Human Mu Class Glutathione S-transferase Research Articles

Using a residue clash map to functionally characterize protein recombination hybrids

In this article, we introduce a rapid, protein sequence database-driven approach to characterize all contacting residue pairs present in protein hybrids for inconsistency with protein family structural features. This approach is based on examining contacting residue pairs with different parental origins for different types of potentially unfavorable interactions (i.e. electrostatic repulsion, steric hindrance, cavity formation and hydrogen bond disruption). The identified clashing residue pairs between members of a protein family are then contrasted against functionally characterized hybrid libraries. Comparisons for five different protein recombination studies available in the literature: (i) glycinamide ribonucleotide transformylase (GART) from Escherichia coli (purN) and human (hGART), (ii) human Mu class glutathione S-transferase (GST) M1-1 and M2-2, (iii) beta-lactamase TEM-1 and PSE-4, (iv) catechol-2,3-oxygenase xylE and nahH, and (v) dioxygenases (toluene dioxygenase, tetrachlorobenzene dioxygenase and biphenyl dioxygenase) reveal that the patterns of identified clashing residue pairs are remarkably consistent with experimentally found patterns of functional crossover profiles. Specifically, we show that the proposed residue clash maps are on average 5.0 times more effective than randomly generated clashes and 1.6 times more effective than residue contact maps at explaining the observed crossover distributions among functional members of hybrid libraries. This suggests that residue clash maps can provide quantitative guidelines for the placement of crossovers in the design of protein recombination experiments.

A T2517C polymorphism in the GSTM4 gene is associated with risk of developing lung cancer

The human Mu class Glutathione S-Transferases is a family of genes encoding phase II detoxifying enzymes thus playing a significant role in the detoxification of potential carcinogens. While there are many contradicting reports on the association of GSTM1 polymorphisms and cancer development, no studies exist to date describing polymorphisms in GSTM4. We have identified a new C-T polymorphism in intron 6 of the GSTM4 gene (T2517C, Genebank sequence accession number X68677) and termed the allele carrying T at this position allele *A and the allele carrying C, allele *B. Screening a population sample in Merseyside, England, revealed 23 carriers of the *B allele out of 156 healthy control individuals but only 12 carriers of the *B allele out of 163 individuals with lung cancer (O.R.=2.23, Fisher's test P=0.026). The polymorphism did not demonstrate any associations with tumour type, gender, and age at presentation. This is the first report on the implication of a polymorphism in the GSTM4 gene in lung cancer risk. Further studies are required to investigate the relation of this polymorphism to cancer risk to substantiate these findings.

The Enhanced Affinity for Thiolate Anion and Activation of Enzyme-bound Glutathione Is Governed by an Arginine Residue of Human Mu Class Glutathione S-Transferases

A series of chimeric human Mu class glutathione S-transferases were designed to determine mechanisms by which they activate enzyme-bound glutathione (GSH) for reaction with electrophilic substrates. In view of evidence that the His(107) residue of hGSTM1a-1a is important for catalysis (Patskovsky, Y. V., Patskovska, L. N., and Listowsky, I. (1999) Biochemistry 38, 1193-1202), the cognate Arg(107) residue of the hGSTM2 subunit was replaced (R107N or R107H) and arginine residues were also incorporated into position 107 of hGSTM1 (H107R) and hGSTM4 (S107R) subunits. The major distinguishing kinetic properties invariably associated with enzymes containing an Arg(107) residue include an inverse dependence of k(cat) on viscosity and lower K(m(GSH values relative to enzymes with other residues at that position. Moreover, affinities for GSH thiolate anion binding are greater for enzymes containing Arg(107))), with K(d) values of 20-50 microM that are consistent with the K(m(GSH values (10-25 microM) obtained by steady-state kinetic analyses. Both thermodynamic and kinetic and data indicate that the Arg(107))) residue is specifically involved in enhancing the binding affinity of GSH thiolate anion relative to that of the protonated form. These enzymes therefore, can be more effective at lower GSH concentrations. Combined mutations indicate that both Arg(107) and Tyr(6) residues are required for thiolate anion formation and stabilization. The three-dimensional structure of ligand-free hGSTM2-2 determined by x-ray crystallography suggests that Arg(107) maintains an electrostatic interaction with the Asp(161) side chain (3 A apart), but is distant from the GSH-binding site. However, an alternative energetically favorable model places the guanidino group 4 A from the sulfur atom of bound GSH. It is suggested therefore, that in solution, motion of the positively charged arginine into the catalytic pocket could provide a counter ion to promote ionization of the sulfhydryl group of GSH, thereby accounting for the observed greater affinity of enzymes containing Arg(107) for binding of thiolate anion.

Human Mu-class glutathione S-transferases present in liver, skeletal muscle and testicular tissue

The major human Mu-class glutathione S-transferases (GST) have been purified to allow comparisons of their catalytic, physicochemical and immunochemical properties. GST isoenzymes, purified from hepatic, testicular and skeletal muscle tissue were found to comprise three distinct subunits (M1, M2 and M3) which may combine to form both homodimeric and heterodimeric proteins. Two distinct subunits, M1a and M1b, which represent allelic charge variants have been isolated but no polymorphic forms encoded at the GST M2 and M3 loci have been observed. Three GST isoenzymes (M1a-1a, M1a-1b and M1b-1b) have been purified from a single liver specimen. In addition, GST M1a-2, M1b-2, M2-2 and M2-3 have been isolated from muscle, whilst the M3-3 homodimer has been purified from human testis. The homodimeric enzymes GST M1a-1a, M1b-1b, M2-2 and M3-3 have p I values of 6.1, 5.5, 5.3 and 5.0, whilst SDS-PAGE indicated that M1a, M1, M2 and M3 have molecular masses of 26.7, 26.6, 26.0 and 26.3 kDa, respectively. The M1, M2 and M3 subunits isolated from either liver, skeletal muscle or testis, are catalytically distinct. Both M1-type subunits (M1a and M1b) possess a high activity for trans-4-phenyl-3-buten-2-one, whereas, the skeletal muscle subunit M2 has a high activity towards 1,2-dichloro-4-nitrobenzene. By contrast, the testicular GST subunit M3 has no detectable activity towards either of these substrates. However, all three Mu-class subunits are active towards the compounds 4-hydroxynonenal and 4-hydroxydecinal, possible endogenous substrates which are produced by lipid peroxidation. The human Mu-class subunits can be distinguished immunochemically; antisera raised against the testicular GST M3-3 showed no reactivity towards either the M1 or M2 subunits. The M3 subunit has a blocked N-terminus but automated amino-acid sequencing of a CNBr-derived peptide allowed 14 residues of the M3 subunit to be identified. These data indicated that testicular GST M3-3 is likely to correspond to the brain/testis Mu-class GST cDNA described by Campbell et al. (Campbell E., Takahashi Y., Abramovitz M., Peretz M., & Listowsky I. (1990) J. Biol. Chem. 265, 9188–9193).

Chromosomal Mapping of the Human Mu Class Glutathione S-Transferases to 1p13

The chromosomal localization of the human Mu class glutathione S-transferase (GST) genes has been complicated by two factors; the total number of genes is unknown and there is a polymorphism that results from the presence or absence of the GSTM1 gene. Three human Mu class glutathione S-transferase isoenzymes, GSTM1, GSTM2, and GSTM3, have been characterized previously, and we have recently cloned and characterized GSTM4, another member of this class. Here we report that a probe derived from GSTM4 cross-hybridizes with the other three known human Mu class GST genes. In situ hybridization with the GSTM4 probe localized a major region of hybridization on chromosome band 1p13. Although there is a region of very weak hybridization on chromosome 6, these data indicate that the human Mu class gene family is largely clustered and not dispersed on different chromosomes. The identical hybridization patterns in individuals with or without the GSTM1 gene suggest that this locus is a component of the Mu class GST gene cluster.

Molecular cloning and heterologous expression of an alternatively spliced human Mu class glutathione S-transferase transcript.

Two cDNA clones encoding a new Mu class glutathione S-transferase (GST) have been isolated from a human testis cDNA library. Both clones are incomplete and appear to result from alternative splicing. One clone is missing the sequence encoding exon 4 and the other is missing exon 8. The complete sequence of the previously undescribed isoenzyme can be deduced from the two cDNA clones. This is the first report of alternative splicing in a GST transcript and may represent either a novel form of regulation in this multigene family or illegitimate transcription and experimental alternative splicing as part of the evolutionary process. By combining components from each clone a complete cDNA has been constructed and the encoded protein expressed in Escherichia coli. In general, the recombinant enzyme has relatively low activity when compared with all the previously described human Mu class GST isoenzymes.

Isolation and analysis of the gene and cDNA for a human Mu class glutathione S-transferase, GSTM4

We have isolated the human gene and cDNA that encode the fourth member of the Mu class glutathione S-transferases (GSTs). The complete gene sequence (GenBank M96233) and the cDNA sequence (M96234) are reported and named GSTM4. The gene, located on chromosome 1, is comprised of eight exons with an organization similar to that of other Mu class genes. GSTM4 differs from a partial gene sequence, termed GSTmu2 (Taylor, J. B., Oliver, J., Sherrington, R., and Pemble, S. E. (1991) Biochem. J. 274, 587-593), in exons 3 through 5 by only 11 nucleotides within introns. The cDNA for GSTM4 was isolated from a library derived from the cervical carcinoma cell line, HeLa. Northern blots demonstrate the presence of GSTM4 mRNA in human heart, placenta, lung, brain, liver, skeletal muscle, pancreas, testis, cerebral cortex, uterus, ovary, a lymphoblastoid cell line, and four carcinoma cell lines. The deduced amino acid sequence of GSTM4 is 87% (GSTM1), 83% (GSTM2), and 70% (GSTM3) identical to the previously described human Mu class GSTs. The polypeptide encoded by the GSTM4 cDNA, produced in a bacterial expression system, conjugates 1-chloro-2,4-dinitrobenzene to GSH with a specific activity of 1.39 +/- 0.21 mumol/min/mg.

A basis for differentiating among the multiple human Mu-glutathione S-transferases and molecular cloning of brain GSTM5.

Specific cDNA probes and antisera were employed to interpret genetic polymorphisms of human Mu-class glutathione S-transferases and to provide a basis for identifying individual forms in human tissues. A cDNA probe that cross-hybridized with various human and rodent Mu-glutathione S-transferase transcripts, hybridized with at least three discrete components by Northern analysis of RNA from human tissue. The smallest (1.3 kb) transcript was identified as the one that encodes GSTM3-3 subunits. A form designated GSTM5, was cloned from a human brain cDNA library and its sequence determined. The open reading frame of GSTM5 shared a high degree of homology with the sequences of other Mu-class glutathione S-transferases, but its 846-nucleotide 3'-noncoding region was unique and considerably larger than that of any of the other Mu forms. Specific synthetic peptide antigens were utilized to distinguish among Mu-class glutathione S-transferases in different tissues of representative individuals. The primary hepatic transcript was that encoding GSTM1-1 with much lesser amounts of GSTM3-3, but livers were devoid of GSTM2-2, and GSTM5-5. Immunoblots confirmed that null-phenotype individuals lacked the GSTM1 gene rather than its GSTM2 homologue that is nearly identical in its exon sequences. The null phenotype therefore was conspicuous in liver, where GSTM1-1 ordinarily was the predominant Mu transcript, but brain and testis contained all four forms. A general strategy was devised to distinguish among and assign primary structures to individual glutathione S-transferases from human tissue.