography. Two closely migrating species of Mr 26,000 and 26,500 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several species resolved by two-dimensional gel analysis, indicating substantial heterogeneity among the GSTs. N-terminal amino acid sequencing revealed one core sequence containing three polymorphisms, whereas the sequence of GST peptides implied a minimum of three different GSTs. The amino acid sequence data assigned the F. hepatica GSTs to the mu class of GSTs with high similarities to these proteins in other helminths and mammals. The native GSTs of F. hepatica appeared to behave as dimers as determined by molecular sieving chromatography. The observation that the GSTs of F. hepatica are heterogeneous in sequence and behave as dimers in the native state suggest that these isoenzymes may exhibit considerable functional heterogeneity which may be of importance to the parasite. Immunocytochemical studies suggest that the main source of GST in F. hepatica are the parenchymal cells and peripheral tissues of the parasite. Some extracellular GST is associated with the lamellae of the intestinal epithelium. The identification of an intestinal GST is unique among trematodes studied to date.     

Molecular cloning and amino acid sequencing of rat liver class theta glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters of carcinogenic arylmethanols.

A cDNA containing the entire coding sequence for the subunit protein of rat liver class theta glutathione S-transferase (GST) Yrs-Yrs was isolated from a rat liver lambda gt11 cDNA library. The cDNA, designated GST theta-1, consisted of 1,258 bp which had an open reading frame of 732 bp encoding a polypeptide of 244 amino acid (AA) residues, including the leading AA Met to be removed on expression. The authenticity of the cDNA structure was supported by matching its deduced AA sequence with N-termini of Yrs and peptides obtained thereof by tryptic digestion as well as by CNBr cleavage. The deduced AA sequence of the subunit Yrs (M.W. 27,311) had only a weak homology (19-23%) with those of rat liver classes alpha, mu, and pi GST isozymes. Thus, the first evidence for the molecular cloning of the class theta GST was provided.     

Glutathione transferase isoenzymes from frog (Xenopus laevis) liver and embryo

The expression of glutathione transferase isoenzymes has been investigated in embryo and adult liver of the frog Xenopus laevis. By analysing the GST isoenzymes recovered from GSH-affinity chromatography in terms of electrophoretic mobility, HPLC elution profile, immunological reactivity, N-terminal amino acid sequence and mass spectrometry molecular mass no significant difference in the GST subunit composition between embryos and liver was found. In both tissues the same three subunits, showing similarity to mu, alpha and sigma class GSTs, are present. These results, together with those previously reported for toad (Bufo bufo), strongly support the notion that the transition from an aquatic environment to a terrestrial atmosphere containing high oxygen concentration has accompanied specific GST gene expression.     

Glutathione S-transferase isozymes in Aedes aegypti: Purification,characterization, and isozyme-specific regulation

Glutathione S-transferase (GST) isozymes were purified from the GG strain of Aedes aegypti, a strain having ≥4-fold higher total GST activity compared to the wild-type lab strain. Purification involved S-hexyl-glutathione affinity chromatography in high salt buffer, and GST specific elution with S-(p-bromobenzyl)-glutathione. Final purification was accomplished on DEAE-Sepharose. Two isozymes, GST-1b and GST-2 were purified using this procedure, and an additional isozyme, GST-1a, was partially purified. The GST-2 isozyme has one of the highest specific activities reported for a GST, with a specific activity of 739 μmol/min/mg using 1-chloro-2,4-dinitrobenzene (CDNB), and 16.4 μmol/min/mg using 3,4-dichloronitrobenzene (DCNB) as substrates. GST-2, GST-1a, and GST-1b were analyzed for amino acid composition and subjected to N-terminal sequencing. All three GSTs showed amino acid differences, especially among the nonpolar and polar amino acids. The amino acid composition of GST-1b was found to be more similar to GST 1-1 from Drosophila melanogaster than to GST-2 or GST-1a from Aedes aegypti. Only GST-2 gave N-terminal sequence data, raising the possibility that GST-1a and 1b are N-terminally blocked. The A. aegypti GST-2 showed amino acid sequence identity or similarity in all but one residue between residue numbers 31 through 41 compared to the D. melanogaster and Musca domestica GST 1-1 isozymes. The pattern of GST isozyme expression was analyzed in various tissues and stages of development of the GG and wild type strains using isozyme-specific antisera and substrates. GST-1a was constitutively overexpressed in all tissues examined in the GG strain compared to the wild type strain. The expression of GST-1b was similar in both strains for all tissues and developmental stages examined. GST-2 was constitutively overexpressed in head, thorax and abdomen, but was not detected in ovaries of the GG strain. These results suggest that elevated GST activity in the GG strain is due to constitutive overexpression of GST-2 and GST-1a. GST-1a, GST-1b and GST-2 apparently are the products of 3 independently regulated genes and appear to be expressed in a tissue-specific manner.     

Allotypic differences in murine mu genes.

We report the complete DNA sequence of a c-DNA clone of the heavy chain mu b allele of the C57BL/6 mouse. Comparisons have been made with the nucleotide sequences of the germ line BALB/c mu a and the plasmacytoma TEPC-183 mu a alleles reported elsewhere over the entire length of the coding and the 3' untranslated region. In contrast to the extensive differences between the gamma 2a a and b alleles we have reported earlier we see a very high degree of homology between the mu alleles. Only one of the nucleotide differences between C57BL/6 mu b and BALB/c mu a leads to an amino acid substitution. This single amino acid exchange must form the allotypic determinant of the mu b allele. A comparison of four different DNA sequences indicates that they are all distinct IgM alleles.     

The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.     

A new class of rat glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters as metabolites of carcinogenic arylmethanols

A glutathione (GSH) S-transferase (GST), catalyzing the inactivation of reactive sulfate esters as metabolites of carcinogenic arylmethanols, was isolated from the male Sprague-Dawley rat liver cytosol and purified to homogeneity in 12% yield with a purification factor of 901-fold. The purified GST was a homo-dimeric enzyme protein with subunit Mr 26,000 and pI 7.9 and designated as Yrs-Yrs because of its enzyme activity toward "reactive sulfate esters." GST Yrs-Yrs could neither be retained on the S-hexylglutathione gel column nor showed any activity toward 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and 1,2-epoxy-3-(4'-nitrophenoxy)propane. 1-Chloro-2,4-dinitro-benzene was a very poor substrate for this GST. 1-Menaphthyl sulfate was the best substrate for GST Yrs-Yrs among the examined mutagenic arylmethyl sulfates. The enzyme had higher activities toward ethacrynic acid and cumene hydroperoxide. N-terminal amino acid sequence of subunit Yrs, analyzed up to the 25th amino acid, had no homology with any of the known class alpha, mu, and pi enzymes of the Sprague-Dawley rat. Anti-Yrs-IgG raised against GST Yrs-Yrs showed no cross-reactivity with any of subunits Ya, Yc, Yb1, Yb2, and Yp. Anti-IgGs raised against Ya, Yc, Yb1, Yb2, and Yp also showed no cross-reactivity with GST Yrs-Yrs. The purified enzyme proved to differ evidently from the 12 known cytosolic GSTs in various tissues of the rat in all respects. Immunoblot analysis of various tissue cytosols of the male rat indicated that apparent concentrations of the GST Yrs-Yrs protein were in order of liver greater than testis greater than adrenal greater than kidney greater than lung greater than brain greater than skeletal muscle congruent to heart congruent to small intestine congruent to spleen congruent to skin congruent to 0.     

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV.

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.     

Glutathione S-transferase isozymes in rat lens.

Rat lens contains two classes of glutathione S-transferase (GST) isozymes; one is class mu, Yb1-Yb1, and the other is class pi, Yp-Yp, judged from their molecular weights, immunological properties and N-terminal amino acid sequences. The expression pattern of GST isozymes in the rat lens is different from that in pig and bovine lenses which have only class pi and class mu isozymes, respectively.     

Amino acid sequence of the major form of toad liver glutathione transferase

To investigate structural relationship between amphibian and mammalian GSTs the complete amino acid sequence of the major form of glutathione transferase present in toad liver (Bufo bufo) was determined. The enzyme subunit is composed of 210 amino acid residues corresponding to a molecular mass of 24,178 Da. In comparison with the primary structure of amphibian bbGSTP1-1, toad liver GST showed 54% sequence identity. On the other hand, toad liver GST showed about 45-55% sequence identity when compared with other pi class GST and less then 25% identity with GST of other classes. Amino acid residues involved in the H site and in the key and lock structure of the toad enzyme are significantly different from those of bbGSTP1-1 and other mammalian pi class GST. On the basis of its structural and immunological properties the toad liver GST, indicated as bbGSTP2-2, could represent the prototype of a subset of the pi family.     

Molecular cloning, sequencing, and expression of human myocardial fatty acid ethyl ester synthase-III cDNA

Fatty acid ethyl ester synthase-III (FAEES-III), previously purified to homogeneity from human heart, metabolizes ethanol nonoxidatively. Using a derived partial amino acid sequence and corresponding oligonucleotide probes, the cDNA for this enzyme has been cloned from a human heart lambda gtll library. Of the five positive clones obtained, one contained a complete coding region (630 base pairs) and the entire 3'-noncoding region (41 base pairs). From this nucleotide sequence the complete 210 amino acid sequence of FAEES-III (Mr 23,307) is reported. Comparison of its amino acid sequence with that of glutathione S-transferase pi-1 suggests that they belong to the same gene family since they differ in only six nucleotides and four amino acids. The sequence of FAEES-III was also compared with those of placental glutathione S-transferase and the basic glutathione S-transferase. FAEES-III was 84% homologous with placental glutathione S-transferase but only less than 10% homologous with the basic glutathione S-transferase. Northern blots demonstrate expression of FAEES-III mRNA in normal human liver, placenta, and heart. In all cases, the mRNA for the enzyme is 0.7 kilobase in size. MCF-7 cells transfected with FAEES-III cDNA have a 14-fold increase in synthase activity and a 12-fold increase in glutathione S-transferase (GST) activity compared with control cells. MCF-7 cells transfected with GST pi-1 cDNA have a 13-fold increase in GST activity compared with control cells but no increase in synthase activity. When the supernatant of COS-7 cells transfected with FAEES-III cDNA were immunoblotted with rabbit FAEES-III antibody, a band at 24 kilodaltons was demonstrated. Thus, we have obtained the first cDNA and amino acid sequence for a human FAEES-III which also has significant GST activity, and we have identified 4 residues potentially responsible for conferring ethanol recognition to GSTs.     

Complete nucleotide sequence of mouse immunoglobulin mu gene and comparison with other immunoglobulin heavy chain genes

We have determined the complete nucleotides sequence (2168 bases) of the immunoglobulin mu gene cloned from newborn mouse DNA. The cloned 13kb fragment contained the entire constant region gene sequence that is interrupted by three intervening sequences at the junction of domains as previously shown in the gamma 1, gamma 2 b and alpha genes. The amino acid sequence predicted by the nucleotide sequence agrees with that of the mu chain secreted by a myeloma MOPC104E except for 8 residues out of 448 residues. The homologous domains of the mu, gamma 1 and gamma 2b genes are more similar to each other than the different domains of the mu genes are. The result implicates that the class of the immunoglobulin heavy chain genes diverged after the heavy chain genes established the multi-domain structure. The short intervening sequences of the mu and gamma genes are more conserved than the coding sequences except for the COOH-terminal domains. The results implicate that the nucleotide sequence of the intervening sequence is under selective pressure, possibly to maintain a secondary structure of the nuclear RNA to be spliced.     

Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family

We have isolated from a constructed lambda gt11 expression library two classes of cDNA clones encoding the entire sequence of the maize GSH S-transferases GST I and GST III. Expression of a full-length GST I cDNA in E. coli resulted in the synthesis of enzymatically active maize GST I that is immunologically indistinguishable from the native GST I. Another GST I cDNA with a truncated N-terminal sequence is also active in heterospecific expression. Our GST III cDNA sequence differs from the version reported by Moore et al. [Moore, R. E., Davies, M. S., O'Connell, K. M., Harding, E. I., Wiegand, R. C., and Tiemeier, D. C. (1986) Nucleic Acids Res. 14:7227-7235] in eight reading frame shifts which result in partial amino acid sequence conservation with the rat GSH S-transferase sequences. The GST I and GST III sequences share approximately 45% amino acid sequence homology. Both the GST I and the GST III mRNAs contain different repeating motifs in front of the initiation codon ATG. Multiple poly(A) addition sites have been identified for these two classes of maize GSH S-transferase messages. Genomic Southern blotting results suggest that both GST I and GST III are present in single or low copies in the maize (GT112 RfRf) genome.     

Molecular cloning, expression and characterization of glutathione S-transferase from Mytilus edulis

The gene coding for glutathione S-transferase (GST) has been isolated from the Mytilus edulis hepatopancreas. Open reading frame analysis indicated that the M. edulis GST (meGST) gene encodes a protein of 206 amino acid residues with a calculated molecular mass of 23.68 kDa. The deduced amino acid sequence showed high sequence similarity with the sequence of the pi class GST. The meGST was expressed in Escherichia coli, and the recombinant meGST was purified by affinity chromatography and characterized. The recombinant meGST exhibited high activity towards the substrates ethacrynic acid (ECA) and 1-chloro-2,4-dinitrobenzene (CDNB). Kinetic analysis with respect to CDNB as substrate gave a K(m) of 0.68 mM and a V(max) of 0.10 mmol/min per mg protein. The recombinant meGST had a maximum activity at approximately pH 8.5, and its optimum temperature was 39 degrees C. The predicted three-dimensional structure of the meGST revealed the N-terminal domain possesses a thioredoxin fold and the six helices of the C-terminal domain make a alpha-helical bundle. These features indicate that the meGST belongs to pi class GST.     

Purification and partial characterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus.

This paper deals with the purification and the partial characterization of glutathione S-transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)-agarose and S-hexyl GSH-agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and Vmax for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti-pi, anti-mu and anti-alpha mammalian GST classes. Three isoforms (3-3, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera to pi and alpha classes and the isoform 5-5 cross-reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N-terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/m micro /pi GST from Fasciola hepatica.