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Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

ABSTRACT: The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis.

SUBMITTER: Fotouhi-Ardakani N

PROVIDER: S-EPMC1220205 | biostudies-other | 1999 May

REPOSITORIES: biostudies-other

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Project description:BackgroundBaylisascaris schroederi, an intestinal nematode of the giant panda, is the cause of the often fatal disease, baylisascariasis. Glutathione S-transferases (GSTs) are versatile enzymes that can affect parasite survival and parasite-host interactions and, are therefore, potential targets for the development of diagnostic tests and vaccines.MethodsIn this study, we identified a full-length cDNA that encoded a novel, secretory sigma-like GST (Bsc-GSTσ) from a B. schroederi-omic dataset. Following cloning and sequencing, sequence and structural analyses and comparative modeling were performed using online-bioinformatics and proteomics tools. The recombinant Bsc-GSTσ (rBsc-GSTσ) protein was prokaryotically expressed and then used to detect antigenicity and reactivity using immunoblotting assays. In addition, the native protein in female adult B. schroederi was located via immunofluorescence techniques, while the preliminary ELISA-based serodiagnostic potential of rBsc-GSTσ was assessed in native and infected mouse sera.ResultsBsc-GSTσ contained a 621-bp open reading frame that encoded a polypeptide of 206 amino acids with two typical sigma GST domain profiles, including a GST_N_Sigma_like at the N-terminus and a GST_C_Sigma_like at the C-terminus. The presence of an N-terminal signal sequence indicated that Bsc-GSTσ was a secretory protein. Sequence alignment and phylogenetic analyses showed that Bsc-GSTσ was a nematode-specific member of the Sigma class GSTs and shared the closest genetic distance with its homologue in Ascaris suum. Further comparative structure analyses indicated that Bsc-GSTσ possessed the essential structural motifs (e.g., βαβαββα) and the consensus secondary or tertiary structure that is typical for other characterized GSTσs. Immunolocalization revealed strong distributions of native Bsc-GSTσ in the body hypodermis, lateral chords, gut epithelium, gut microvilli, oviduct epithelium, and ovaries of adult female worms, similar to its homologue in A. suum. Building on good immunogenic properties, rBsc-GSTσ-based ELISA exhibited a sensitivity of 79.1% and a specificity of 82.0% to detect anti-B. schroederi IgG antibodies in the sera of experimentally infected mice.ConclusionThis study presents a comprehensive demonstration of sequence and structural-based analysis of a new, secretory sigma-like GST from a nematode, and its good serodiagnostic performance suggests that rBsc-GSTσ has the potential to detect B. schroederi and, therefore, could be used to develop an ELISA-based serological test to diagnose baylisascariasis in giant pandas.

Project description:A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

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Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

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