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A subclass of glutathione S-transferases (GSTs), exemplified by the human hGSTM3 and rodent GSTM5 subunits, has properties that distinguish it from other Mu class GSTs. Thus, they originate from single copy genes that are in an inverted order and, apart from the coding regions, share little sequence homology relative to the others in the Mu cluster. The genes for this M3/M5 subgroup encode for proteins that are in many ways unique, including their extended lengths with key amino acid substitutions. The M3/M5 subclass is selectively expressed in testis and brain and could function differently from the other GSTs.

Cytosolic glutathione S-transferase (GST) subunits from human testis were resolved by HPLC and unambiguously identified by combined use of peptide sequence-specific antisera and electrospray ionization mass spectrometry (ESI MS). Allelic variants of hGSTP1, hGSTM1 and hGSTA2 were distinguished on the basis of observed differences in their molecular masses. Relative amounts of the multiple different subunit types in various human tissues were determined from HPLC profiles. From this type of analysis, tissues from hGSTM1 null allele individuals were readily discerned at the protein level; liver was the only tissue in which the hGSTM1 subunit was the major mu-class GST. hGSTM4 and hGSTM5 subunits were found at very low levels in all tissues examined. By far the tissue richest in the unique hGSTM3 subunit was testis, although brain also has significant levels.

A rat testicular Mu-class glutathione S-transferase (GST) resolved by reversed-phase high performance liquid chromatography cross-reacted with peptide sequence-specific antisera raised against the human hGSTM3 subunit. Electrospray ionization mass spectrometry indicated that this rat GST subunit (designated rGSTM5 in this report) has a significantly greater molecular mass (26,541 Da) than the other rat GST subunits. The mouse homologue (mGSTM5 subunit) was also identified and characterized by high performance liquid chromatography and electrospray ionization mass spectrometry. Sequence analysis of rGSTM5 peptide fragments and the sequence deduced from a cDNA clone showed that the protein is highly homologous to the hGSTM3 and murine mGSTM5 subunits. All three GSTs of this subclass have N- and C-terminal extensions with C-terminal cysteine residues, but the two penultimate amino acids near the C terminus are divergent in the three species. The proteins of this class Mu subfamily have similar catalytic specificities and mechanisms, are all cysteine rich, are found mainly in testis, and share characteristics that distinguish them from other GSTs. Moreover, the rGSTM5 subunit isolated from rat testis was not found in heterodimeric combination with other common Mu-class GST subunits. As the rGSTM5, mGSTM5, and hGSTM3 subunits are structurally more closely related to each other than they are to other Mu GSTs, it is proposed that they be considered a functionally distinct and separate subfamily within class Mu. The identification of this unique mammalian GST subclass could advance strategies for interspecies comparisons of GSTs and provides a rodent model for studies on functions and regulatory mechanisms for human GSTs.

To gain insight into mechanisms of cell type-specific transcription of class mu-glutathione S-transferase genes, the gene encoding the Yb3 subunit was cloned. Yb3 subunits are selectively expressed at high levels in rat brain and testis but not in liver or kidney. The Yb3 subunit gene spans over 6 kb and consists of 8 exons and 7 introns and a sequence consisting of tandem direct repeat consensus octamer DNA binding motifs separated by a 6 base pair (bp) spacer was identified in its 5'-flanking region. Gel shift assays with a 40 bp segment of DNA containing the two consensus octamer sequences, revealed the presence of specific binding proteins in nuclear extracts of rat brain, testis and C6 glioma cells. DNA binding activity was greatly reduced in liver, kidney and HTC cells. Reporter vectors carrying segments of the 5'-flanking region of the Yb3 subunit gene fused to a luciferase gene were introduced into C6 glioma cells which express high levels of Yb3 subunits, and into HTC cells which do not. The plasmids consisting of the Yb3 gene promoter up to, but not including, the octamer motifs did not support luciferase transcription in the C6 glioma cells, but larger fragments that included the octamer repeat sequences, effectively directed transcription in the C6 glioma cells. With mutated octameric sequences transcriptional activity was greatly reduced, and none of the same Yb3 constructs directed substantial luciferase transcription in the HTC cells. The results show that octamer motifs in the 5'-flanking region of the Yb3 subunit gene are functional and are the principal cis-acting elements that account for its discrete cell type-selective expression. This gene is one of the few known targets for octamer DNA binding transcription factors in brain.

mu-Class glutathione S-transferases (GSTs) were identified in all 13 human testes and 28 brains examined; even subjects whose livers were devoid of mu-GSTs expressed extrahepatic GSTs of this class. Testes and brains from individuals with mu-class GSTs in their livers had additional forms that also reflected the liver phenotypes. An isoenzyme with an isoelectric point of 5.2, which was a major GST in testis and present as well in cerebral cortex but not detected in any livers, was identified and purified. Sequence analysis of peptides derived by cleavage of the testicular mu-class GST by Achromobacter protease I revealed distinct aspects of primary structure not found previously in any mammalian mu-class GSTs. These unique features included a blocked and extended amino terminus and 3 additional residues (Pro-Val-Cys) at the carboxyl terminus. This structure was confirmed by molecular cloning and sequencing of cDNAs derived from human testis and brain libraries. In the coding region the mRNA of the brain-testis mu-class GST was 75% homologous with that of the liver form, and its 3'-untranslated sequence was mostly divergent, indicating that it is the product of a separate gene. Distinct catalytic and structural properties of the testis-brain mu-class GSTs suggest that these GSTs may be uniquely involved in blood-barrier functions common to both organs.

Rat testicular and liver cystols contain ligandin as determined immunologically, and have high glutathione-S-transferase activity. Unlike liver cytosol, testicular cytosol does not contain protein components that bind bilirubin or sulfobromophthalein with high affinity. To investigate these effects, ligandin was purified to homogeneity from rat testis. Whereas rat liver ligandin consists of equal amounts of two subunits with molecular weights of 22,000 (Ya) and 25,000 (Yb), more than 90% of testicular ligandin consists of Yb. Rat testicular ligandin is immunologically similar to liver ligandin, and has identical glutathione-S-transferase activity, but lacks the capacity for high affinity binding of bilirubin and sulfobromophthalein. The amino acid composition and other properties of testicular ligandin are similar to those of the Yb subunit of liver ligandin. Sulfobromophthalein and bilirubin biphasically inhibit the glutathione-S-transferase activity of liver ligandin: initial high affinity inhibition is followed by reduced inhibition. Testicular ligandin has only low affinity inhibition kinetics. These results suggest that Ya is required for high affinity binding, and that reduced organic anion binding by testicular ligandin results from the lower amounts of Ya in testis.