Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.     

A genomics approach to the comprehensive analysis of the glutathione S-transferase gene family in soybean and maize

By BLAST searching a large expressed sequence tag database for glutathione S-transferase (GST) sequences we have identified 25 soybean (Glycine max) and 42 maize (Zea mays) clones and obtained accurate full-length GST sequences. These clones probably represent the majority of members of the GST multigene family in these species. Plant GSTs are divided according to sequence similarity into three categories: types I, II, and III. Among these GSTs only the active site serine, as well as another serine and arginine in or near the "G-site" are conserved throughout. Type III GSTs have four conserved sequence patches mapping to distinct structural features. Expression analysis reveals the distribution of GSTs in different tissues and treatments: Maize GSTI is overall the most highly expressed in maize, whereas the previously unknown GmGST 8 is most abundant in soybean. Using DNA microarray analysis we observed increased expression among the type III GSTs after inducer treatment of maize shoots, with different genes responding to different treatments. Protein activity for a subset of GSTs varied widely with seven substrates, and any GST exhibiting greater than marginal activity with chloro-2,4 dinitrobenzene activity also exhibited significant activity with all other substrates, suggesting broad individual enzyme substrate specificity.     

Purification,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.     

Purification and characterization of human muscle glutathione S-transferases: evidence that glutathione S-transferase zeta corresponds to a locus distinct from GST1, GST2, and GST3.

Human muscle glutathione S-transferase isozyme, GST zeta (pI 5.2) has been purified by three different methods using immunoaffinity chromatography, DEAE cellulose chromatography, and isoelectric focusing. GST zeta prepared by any of the three methods does not recognize antibodies raised against the alpha, mu, or pi class glutathione S-transferases of human tissues. GST zeta has a blocked N-terminus and its peptide fingerprints also indicate it to be distinct from the alpha, mu, or pi class isozymes. As compared to GSTs of alpha, mu, and pi classes, GST zeta displays higher activities toward t-stilbene oxide and Leukotriene A4 methyl ester. GST zeta also expresses GSH-peroxidase activity toward hydrogen peroxide. The Kms of GST zeta for CDNB and GSH were comparable to those reported for other human GSTs but its Vmax for CDNB, 7620 mol/mol/min, was found to be considerably higher than that reported for other human GSTs. The kinetics of inhibition of GST zeta by hematin, bile acids, and other inhibitors also indicate that it was distinct from the three classes of GST isozymes. These studies suggest that GST zeta corresponds to a locus distinct from GST1, GST2, and GST3 and probably corresponds to the GST4 locus as suggested previously by Laisney et al. (1984, Human Genet. 68, 221-227). The results of peptide fingerprints and kinetic analysis indicate that as compared to the pi and alpha class isozymes, GST zeta has more structural and functional similarities with the mu class isozymes. Besides GST zeta several other GST isozymes belonging to pi and mu class have also been characterized in muscle. The pi class GST isozymes of muscle have considerable charge heterogeneity among them despite identical N-terminal sequences.     

Comparative genomics identifies new alpha class genes within the avian glutathione S-transferase gene cluster

Glutathione S-transferases (GSTs: EC2.5.1.18) are a superfamily of multifunctional dimeric enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic chemicals. In most animals and in humans, GSTs are the principal enzymes responsible for detoxifying the mycotoxin aflatoxin B₁ (AFB₁) and GST dysfunction is a known risk factor for susceptibility towards AFB₁. Turkeys are one of the most susceptible animals known to AFB₁, which is a common contaminant of poultry feeds. The extreme susceptibility of turkeys is associated with hepatic GSTs unable to detoxify the highly reactive and electrophilic metabolite exo-AFB₁-8,9-epoxide (AFBO). In this study, comparative genomic approaches were used to amplify and identify the α-class tGST genes (tGSTA1.1, tGSTA1.2, tGSTA1.3, tGSTA2, tGSTA3 and tGSTA4) from turkey liver. The conserved GST domains and four α-class signature motifs in turkey GSTs (with the exception of tGSTA1.1 which lacked one motif) confirm the presence of hepatic α-class GSTs in the turkey. Four signature motifs and conserved residues found in α-class tGSTs are (1) xMExxxWLLAAAGVE, (2) YGKDxKERAxIDMYVxG, (3) PVxEKVLKxHGxxxL and (4) PxIKKFLXPGSxxKPxxx. A BAC clone containing the α-class GST gene cluster was isolated and sequenced. The turkey α-class GTS genes genetically map to chromosome MGA2 with synteny between turkey and human α-class GSTs and flanking genes. This study identifies the α-class tGST gene cluster and genetic markers (SNPs, single nucleotide polymorphisms) that can be used to further examine AFB₁ susceptibility and resistance in turkeys. Functional characterization of heterologously expressed proteins from these genes is currently underway.     

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse,ancient protein super family using motif search and structural modeling

Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor 1γ (EF1γ) contains an N-terminal domain related to class θ glutathione S-transferases (GST). GST-like proteins related to class θ comprise a large group including, in addition to typical GSTs and EF1γ, stress-induced proteins from bacteria and plants, bacterial reductive dehalogenases and β-etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (α, μ, and π). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST-related domain of EF1γ is likely to form a fold very similar to that in the known structures of class α, μ, and π GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GST-like domain in EF1γ is enzymatically active and that to exhibit GST activity, EF1γ has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EF1 and aminoacyl-tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.     

Response of glutathione S-transferase (GST) genes to cadmium exposure in the marine pollution indicator worm, Perinereis nuntia

Glutathione S-transferase (GST) is a phase II enzyme that functions as a detoxicant by catalyzing the conjugation of reduced glutathione with a variety of xenobiotics via cysteine thiol. Molecular genetic approaches using gene biomarkers show substantial relevance as sensitive biomarkers for the indication of pollution levels. In order to use GSTs as molecular biomarkers for marine pollution monitoring, we cloned and sequenced the full-length cDNA of seven GST genes from the marine polychaete Perinereis nuntia. The deduced amino acid sequence of Pn-GSTs showed a high similarity to those of other species that clustered into the same clades in a phylogenetic analysis. In addition, to evaluate Pn-GSTs as useful biomarkers on effects after cadmium (Cd) exposure, we exposed sublethal concentrations of Cd (5, 50, and 500 μg/L) to P. nuntia, and they showed relatively different but significantly increases, depending on exposure time and Cd concentrations. Particularly, Pn-GST-omega and Pn-GST-sigma genes were highly sensitive with a clear dose-dependent manner on mRNA expression. The total GST activities also have significantly increased levels at higher concentrations of Cd exposure. These results indicate that Pn-GSTs play important roles in Cd-induced oxidative stress in terms of the physiological changes relating to metabolism and cell protection, and those genes would have great potential as molecular biomarkers to monitor marine environmental health.     

Charcot‐Marie‐Tooth disease‐associated mutants of GDAP1 dissociate its roles in peroxisomal and mitochondrial fission

Mitochondria and peroxisomes can be fragmented by the process of fission. The fission machineries of both organelles share a set of proteins. GDAP1 is a tail‐anchored protein of mitochondria and induces mitochondrial fragmentation. Mutations in GDAP1 lead to Charcot‐Marie‐Tooth disease (CMT), an inherited peripheral neuropathy, and affect mitochondrial dynamics. Here, we show that GDAP1 is also targeted to peroxisomes mediated by the import receptor Pex19. Knockdown of GDAP1 leads to peroxisomal elongation that can be rescued by re‐expressing GDAP1 and by missense mutated forms found in CMT patients. GDAP1‐induced peroxisomal fission is dependent on the integrity of its hydrophobic domain 1, and on Drp1 and Mff, as is mitochondrial fission. Thus, GDAP1 regulates mitochondrial and peroxisomal fission by a similar mechanism. However, our results reveal also a more critical role of the amino‐terminal GDAP1 domains, carrying most CMT‐causing mutations, in the regulation of mitochondrial compared to peroxisomal fission.     

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.     

A novel glutathione transferase from Haemophilus influenzae which has high affinity towards antibiotics

Cytosolic glutathione transferase (GSTs) are a family of multi-functional proteins which catalyse the conjugation of glutathione (GSH) to a large variety of endogenous and exogenous electrophilic compounds. Much is known about cytosolic mammalian GSTs, however, the presence of GSTs in several aerobic and anaerobic micro-organisms has also been demonstrated. Several findings seem to suggest that bacterial GSTs are involved in processes of biodegradation of xenobiotics, including antibiotics. However, the function played by these enzymes in the bacterial cell still remains to be clarified. At present, it is ill-defined whether bacterial GST can be classified, as in the case of mammalian enzymes, into several distinct classes.Here we report the purification of a GST isoform from Haemophilus influenzae using GSH-affinity chromatography. The purified protein was characterised by immunological and kinetic properties different from other known GSTs. The dissociation constants of chloramphenicol, ampicillin, rifampicin and tetracycline to the purified enzyme were 0.62, 9.06, 4.08 and 1.77 microM, respectively, as determined by following the quenching of the protein intrinsic fluorescence. These values were much lower than those previously determined for the same drugs with other mammalian or bacterial GSTs.The present results indicate that the enzyme purified from H. influenzae is a novel GST isoform well distinguished from other known mammalian or bacterial GSTs.     

Molecular evolution of glutathione S-transferases in the genus Drosophila

As classical phase II detoxification enzymes, glutathione S-transferases (GSTs) have been implicated in insecticide resistance and may have evolved in response to toxins in the niche-defining feeding substrates of Drosophila species. We have annotated the GST genes of the 12 Drosophila species with recently sequenced genomes and analyzed their molecular evolution. Gene copy number variation is attributable mainly to unequal crossing-over events in the large delta and epsilon clusters. Within these gene clusters there are also GST genes with slowly diverging orthologs. This implies that they have their own unique functions or have spatial/temporal expression patterns that impose significant selective constraints. Searches for positively selected sites within the GSTs identified G171K in GSTD1, a protein that has previously been shown to be capable of metabolizing the insecticide DDT. We find that the same radical substitution (G171K) in the substrate-binding domain has occurred at least three times in the Drosophila radiation. Homology-modeling places site 171 distant from the active site but adjacent to an alternative DDT-binding site. We propose that the parallel evolution observed at this site is an adaptive response to an environmental toxin and that sequencing of historical alleles suggests that this toxin was not a synthetic insecticide.     

Frequencies of GSTM1, GSTT1, and GSTP1 polymorphisms in a Brazilian population

The glutathione S-transferase (GST) family of enzymes has a vital role in phase II of biotransformation of environmental carcinogens, pollutants, drugs and other xenobiotics. GSTs are polymorphic, with the type and frequency of polymorphism being ethnic dependent. Polymorphisms in GST genes have been shown to be associated with susceptibility to disease and disease outcome. We determined the frequencies of GSTM1, GSTT1 and GSTP1 polymorphisms in 591 volunteers who had been residents of Rio de Janeiro for at least six months. Blood was collected and DNA extracted by proteinase K/SDS digestion. Information about social habits and health problems was also recorded. GSTM1 and GSTT1 polymorphisms were analyzed by a PCR-Multiplex procedure, whereas GSTP1 polymorphism was analyzed by PCR-RFLP. We found that 42.1% (48.9% of whites and 34.2% of non-whites) of the individuals had the GSTM1 null genotype, whereas 25.4% (25.1% of whites and 25.7% of non-whites) had the GSTT1 null genotype. The genotypic distribution of GSTP1 was 49.7% I/I, 38.1% I/V, and 12.2% V/V, whereas the allelic frequencies were 0.69 for the Ile allele, and 0.31 for the Val allele. The frequencies of GST polymorphisms in this Brazilian population were found to be different from those observed in other populations, particularly of other South American countries.     

An Inducible Glutathione S-Transferase in Soybean Hypocotyl Is Localized in the Apoplast

Glutathione S-transferases (GSTs) with additional activities as fatty acid hydroperoxidases were investigated in soybean (Glycine max L.) hypocotyls. Aside from the GSTs present in total soluble tissue extracts, enzyme activities and distinct immunoreactive GST polypeptides were also detected in the intercellular washing fluid. Whereas the intracellular isoenzymes were both constitutive and inducible, apoplastic GST and glutathione peroxidase was detectable only in tissues treated with the known GST inducer 2,3,5-triiodobenzoic acid. Monensin inhibited the induced accumulation of apoplastic GST but did not affect the intracellular isoforms. The discovery of apoplastic inducible GST will be discussed in light of the putative function of these enzymes in plants.     

The Suppressor of Killer of prune, a unique glutathione S-transferase

The prune-Killer of prune conditional dominant, lethal interaction in Drosophila was identified in the 1950s, but its mechanism remains unknown. We undertook a genetic screen for suppressors of this lethal interaction and identified a gene we named, Suppressor of Killer of prune Su(Kpn). Su(Kpn) is a unique protein with four N-terminal FLYWCH zinc-finger domains, an acidic domain and a C-terminal glutathione S-transferase (GST) domain. The GST domain of Su(Kpn) is of particular interest because GSTs are usually independent of other protein domains. While GSTs are generally thought of as detoxifying enzymes, they are also associated with cellular toxicity. We predict that the GST domain of the Su(Kpn) creates a toxic product in prune-Killer of prune flies that is lethal. The substrate of the Su(Kpn) remains unknown.     

Glutathione transferase-like proteins encoded in genomes of yeasts and fungi: insights into evolution of a multifunctional protein superfamily

Most fungal glutathione transferases (GSTs) do not fit easily into any of the previously characterised classes by immunological, sequence or catalytic criteria. In contrast to the paucity of studies on GSTs cloned or isolated from fungal sources, a screen of databases revealed 67 GST-like sequences from 21 fungal species. Comparison by multiple sequence alignment generated a dendrogram revealing five clusters of GST-like proteins designated clusters 1, 2, EFIBgamma, Ure2p and MAK16, the last three of which have previously been related to the GST superfamily. Surprisingly, a relatively small number of fungal GSTs belong to mainstream classes and the previously-described fungal Gamma class is not widespread in the 21 species studied. Representative crystal structures are available for the EFIBgamma and Ure2p classes and the domain structures of representative sequences are compared with these. In addition, there are some "orphan" sequences that do not fit into any previously-described class, but show similarity to genes implicated in fungal biosynthetic gene clusters. We suggest that GST-like sequences are widespread in fungi, participating in a wide range of functions. They probably evolved by a process similar to domain "shuffling".