The impact of glutathione transferase kappa deficiency on adiponectin multimerisation in vivo.

Glutathione transferase Kappa (GSTK1-1) also termed disulfide bond-forming oxidoreductase A-like protein (DsbA-L) has been implicated in the post-translational multimerization of adiponectin and has been negatively correlated with obesity in mice and humans. We investigated adiponectin in Gstk1(-/-) mice and surprisingly found no difference in the levels of total serum adiponectin or the level of high molecular weight (HMW) multimers when compared with normal controls. Non-reducing SDS-polyacrylamide gel electrophoresis and western blotting also showed a similar distribution of low, middle and HMW multimers in normal and Gstk1(-/-) mice. Variation in adiponectin has been correlated with glucose tolerance and with the levels of phosphorylated AMP-kinase but we found similar glucose tolerance and similar levels of phospho 5-AMP-activated protein kinase in normal and Gstk1(-/-) mice. Consequently, our findings suggest that GSTK1-1 is not absolutely required for adiponectin multimerization in vivo and alternate pathways may be activated in GSTK1-1 deficiency.

GSTZ1d: a new allele of glutathione transferase zeta and maleylacetoacetate isomerase.

The zeta class glutathione transferases (GSTs) are known to catalyse the isomerization of maleylacetoacetate (MAA) to fumarylacetoacetate (FAA), and the biotransformation of dichloroacetic acid to glyoxylate. A new allele of human GSTZ1, characterized by a Thr82Met substitution and termed GSTZ1d, has been identified by analysis of the expressed sequence tag (EST) database. In European Australians, GSTZ1d occurs with a frequency of 0.16. Like GSTZ1b-1b and GSTZ1c-1c, the new isoform has low activity with dichloroacetic acid compared with GSTZ1a-1a. The low activity appears to be due to a high sensitivity to substrate inhibition. The maleylacetoacetate isomerase (MAAI) activity of all known variants was compared using maleylacetone as a substrate. Significant differences in activity were noted, with GSTZ1a-1a having a notably lower catalytic efficiency. The unusual catalytic properties of GSTZ1a-1a in both reactions suggest that its characteristic arginine at position 42 plays a significant role in the regulation of substrate access and/or product release. The different amino acid substitutions have been mapped on to the recently determined crystal structure of GSTZ1-1 to evaluate and explain their influence on function.

Identification, characterization, and crystal structure of the Omega class glutathione transferases.

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-A resolution and has a characteristic GST fold (Protein Data Bank entry code ). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.

Structure and organization of the human theta-class glutathione S-transferase and D-dopachrome tautomerase gene complex.

The structure and organization of the human Theta-class glutathione S-transferase (GST) genes have been determined. GSTT1 and GSTT2 are separated by approx. 50 kb. They have a similar structure, being composed of five exons with identical exon/intron boundaries. GSTT1 is 8.1 kb in length, while GSTT2 is only 3.7 kb. The GSTT2 gene lies head-to-head with a gene encoding d-dopachrome tautomerase (DDCT), which extends over 8.5 kb and contains four exons. The sequence between GSTT2 and DDCT may contain a bidirectional promoter. The GSTT2 and DDCT genes have been duplicated in an inverted repeat. Sequence analysis of the duplicated GSTT2 gene has identified an exon 2/intron 2 splice site abnormality and a premature translation stop signal at codon 196. These changes suggest that the duplicate gene is a pseudogene, and it has been named GSTT2P.

Genetic heterogeneity of the structure and function of GSTT2 and GSTP1.

In this study new methods for the detection of two polymorphic sites in the GSTP1 coding region have been developed. Both sites are polymorphic in several racial groups and there are significant differences between groups, in the gene frequency at each site. Although previous studies of recombinant GSTP1-1 have suggested that there are significant differences in the specific activity and stability of the I105 or V105 isoforms, no differences in the distribution of GSTP1-1 activities in normal blood donors with different GSTP1 genotypes were detected in this study. These data were obtained with CDNB as a substrate and greater differences may be apparent with different substrates. The structure and organization of the GSTT2 gene was also investigated and a pseudogene that occurs at a polymorphic frequency in European Australians was discovered. This pseudogene can be detected by PCR/RFLP analysis.

Polymorphism of the Pi class glutathione S-transferase in normal populations and cancer patients.

Deficiencies of the glutathione transferase isoenzymes GSTM1-1 and GSTT1-1 have been shown to be risk modifiers in a number of different cancers but there have been no similar studies with GSTP1-1, the only member of the Pi class of glutathione S-transferases expressed in humans. Over-expression of GSTP1-1 in tumours suggests that it may be a significant factor in acquired resistance to certain anticancer drugs. We previously identified a cDNA clone with two amino acid substitutions (I105V, A114V). This clone suggests that the GSTP1 gene is polymorphic and it is possible that the different genotypes may be associated with altered cancer risk or drug resistance. In the present study, we report methods for genotyping individuals at codons 105 and 114 of GSTP1 and demonstrate that these two loci are polymorphic in several different racial groups. We also detected significant linkage disequilibrium between these two loci. To determine if either of the alleles at these two loci were associated with altered cancer susceptibility, we genotyped individuals with colorectal cancer or lung cancer. A total of 131 colorectal and 184 lung cancer patients were compared with 199 control individuals. Overall, there were no significant associations between the GSTP1 polymorphisms and either form of cancer.

Application of HUMF13A01 (AAAG)n STR polymorphism to the genetic diagnosis of coagulation factor XIII deficiency.

Deficiency of the A subunit of coagulation factor XIII causes a severe bleeding disorder requiring life long replacement therapy. The mutations causing A subunit deficiency appear to be very heterogeneous, and it is impractical to identify each mutation before genetic counselling or prenatal diagnosis can be attempted. In this study we have shown that a highly polymorphic short tandem repeat element, HUMF13A01 (AAAG)n that occurs in the 5' flanking sequence of the factor XIII A subunit gene, can be used to follow the segregation of deficiency causing mutations. We studied 6 families with factor XIII A subunit deficiency from 5 different ethnic groups. All parents were heterozygous for the repetitive element and therefore all the families were informative. The linked polymorphism was used to carry out the first prenatal diagnosis of factor XIII A subunit deficiency. The analysis of this polymorphism by the polymerase chain reaction is rapid, reliable, requires little DNA and is ideal for the genetic analysis of factor XIII A subunit deficiency.

Chromosomal localization of the gene for the human theta class glutathione transferase (GSTT1).

Two loci encoding Theta class glutathione transferases (GSTs) have been identified in humans. In situ hybridization studies have localized the GSTT1 gene to 22q11.2. This is the same band to which we previously localized the GSTT2 gene. This finding confirms the trend for human GST genes to be found in class-specific clusters.

Evidence for an essential serine residue in the active site of the Theta class glutathione transferases.

A consistent feature of the Alpha-, Mu- and Pi-class glutathione transferases (GSTs) is the presence near the N-terminus of a tyrosine residue that contributes to the activation of glutathione. While this residue appears to be conserved in many Theta-class GSTs, its absence in some suggested that the Theta-class GSTs may have a significantly different structure or catalytic mechanism. The elucidation of the crystal structure of the Theta-class GST from the Australian sheep blowfly, Lucilia cuprina, has indicated that a serine residue rather than a tyrosine residue can form a hydrogen bond with the glutathionyl sulphur atom. The present studies show that mutation of Ser-9 to alanine substantially inactivates the L. cuprina GST, confirming its importance in the reaction mechanism. As this serine is conserved in all Theta-class enzymes reported so far, it seems that an active-site serine is a significant factor that distinguishes the Theta-class GSTs from members of the Alpha-, Mu- and Pi-class isoenzymes.

Glutathione S-transferase M1 and T1 polymorphisms: susceptibility to colon cancer and age of onset.

The M1 member of the Mu subclass of glutathione S-transferase (GSTM1) is only expressed in about 50% of individuals. In contrast, GSTT1, a member of the theta class which has been recently shown to be polymorphic, is expressed in 85% of Australian individuals. Previous studies have shown a significant excess of homozygous null GSTM1 genotypes among individuals with colorectal cancer, particularly those with proximal tumours. This suggests that GSTM1 plays a role in susceptibility to this neoplasm. In this study of 132 individuals with colorectal cancer and 200 controls, no significant excess of GSTM1 homozygous null genotypes was found among colorectal cancer patients with either a proximal or distal tumour. This suggests that the association between GSTM1 homozygous null genotypes and colorectal cancer is of smaller effect than has been reported previously using larger sample sizes. We have also examined the frequency of homozygous null GSTT1 genotypes in patients with colorectal cancer. Although the frequency was not significantly different in cases compared to control individuals, GSTT1 null homozygotes were significantly more common in patients who were diagnosed before the age of 70 years than in those who were diagnosed at an older age. This suggests that the GSTT1 genotype, and perhaps also the GSTM1 genotype for which a similar, but non-significant effect was seen, might influence the age of onset of colorectal cancer.

Mutations causing coagulation factor XIII subunit A deficiency: characterization of the mutant proteins after expression in yeast.

We identified the mutations causing factor XIII A subunit deficiency in two families. Two distinct mutations were identified in the S family: the nonsense mutation Tyr 441-->stop in exon 11, inherited through the paternal line, and the missense mutation Asn 60-->Lys in exon 3, inherited through the maternal line. Two members of the J family were heterozygous for the previously described type 3 A subunit. The substitution giving rise to the type 3 variant was found to be Gly 501-->Arg in exon 12. The Asn 60-->Lys and Gly 501-->Arg mutations were constructed in cDNA clones and expressed in yeast (Saccharomyces cerevisiae AH22). Although mRNA could be detected, protein containing the Asn 60-->Lys substitution could not be detected, suggesting extreme instability or susceptibility to proteolysis. A subunits containing the Gly 501-->Arg substitution were expressed and found to be enzymatically active in fresh yeast lysates. This variant has thermal instability and lost activity during storage or purification. Gel filtration studies suggested that the type 3 variant assembled as a dimer, as do normal A subunits. The data suggest that the Gly 501-->Arg (Type 3 variant) would cause severe factor XIII deficiency if inherited in the homozygous form or as a compound heterozygote with another deleterious mutation.

Localization of the human UBA52 ubiquitin fusion gene to chromosome band 19p13.1-p12.

Because of the conservation of the ubiquitin coding sequence and the number of transcriptionally active genes and reverse-transcribed pseudogenes, it has not been possible to use ubiquitin cDNA clones to map the functional ubiquitin genes. The UBB and UBC polyubiquitin genes have previously been mapped by the use of specific intron or 5' flanking sequence probes. In this study, we have used an intron sequence from the UBA52 gene for chromosome mapping studies. Analysis of somatic cell hybrids containing individual human chromosomes indicated that the UBA52 gene is located on chromosome 19. In situ hybridization studies confirmed the chromosomal localization but showed two peaks of hybridization: a major one over 19p13.1-p12 and a secondary one over 19q12-q13.11. Because the peak of hybridization over 19p13.1-p12 was consistently the strongest in five individuals, it is likely that this is the location of the UBA52 gene. Thus far, three of the four transcriptionally active ubiquitin genes have been assigned to separate chromosomes.

Evidence against a relationship between fatty acid ethyl ester synthase and the Pi class glutathione S-transferase in humans.

Recently, Bora et al. (Bora, P. S., Bora, N. S., Wu, X., and Lange, L. G. (1991) J. Biol. Chem. 266, 16774-16777) reported the cloning and expression of a human fatty acid ethyl ester synthase III (FAEES-III) cDNA that has only four amino acid substitutions compared with human glutathione S-transferase (GST) GSTP1-1, and, when expressed in MCF-7 cells, the protein has both FAEES and GST activities. By site-directed mutagenesis of a GSTP1 cDNA, we have constructed a clone that encodes the FAEES-III protein described by Bora et al. (1991). The recombinant FAEES-III protein was expressed in Escherichia coli and has been shown to be devoid of FAEES and GST activities. The recombinant FAEES-III protein does not bind to a glutathione agarose affinity matrix, presumably because two of the substituted amino acids, Trp-39-->Cys and Gln-52-->Glu, are thought to contribute to the GST glutathione binding site. One of the base substitutions in the FAEES-III cDNA encodes an extra SacI site not found in the GSTPI cDNA. Polymerase chain reaction amplification of human genomic DNA has identified the GSTPI gene, but no DNA from the proposed FAEES gene with a diagnostic SacI site has been detected. Evaluation of the hybridization pattern of HindIII genomic restriction fragments has identified fragments that contain the GSTPI gene and a pseudogene (Board et al. 1992), and there do not appear to be any hybridizing fragments that could contain the FAEES-III gene. Our results do not provide any evidence in support of a relationship between FAEES-III and GST, and the cDNA reported by Bora et al. (1991) may have resulted from a cloning artifact.

The human Pi class glutathione transferase sequence at 12q13-q14 is a reverse-transcribed pseudogene.

A previous in situ hybridization study with a Pi class glutathione S-transferase cDNA probe revealed the presence of hybridizing sequences on the long arms of chromosomes 11 and 12. Since the GSTP1 gene is known to be on chromosome 11 and since it is thought that chromosomes 11 and 12 arose from an ancient tetraploidization event, it was of interest to determine if the gene on chromosome 12 encoded a closely related Pi class glutathione S-transferase isoenzyme. This gene has now been cloned and sequenced. The results are surprising and indicate that the gene is a partial reverse-transcribed pseudogene that has been inserted into the genome at 12q by chance and has not resulted from the prior tetraploidization of the human genome.

Identification of a point mutation in factor XIII A subunit deficiency.

Oligonucleotide primers have been designed for the amplification of all 15 exons of the human coagulation factor XIII A subunit gene. Each exon and its intron flanking regions has been amplified and sequenced from a patient with severe A subunit deficiency. A single G to A transition in the last base of exon 14 has been identified in the homozygous proband and in his heterozygous parents. The mutation would result in the substitution 681 Arg to His in the mature protein product. However, because the mutation is at a splice junction, the deficiency may result from a defect in pre-messenger RNA splicing.

Localization of the human UBC polyubiquitin gene to chromosome band 12q24.3.

The localization of specific human ubiquitin genes has not been straightforward because of the conservation of the ubiquitin coding sequence and the number of processed pseudogenes. An congruent to 1.4-kb sequence from the 5'-flanking region of the UBC gene has been shown to be unique to that locus and free from dispersed repeat elements. The cloned 5'-flanking fragment has been used to probe Southern blots of DNA obtained from somatic cell hybrid cell lines. These data indicate that the UBC gene is located on chromosome 12. In situ hybridization with the 5'-flanking probe has refined the assignment to the broad chromosomal subband 12q24.3. These data show that the active ubiquitin genes are not clustered and are located on separate chromosomes. In addition, these studies demonstrate the utility of intron or flanking sequence probes in the specific chromosomal assignment of members of highly conserved gene families.

Expression of functional coagulation factor XIII in Escherichia coli.

Coagulation factor XIII is a zymogen that can be activated by thrombin cleavage to a transglutaminase that catalyses the formation of covalent crosslinks between fibrin chains in the final stages of the blood clotting cascade. Although circulating factor-XIII is composed of A and B subunits the catalytic activity is a property of the A subunits. In this study we have constructed a plasmid (pKKF13A) that contains a cDNA encoding the A subunit positioned downstream of a tac promoter. Escherichia coli containing this plasmid produce A subunit protein when grown in the presence of IPTG. The cloned A subunit has been partially purified and characterized. Comparison with A subunits purified from plasma showed that the cloned A subunits were of the same size, assembled as dimers, and had the same native electrophoretic mobility. The cloned A subunits expressed transglutaminase activity with putrescine, dansylcadaverine and casein as substrates, and were able to crosslink fibrin in clots formed from A subunit deficient plasma. These studies have demonstrated that functional recombinant factor XIII A subunit can be produced in E. coli and suggest that recombinant factor XIII can potentially provide a safe and inexhaustible supply for therapeutic use.

Genetic heterogeneity of the human glutathione transferases: a complex of gene families.

The glutathione transferases (GSTs) are involved in the metabolism of a wide range of compounds of both exogenous and endogenous origin. There is evidence that deficiency of GST may increase sensitivity to certain environmentally derived carcinogens. In contrast, elevated expression has been implicated in resistance to therapeutic drugs. The GSTs are the products of several gene families. This review summarizes the present knowledge of the genetic interrelationships between the various isoenzymes, their deficiencies and the physical locations of their genes.

Molecular genetics of the human glutathione S-transferase.

Multiple human cytosolic glutathione transferases have been described. These enzymes are the products of multiple genes that can be classified into at least four evolutionary classes. The genes encoding each class appear to be clustered on distinct chromosomes. Over-expression of glutathione S-transferase (GST) isoenzymes has been implicated in drug resistance and, conversely, deficiency of GST isoenzymes has been implicated in susceptibility to carcinogens. Some GST genes are expressed at varying levels in different individuals, and there is a frequent deficiency of the Mu class GST1 isoenzyme in all the racial groups studied so far. This deficiency is due to a deletion of the GST 1 gene. The Alpha class genes are located on the short arm of chromosome 6 and are closely linked, with less than 2 kb separating some genes. There is evidence for the existence of several pseudogenes in this cluster. A complete Alpha class gene has 7 exons and extends over 13 kb. The 5' flanking region of the gene encoding the GST2 type 1 isoenzyme has been cloned and sequenced. This region contains a number of putative promoter and enhancer elements that are similar to those found in rat and mouse Alpha class genes.