Expression and characterization of a novel class of glutathione S-transferase from Anopheles dirus

A new Anopheles dirus glutathione S-transferase (GST) has been obtained and named adGST4-1. Both genomic DNA and cDNA for heterologous expression were acquired. The genomic sequence was 3188bp and consisted of the GST gene as well as flanking sequence. The flanking sequence was analyzed for possible regulatory elements that would control gene expression. In Drosophila several of these elements have been shown to be involved in development and cell differentiation. The deduced amino acid sequence has low identity compared with the four alternatively spliced enzymes, adGST1-1 to 1-4, from another An. dirus GST gene adgst1AS1. The percent identities are 30--40% and 11--12% comparing adGST4-1 to insect GSTs from Delta and Sigma classes, respectively. Enzyme characterization of adGST4-1 shows it to be distinct from the other An. dirus GSTs because of low enzyme activity for customary GST substrates including 1-chloro-2, 4-dinitrobenzene (CDNB). However, this enzyme has a greater affinity of interaction with pyrethroids compared to the other An. dirus GSTs.     

Molecular cloning and characterization of theta-class glutathione S-transferase (GST-T) from the hermaphroditic fish Rivulus marmoratus and biochemical comparisons with alpha-class glutathione S-transferase (GST-A)

We cloned and sequenced full-length cDNA of a theta-class-like glutathione S-transferase (GST-T) from liver tissue of the self-fertilizing fish Rivulus marmoratus. The full-length cDNA of rm-GST-T was 907 bp in length containing an open reading frame of 666 bp that encoded a 221-amino acid putative protein. Its derived amino acid sequence was clustered with other vertebrate theta-class GSTs in a phylogenetic tree. The deduced amino acid sequence of theta-like rm-GST (rm-GST-T) was compared with both classes (alpha and theta) of GST and alpha-class rm-GST (rm-GST-A). Tissue-specific expression of two rm-GST mRNAs was investigated using real-time RT-PCR. To further characterize the catalytic properties of this enzyme along with rm-GST-A, we constructed the recombinant theta-like rm-GST plasmid with a 6 x His-Tag at the N-terminal of rm-GST-T cDNA. Recombinant rm-GST-T was highly expressed in transformed Escherichia coli, and its soluble fraction was purified by His-Tag affinity column chromatography. The kinetic properties and effects of pH and temperature on rm-GST-T were further studied, along with enzyme activity and inhibition effects, and compared with recombinant rm-GST-A. These results suggest that recombinant rm-GSTs such as rm-GST-A and rm-GST-T play a conserved functional role in R. marmoratus.     

Heterologous expression and functional characterization of avian mu-class glutathione S-transferases

Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B₁ (AFB₁), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB₁-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB₁, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB₁ detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8 kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA). However, none of the GSTM proteins had AFBO detoxification capability, in contrast to recombinant alpha-class GSTs shown in our recent study to possess this important activity. In total, our data indicate that although turkey hepatic GSTMs may contribute to xenobiotic detoxification, they probably play no role in detoxification of AFBO in the liver.     

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.     

The 110kDa glutathione transferase of Yarrowia lipolytica is encoded by a homologue of the TEF3 gene from Saccharomyces cerevisiae: cloning, expression, and homology modeling of the recombinant protein

The TEF4 gene of the non-saccharomyces yeast Yarrowia lipolytica encodes an EF1Bgamma protein with structural similarity to the glutathione transferases (GSTs). This 1203bp gene was cloned, over-expressed in Escherichia coli, and the recombinant protein characterized. DNA sequencing of the cloned gene agreed with the recently completed Y. lipolytica genome and showed 100% identity to a previously reported 30-residue N-terminal sequence for a 110kDa Y. lipolytica GST, except that it encoded two additional N-terminal residues (N-Met-Ser-). The recombinant protein (subunit M(r) 52kDa) was found not to possess GST activity with 1-chloro-2,4-dinitrobenzene. Partial tryptic digestion released two fragments of M(r) 22 and 18kDa, which we interpret as N- and C-terminal domains. Homology modeling confirmed that the N-terminal domain of Y. lipolytica TEF4 encodes a GST-like protein.     

Characterization and molecular cloning of a glutathione S-transferase from the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)

Glutathione S-transferases (GST) catalyzing the conjugation of reduced glutathione to a vast range of xenobiotics including insecticides were characterized in the whitefly Bemisia tabaci. GST activities were determined in susceptible and resistant strains of B. tabaci towards artificial substrates, i.e. 1-chloro-2,4-dinitrobenzene (CDNB) in a photometric microplate assay and monochlorobimane (MCB) in a fluoroemtric microplate assay and characterized by their Michaelis-Menten kinetics. The inhibitory potential of ethacrynic acid was very effective with IC50-values between 0.9 and 5.8 microM depending on substrate and strain. The inhibitory effect of dicumarol was 10 times lower. Glutathione-affinity chromatography purified GST enzymes of two different B. tabaci strains appeared as a single band on SDS-PAGE and had a molecular mass of 23.5 kDa determined by MALDI mass spectrometry. The N-terminus of the purified enzyme was sequenced by Edman degradation. The nearly full-length cDNA of the enzyme was isolated by RT-PCR using a degenerate primer derived from the N-terminal amino acid sequence and contained an open reading frame encoding a 194-amino-acid protein. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to insect class sigma GSTs.     

Identification of alpha-lactalbumin and beta-lactoglobulin in cynomolgus monkey (Macaca fascicularis) milk.

1. An electrophoretic analysis of whey protein from cynomolgus monkey milk revealed that its constituents are more similar to bovine milk than human milk, i.e. cynomolgus monkey milk whey contains, besides alpha-lactalbumin-like protein (LaP), another predominant component similar to bovine beta-lactoglobulin (LgP), in its electrophoretic behavior on both disc- and SDS-polyacrylamide gel electrophoreses. 2. The amino acid composition of LaP shows close similarity to that of human alpha-lactalbumin, and LaP forms an immunoprecipitin line with anti-human alpha-lactalbumin rabbit antiserum. The homology between LaP and alpha-lactalbumin was further confirmed by an analysis of the N-terminal amino acid sequence. 3. LgP is not immunologically identical to bovine beta-lactoglobulin, but its amino acid composition is similar. The result of the N-terminal amino acid sequence analysis of LgP (up to the 26th residue) strongly suggests homology between this protein and beta-lactoglobulin.     

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.     

Lectins from seeds of Crotalaria pallida (smooth rattlebox)

A lectin from the seeds of Crotalaria pallida (CPL), with an apparent molecular mass of 30 kDa, determined by SDS-polyacrylamide gel electrophoresis, showed human type A and B erythrocytes agglutination activity, which is inhibited by raffinose and galactose. The lectin requirement for divalent cation was demonstrated with EDTA/EGTA blocking hemagglutination activity. Although the N-terminal amino acid sequence of CPL is identical to another lectin from Crotalaria striata, which is taxonomically synonymous to Crotalaria pallida, these lectins differ in amino acid composition and hemagglutination properties.     

The isolation and characterization of the major glutathione S-transferase from the squid Loligo vulgaris

1. The major glutathione S-transferase (GST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8. 2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane. 3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs. 4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species.     

Plasmodium vivax: molecular cloning, expression and characterization of glutathione S-transferase

Malaria parasite glutathione S-transferases (GSTs) are postulated to be essential for parasite survival by protecting the parasite against oxidative stress and buffering the detoxification of heme-binding compounds; therefore, GSTs are considered potential targets for drug development. In this study, we identified a Plasmodium vivax gene encoding GST (PvGST) and characterized the biochemical properties of the recombinant enzyme. The PvGST contained 618 bp that encoded 205 amino acids and shared a significant degree of sequence identity with GSTs from other Plasmodium species. The recombinant homodimeric enzyme had an approximate molecular mass of 50kDa and exhibited GSH-conjugating and GSH-peroxidase activities towards various model substrates. The optimal pH for recombinant PvGST (rPvGST) activity was pH 8.0, and the enzyme was moderately unstable at 37 degrees C. The K(m) values of rPvGST with respect to GSH and CDNB were 0.17+/-0.09 and 2.1+/-0.4mM, respectively. The significant sequence homology and similar biochemical properties of PvGST and Plasmodium falciparum GST (PfGST) indicate that they may have similar molecular structures. This information may be useful for the design of specific inhibitors for plasmodial GSTs as potential antimalarial drugs.     

Molecular cloning and characterization of alpha-class glutathione S-transferase genes from the hepatopancreas of red sea bream, Pagrus major

Two distinct cDNAs corresponding to GSTA1 and GSTA2 genes encoding glutathione S-transferases (GSTs) from the hepatopancreas of red sea bream, Pagrus major were cloned and sequenced. A comparison of the nucleotide sequences of GSTA1 and GSTA2 revealed 98% identity and their derived amino acid sequences had 96% similarity. Both genes could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. Genomic DNA cloning showed that both GSTA1 and GSTA2 genes consisted of six exons and five introns. In a comparison of genomic DNAs, the structures of GSTA1 and GSTA2 differed. In addition, Southern-blot analysis indicated that at least two kinds of alpha-class GSTs existed in the P. major genome. In order to biochemically characterize the recombinant enzymes (pmGSTA1-1 and pmGSTA2-2), both clones were highly expressed in Escherichia coli. The purified pmGSTA1-1 and pmGSTA2-2 exhibited glutathione conjugating activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide, while neither pmGSTs show detectable activity toward 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal, or p-nitrobenzyl chloride. Despite their high level of amino acid sequence identity, the pmGSTs had quite different enzyme-kinetic parameters.     

Characterization of glutathione S-transferases from day-old chick livers

Glutathione S-transferases (GSTs, EC 2.5.1.18) were isolated from the liver cytosolic fraction of 1 day old Leghorn chicks by S-hexylglutathione and glutathione affinity columns arranged in tandem. After sample loading, the affinity columns were detached from each other and developed separately. Four groups of GSTs (CL 1, 2, 3, and 4) were eluted from the hexylglutathione column, and an additional group of GSTs (CL 2 and 5) was eluted from the glutathione affinity column. CL 2, CL 3, and CL 5 were further purified to homogeneity by chromatofocusing, and the substrate specificities of each group were determined. Fractions from the chromatofocusing column were analyzed by native IEF electrophoresis. Protein bands were electroblotted onto PVDF membrane for N-terminal sequence analysis or extracted from IEF gel and rerun on SDS-PAGE to determine the subunit composition of each GST dimer. CL 2, CL 3, and CL 5 can form homodimers, whereas CL 1 and CL 4 exist only as CL 1-2 and CL 3-4 heterodimers. CL 2 and CL 5 have N-terminal amino acid sequences homologous to rat liver Yb and Ya GSTs, respectively. CL 1 has a unique N-terminal sequence that is not homologous to any known GSTs.     

Glutathione S-transferases in the Japanese quail: Tissue distribution and purification of the liver isozymes

Cytosolic glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EA), 1,2-epoxy-3-(p-nitrophenoxyl)propane (EPNP), trans-4-phenyl-3-buten-2-one (t-PBO), δ³-androstene-3,17-dione (ASD) and trans-stilbene oxide (t-SO); cytosolic glutathione peroxidase activity toward cumene hydroperoxide (CuOOH); and microsomal GST activity toward CDNB were examined in liver, kidney, brain, and lung of adult male and female Japanese quail. In all cases, the renal specific activity per milligram protein was higher than the hepatic activity and was the highest among the four tissues examined. No consistent sex differences in GST activity were observed. The GSTs were purified from quail liver cytosol by S-hexylglutathione and glutathione affinity chromatography. Total GSTs eluted from the S-hexylglutathione affinity column were further separated by chromatofocusing, and the microheterogeneity of the GST isozymes was shown by high-resolution native isoelectrofocusing (IEF) in polyacrylamide slab gels and by SDS-PAGE. Five subunits were identified: QL1 (30.5 kDa), QL2 (27.2 kDa), QL3a (26.8 kDa), QL3b (26.5 kDa), and QL4 (25.5 kDa). Western blot analysis revealed that QL1 and QL2 reacted with antibodies raised against the rat Mu class GSTs (Yb1 and Yb2), and QL3a and QL3b reacted with those raised against the Alpha class (rat Ya and mouse a). Substrate specific activity of each isoform was determined with CDNB, DCNB, CuOOH, EA, t-PBO, ASD, and t-SO. QL3a and QL3b have high reactivity toward CuOOH, while QL1 and QL2 showed high activity toward t-SO. The N-terminal amino acid sequence of QL2 was identical to that of the chicken Mu class GST subunit CL2. However, no sequence was obtained with QL1 due to possible N-terminal blockage. © 1996 John Wiley & Sons, Inc.     

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis)

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35 kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35 kDa protein was ~ 98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates.     

Isolating and characterising a lectin from Galactia lindenii seeds that recognises blood group H determinants

A lectin was isolated from Galactia lindenii seeds and characterised. The lectin, purified by affinity chromatography, readily agglutinated O(H) human erythrocytes and interacted weakly with rabbit and rat erythrocytes. Specificity towards blood group H-type determinants was established; among them H-type 2 (alpha-L-Fuc (1-2)-beta-D-Gal (1-4)-beta-D-GlcNAc-O-R) was recognised by the lectin. The binding to the glycoconjugate was partially inhibited by GalNAc and Me-beta-Gal. The protein is an M=104,256 tetramer which dissociates into identical M=26,064 subunits under non-reducing conditions. Its amino acid composition, pI, A(1%), and N-terminal sequence (23 residues) were determined. The N-terminal region showed a unique sequence found hitherto only in some lectins (designated type-II) from the Dioclea genus. This work presents the evidence concerning a distinct type of lectin found in the Diocleinae tribe able to recognise the H-type 2 human blood group determinant and clearly different from the Glc/Man-specific lectins. The protein is a potential tool in cellular and histochemical studies.