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.     

Expression of Yb1 glutathione S-transferase using a baculovirus expression system

A full-length cDNA clone was isolated for rat liver Yb1 glutathione S-transferase (EC 2.5.1.18). The coding sequence of Yb1 cDNA was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The enzymatically active recombinant Yb1 glutathione S-transferase protein has a native molecular weight of 42,000 daltons (by molecular sieve chromatography), a subunit molecular weight of 26,500 daltons (by SDS-polyacrylamide gel electrophoresis), a pI of 8.4 and an extinction coefficient E1%280 of 5.6 +/- 0.4.     

Expression of an enzymatically active Yb3 glutathione S-transferase in Escherichia coli and identification of its natural form in rat brain

Glutathione S-transferases containing Yb3 subunits are relatively uncommon forms that are expressed in a tissue-specific manner and have not been identified unequivocally or characterized. A cDNA clone containing the entire coding sequence of Yb3 glutathione S-transferase mRNA was incorporated into a pIN-III expression vector used to transform Escherichia coli. A fusion Yb3-protein containing 14 additional amino acid residues at its N terminus was purified to homogeneity. Recombinant Yb3 was enzymatically active with both 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates but lacked glutathione peroxidase activity. Substrate specificity patterns of recombinant Yb3 were more limited than those of glutathione S-transferase isoenzymes containing Yb1- or Yb2-type subunits. Peptides corresponding to unique amino acid sequences of Yb3 as well as a peptide from a region of homology with Yb1 and Yb2 subunits were synthesized. These synthetic peptides were used to raise antibodies specific to Yb3 and others that cross-reacted with all Yb forms. Immunoblotting was utilized to identify the natural counterpart of recombinant Yb3 among rat glutathione transferases. Brain and testis glutathione S-transferases were rich in Yb3 subunits, but very little was found in liver or kidney. Physical properties, substrate specificities, and binding patterns of the recombinant protein paralleled properties of the natural isoenzyme isolated from brain.     

Rat glutathione S-transferases supergene family. Characterization of an anionic Yb subunit cDNA clone

High multiplicity of 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. Subunit composition analysis of rat liver GSH S-transferases indicated that heterodimer associations were not random, limiting the generation of GST isozyme multiplicity. We have analyzed a Yb subunit cDNA clone, pGTR187, that may correspond to an anionic Yb subunit sequence. Comparison with other GSH S-transferase cDNA sequences and blot hybridization results indicates that the multiple Yb subunits are encoded by a multigene family. This Yb subunit sequence has very limited homology to Ya and Yc subunit cDNAs, but slightly more sequence homology to the Yp subunit cDNA. More consistent sequence homology is found at the amino acid level with 28% conservation throughout the coding sequences. These results and results published from other laboratories clearly indicate that rat GSH S-transferases are products of at least four different gene families that constitute a supergene family. Conceptually, the supergene family may encode GSH S-transferases of very different structures that are essential to metabolize a multitude of xenobiotics in addition to serving other physiologically important functions.     

The human liver glutathione S-transferase gene superfamily: expression and chromosome mapping of an Hb subunit cDNA.

We have isolated from a lambda gt10 cDNA library a clone lambda GTH4 which encodes a human liver glutathione S-transferase Hb subunit, designated as subunit 4. Expression of this cDNA in E. coli and subsequent purification and immunoblotting analysis provided a definitive assignment of a structure and function relationship. RNA blot hybridization with human liver poly(A) RNA revealed a single band of approximately 1200 nucleotides, comparable in size to the rat brain Yb3 mRNA. Divergence analysis of amino acid replacement sites in subunit 4 relative to the four rat Yb subunits revealed that it is most closely related to the brain-specific Yb3 subunit. This conclusion is further substantiated by the nucleotide sequence homology between lambda GTH4 and the Yb3 cDNA in their 3' untranslated region. In situ chromosome mapping has located this glutathione S-transferase gene in the region of p31 on chromosome 1. Results from many laboratories, including ours, indicate that the human glutathione S-transferases are encoded by a gene superfamily which is located on at least two different chromosomes.     

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.     

Recombinant glutathione S-transferase (GST) expressing cells purified by flow cytometry on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane.

COS cells transiently expressing glutathione S-transferase (GST) pi, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (+/-)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On-line reagent addition ensured that all cells were exposed to 25 microM mClB for 30-35 s during transit before being analyzed for fluorescence intensity and sorted. The apparent Km for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 microM. Stained GST Ya+ or Yb1+ cells catalyzed the conjugation 2 or 5 times more effectively than GST pi+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 +/- 0.3-fold greater than that of the control (80 +/- 4 nmol/min/mg protein). Upon a 5-fold purification of GST pi+ cells in the electroporated population, resistance to anti-BPDE in colony-forming assays increased 5 times, from 1.1-fold (unsorted) to 1.5-fold (sorted) (P less than 0.001).     

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.     

Inhibition of human glutathione transferases by multidrug resistance chemomodulators in vitro

Reversal of the drug-resistance phenotype in cancer cells usually involves the use of a chemomodulator that inhibits the function of a resistance-related protein. The aim of this study was to investigate the effects of MDR chemomodulators on human recombinant glutathione S-transferase (GSTs) activity. IC50 values for 15 MDR chemomodulators were determined using 1-chloro-dinitrobenzene (CDNB), cumene hydroproxide (CuOOH) and anticancer drugs as substrates. GSTs A1, P1 and M1 were inhibited by O6-benzylguanine (IC50s around 30 microM), GST P1-1 by sulphinpyrazone (IC50 = 66 microM), GST Al-1 by sulphasalazine, and camptothecin (34 and 74 microM respectively), and GST M1-1 by sulphasalazine, camptothecin and indomethacin (0.3, 29 and 30 microM respectively) using CDNB as a substrate. When ethacrynic acid (for GST P1-1), CuOOH (for A1-1) and 1,3-bis (2-chloroethyl)-1-nitrosourea (for GST M1-1) were used as substrates, these compounds did not significantly inhibit the GST isoforms. However, progesterone was a potent inhibitor of GST P1-1 (IC50 = 1.4 microM) with ethacrynic acid as substrate. These results suggest that the target of chemomodulators in vivo could be a specific resistance-related protein.     

Production of recombinant rat interleukin-6 in Escherichia coli using a novel highly efficient expression vector pGEX-3T.

Interleukin-6 (IL-6) is one of the most important mediators of the acute phase reaction in liver. For the production of recombinant rat IL-6 in Escherichia coli, a previously isolated cDNA coding for the rat IL-6 was cloned into the modified novel expression vector pGEX-3T. The IL-6 cDNA was highly expressed as a fusion protein with the glutathione S-transferase (GST) at its C-terminus and rat IL-6 at its N-terminus. The GST-IL-6 fusion protein was controlled by a tac-promoter and could be induced very efficiently by isopropyl-beta-D-thiogalactopyranoside. The synthesized GST-IL-6 fusion protein was insoluble and precipitated intracellularly in E. coli. Using an advanced technique, the insoluble protein was solubilized and purified to homogeneity by affinity chromatography using immobilized glutathione in a one-step procedure.     

The gene for the microsomal glutathione S-transferase is on human chromosome 12

The microsomal glutathione S-transferase (GST) is a unique membrane-bound GST structurally distinct from the cytosolic GSTs. A cDNA encoding this 154 amino acid protein has recently been isolated and characterized. Using the cDNA as the hybridization probe, we now report the assignment of the human microsomal GST gene to chromosome 12 through the use of a panel of mouse-human somatic cell hybrid lines. This locus has recently been designated as GST 12. In addition, genomic Southern blotting data suggest that the human microsomal GST is encoded by a single- or very-low-copy gene. Therefore, the human GST gene superfamily resides on at least four separate chromosomes: 1 (GST 1), 6 (GST 2), 11 (GST 3), and 12 (GST 12).     

Small GTP binding proteins: Rab GTPases from the brain of Bombyx mori

From a mRNA of the brain of Bombyx mori, we isolated 8 cDNA clones (BRabs), each of which encodes a different member of Rab-protein family. Four of them have more than 80% amino acid identity to the corresponding members of Drosophila Rab proteins. The other 4 proteins show low sequence similarity to any of the known Rab proteins. However, all of them contain the region conserved in rab protein. Using RACE (Rapid Amplification of cDNA ends), the one full-length cDNA clone (BRab14) was isolated. The clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. After purification, the fusion protein was cut with protease to remove GST-Tag and applied to a glutathione S-Sepharose column. The protein bound [(3)H]-GDP with association constant of 1.02 x 10(11) M(-1). Further, the protein was phosphorylated by protein kinase. This result suggests that Rab protein in the brain of Bombyx mori binds GDP or GTP and its function is regulated by phosphorylation.     

Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli

Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity. Here, we addressed this question by production of such protein. In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica. Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs. 16.6 s(-1)), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity. It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts for purification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity.     

Dichloromethane mediated in vivo selection and functional characterization of rat glutathione S-transferase theta 1-1 variants.

Methylobacterium dichloromethanicum DM4 is able to grow with dichloromethane as the sole carbon and energy source by using a dichloromethane dehalogenase/glutathione S-transferase (GST) for the conversion of dichloromethane to formaldehyde. Mammalian homologs of this bacterial enzyme are also known to catalyze this reaction. However, the dehalogenation of dichloromethane by GST T1-1 from rat was highly mutagenic and toxic to methylotrophic bacteria. Plasmid-driven expression of rat GST T1-1 in strain DM4-2cr, a mutant of strain DM4 lacking dichloromethane dehalogenase, reduced cell viability 10(5)-fold in the presence of dichloromethane. This effect was exploited to select dichloromethane-resistant transconjugants of strain DM4-2cr carrying a plasmid-encoded rGSTT1 gene. Transconjugants that still expressed the GST T1 protein after dichloromethane treatment included rGSTT1 mutants encoding protein variants with sequence changes from the wild-type ranging from single residue exchanges to large insertions and deletions. A structural model of rat GST T1-1 suggested that sequence variation was clustered around the glutathione activation site and at the protein C-terminus believed to cap the active site. The enzymatic activity of purified His-tagged GST T1-1 variants expressed in Escherichia coli was markedly reduced with both dichloromethane and the alternative substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane. These results provide the first experimental evidence for the involvement of Gln102 and Arg107 in catalysis, and illustrate the potential of in vivo approaches to identify catalytic residues in GSTs whose activity leads to toxic effects.     

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.     

vMIP-ⅡN端重组肽的表达纯化及活性鉴定

病毒巨噬细胞炎症蛋白-Ⅱ (viral macrophage inflammatory protein-Ⅱ,vMIP-Ⅱ)由卡波西肉瘤疱疹病毒编码,前期研究证明了vMIP-II N端21肽(NT21MP)选择性的阻断趋化因子CXCR4,从而抑制乳腺癌细胞趋化的活性。通过化学合成法获得编码vMIP-ⅡN末端的基因序列,与pGEX-KG（克隆位点的N 端有谷胱甘肽转移酶GST标签序列）连接构建原核表达载体,重组质粒在大肠杆菌E.coli BL21(DE3)中获得表达,免疫印迹显示重组蛋白GST-NT21MP主要在细菌裂解液上清中表达,可溶性部分经亲和层析、超滤、快速蛋白液相色谱（fast protein liquid chromatography,FPLC）纯化获得高纯度的GST-NT21MP蛋白。利用Transwell趋化试验测定GST-NT21MP的活性。结果显示,重组蛋白GST-NT21MP能够抑制乳腺癌细胞SK-BR-3的趋化活性,可作为治疗乳腺癌转移的潜在性靶向药物。     

Expression and purification of pheophorbidase, an enzyme catalyzing the formation of pyropheophorbide during chlorophyll degradation: comparison with the native enzyme

Formation of pyropheophorbide (PyroPheid) during chlorophyll metabolism in some higher plants has been shown to involve the enzyme pheophorbidase (PPD). This enzyme catalyzes the conversion of pheophorbide (Pheid) a to a precursor of PyroPheid, C-13(2)-carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield PyroPheid a. In this study, expression, purification, and biochemical characterization of recombinant PPD from radish (Raphanus sativus L.) were performed, and its properties were compared with those of highly purified native PPD. Recombinant PPD was produced using a glutathione S-transferase (GST) fusion system. The PPD and GST genes were fused to a pGEX-2T vector and expressed in Escherichia coli under the control of a T7 promoter as a fusion protein. The recombinant PPD-GST was expressed as a 55 kDa protein as measured by SDS-PAGE and purified by single-step affinity chromatography through a GSTrap FF column. PPD-GST was purified to homogeneity with a yield of 0.42 mg L(-1) of culture. The protein purified by this method was confirmed to be PPD by measuring its activity. The purified PPD-GST fusion protein revealed potent catalytic activity for demethylation of the methoxycarbonyl group of Pheid a and showed a pH optimum, substrate specificity, and thermal stability quite similar to the native enzyme purified from radish, except for the Km values toward Pheid a: 95.5 microM for PPD-GST and about 15 microM for native PPDs.