Rationale for Reclassification of a Distinctive Subdivision of Mammalian Class Mu Glutathione S-Transferases That Are Primarily Expressed in Testis*

Jonathan D. RoweDagger , Yury V. Patskovsky, Larysa N. Patskovska, Elena Novikova, and Irving Listowsky§

From the Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Because the multiple forms of glutathione S-transferases (GSTs)1 are usually products of gene superfamilies (1-5), attempts have been made to devise species-independent classification systems. Accordingly, cytosolic mammalian GSTs are grouped in Alpha, Mu, Pi, and Theta classes on the basis of sequence homologies, subunit assembly patterns, and other common properties (6). Multiple subunit types from within each of these classes usually assemble in homo- or heterodimeric combinations. However, with the exception of GST Pi, which is usually the product of a single gene, cross-correlations among the multiple Mu- or Alpha-class GSTs in different species have been of limited success. Consequently, efforts to establish general animal models for study of structure, function, and regulation of expression of specific human GST genes have been impeded.

GSTs function catalytically to conjugate GSH with a variety of electrophilic substrates (7-9), but also may serve as intracellular stoichiometric binding proteins for various lipophilic ligands (2, 10, 11). Individual classes of GSTs exhibit overlapping but distinct substrate and ligand binding specificities (2, 5). Mammalian GSTs are expressed in discrete tissue-specific patterns. For instance livers of some species are rich in Alpha- and some Mu-class GSTs but do not contain Pi, yet some Mu subunits not present in liver are major components in certain extrahepatic tissues (12-14).

The human Mu-class subunit hGSTM32 (see Table I) (15) is distinct from others in this class in that it has less than 75% sequence identity to other Mu-class isoforms, which among themselves usually exhibit close to 90% sequence identities. Moreover, among the Mu-class GSTs, hGSTM3 is structurally unique containing 4-residue N-terminal and 3-residue C-terminal extensions. In addition, the hGSTM3 gene is expressed selectively in testis and brain; otherwise that subunit is found at very low levels in most other tissues (12).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Subunit Nomenclature

The present study was designed to determine if structural features of the hGSTM3 subunit are also found in certain GSTs of other species and whether accordingly this type of GST should be considered as a special class of mammalian GST. Indeed, this study establishes that a labile and poorly characterized rat subunit previously designated as Yo or GST 11 (14, 16, 17) and mouse mGSTM5 are similar to the human hGSTM3 subunit in terms of structure, catalytic mechanisms, tissue-selective expression patterns, and other unique properties.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials

The S-methyl 2-naphthyl GSH and S-octyl-GSH were gifts from Dr. Larry Kauvar, Terrapin Technologies, South San Francisco. Epoxy-linked GSH agarose, GSH, the S-alkyl-GSH derivatives, glutathione sulfonic acid, S-(p-nitrobenzyl)-GSH, and S-(1,2-dicarboxyethyl) GSH, were obtained from Sigma. The 1-chloro-2,4-dinitrobenzene (CDNB) was from Aldrich, Taq polymerase from Life Technologies, Inc., and the pET3a expression vector from Stratagene. The Lys-C protease was a gift from Dr. Y. Burstein, Department of Organic Chemistry, Weizmann Institute.

Methods

Isolation and Characterization of GST-- Cytosolic GSTs were purified from freshly removed rat or mouse testes by epoxy-linked GSH-agarose affinity chromatography (18). GST purification and HPLC analyses were performed in the same day to avoid possible changes in protein properties. Methods for expression and purification of recombinant GSTs are described below.

Electrophoresis and Immunoblotting-- SDS-polyacrylamide gel electrophoresis was performed in 12% polyacrylamide resolving gels which were either stained with Coomassie Blue or prepared for immunoblotting. Resolved proteins were transferred from the polyacrylamide gels to nitrocellulose membranes in 12 mM Tris, 96 mM glycine buffer, pH 8.3, containing 20% methanol, by electrophoresis for 1 h at 35 V using a NOVEX blotting apparatus. The membranes were blocked with 5% w/v non-fat dry milk in 20 mM Tris, 150 mM NaCl buffer, pH 7.6 (TBS), and then incubated with specific antisera for 2 h at room temperature. The peptide sequence-specific antiserum selective for the hGSTM3 subunit was raised as described previously (13) and used at a dilution of 1:500. After extensive washing with TBS, the blots were incubated with a goat anti-rabbit IgG-horseradish peroxidase conjugate for 1 h at room temperature. After an additional wash, blots were developed by use of a Renaissance chemiluminescence developing kit (DuPont) and by exposure to x-ray film.

HPLC Analysis of Purified Testis GSTs-- A Hewlett-Packard HPLC 1090 system was used to resolve rat or mouse testicular GST subunits. Subunit separation was achieved on a Vydac C4 column by elution with acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.08% trifluoroacetic acid. Gradients were developed as follows; the gradient was begun at 20% acetonitrile, and after 10 min the percentage of acetonitrile was then increased to 40% followed by a gradient of 0.5% acetonitrile/min over 60 min. The flow rate was 1 ml/min. A diode array detector was used for measuring absorbances of the protein peaks at 214 and 280 nm. HPLC fractions were collected manually in separate tubes and either desiccated in a Speed-Vac or used directly for mass spectrometric analysis. Desiccated fractions were redissolved in water and used immediately for SDS-polyacrylamide gel electrophoresis and other analytical procedures.

Isoelectric Focusing-- Protein isoelectric focusing was performed with rat testis cytosolic proteins (100 µg) and purified rat testis GSTs (25 µg) in Novex pH 3-10 precast isoelectric focusing slab gels of 5% acrylamide were used. The buffers, current, and other conditions were those recommended by the manufacturer (NOVEX) and electrofocusing was carried out for 2.5 h.

Peptide Maps-- The rat rGSTM5 subunit was reduced in a buffer solution containing 6 M guanidinium chloride, 50 mM Tris-HCl, pH 8.5, and 5 mM dithiothreitol. 2-Iodo[2-14C]acetic acid (Amersham Pharmacia Biotech, 2 mCi/mmol) was added in the presence of excess unlabeled 2-iodoacetate for reaction with cysteine residues. The alkylated protein was dialyzed against of 25 mM Tris-HCl, pH 9.0, 1 mM EDTA. Digestion with Lys-C endoproteinase was then carried out at 37 °C for 8 h in 50 mM Tris-HCl, 3 M urea, pH 9.0, with ratios of protease to GST of 1:100. The peptides generated were resolved and purified by reversed-phase HPLC on a Vydac C18 column. The peptides were collected, assayed by scintillation counting for the presence of 14C-radiolabeled material, and prepared for sequence analysis.

Protein Sequence Analysis-- An Applied Biosystems 477A sequencer equipped with an Applied Biosystems 120A amino acid analyzer was used for sequence analysis of protein and peptide samples purified by HPLC methods.

Mass Spectrometry-- An API-III triple quadrupole mass spectrometer (PE-SCIEX, Ontario, Canada) with the SCIEX Ionspray interface and nitrogen as the nebulizer gas was used for mass spectral analysis. An ionspray voltage of ~3600 volts and an orifice voltage of 85 volts was used. Mass spectral analysis was performed either by on-line HPLC connected to the mass spectrometer or by infusion of the collected fractions using a Harvard Apparatus syringe pump. Molecular masses of individual GST subunits were determined from at least three different preparations.

Kinetic Analyses-- Enzyme assays were performed using CDNB and GSH as substrates in 0.1 M sodium phosphate buffer at pH values in the range of 5.8-8.0. Rates of product formation were determined by measurement of absorbances at 340 nm (18). To determine bisubstrate reaction mechanisms, the concentration of each individual substrate was maintained constant in the range of 0.02-2.0 mM, while the concentration of the other substrate was varied over the same range (0.02-2.0 mM). At these substrate concentrations, hyperbolic kinetics were observed for the reaction. Data were best-fitted to steady-state two substrate rate equations (19), using Milton Roy Inc. (Rochester) software.

Inhibitors were tested at various concentrations at substrate concentration ranges described above. Inhibition studies were carried out at pH 6.2, which was determined to be the apparent pKa of each enzyme from plots of log kcat versus pH. For the GSH conjugates inhibition was determined to be competitive vis à vis GSH, and inhibition constants (Ki) were determined from these data.

Synthesis of S-(2,4-Dinitrophenyl)glutathione (GS-DNB)-- GS-DNB was synthesized enzymatically in the presence of 0.1 mg/ml hGSTM3-3, 1.0 mM CDNB, and 1.0 mM GSH. Unreacted excess CDNB was extracted with ether and the water-soluble conjugate was purified using reversed-phase HPLC with a gradient of 5-65% acetonitrile on a Vydac C18 (10 × 250) column. To verify the structure of the GS-DNB conjugate, the molecular mass of the purified product was determined to be 474 ± 0.8 Da by ESI-MS, which is precisely that of the expected GS-DNB product. The concentration of the conjugate was determined spectrophotometrically on the basis of its molar extinction coefficient at 343 nm of 9.6 mM-1 cm-1.

RT-PCR Analysis-- Total RNA was purified from freshly removed rat and mouse testicular tissues by use of a Promega RNA purification kit. Primers were designed based upon comparison of the reported cDNA sequence of mouse mGSTM5 (20). Sequences of the primers were: primer 1 (sense, 30-mer), 5'-TCC ATC GCT GTA TCC CCG AAG CCA AGA TCG-3'; primer 2 (reverse, 30-mer); 5'-CCC TCA GGG CAA GAT GGC TCA GCA GCA GCG-3'. To synthesize cDNAs, reverse transcriptase (RAV-2, Amersham Pharmacia Biotech) and the reverse primer 2 were used according to published methods (21). An aliquot (2 µl) of the cDNA solution was used as a template for PCR amplification. The reaction mixture contained 0.5 mM each of dNTP, 4 mM MgCl2, 0.16 µmol of primers 1 and 2, and 5 units of Taq DNA polymerase in a total volume of 100 µl. The PCR reaction was performed in 35 cycles, each one consisting of steps for denaturation (1 min, 94 °C), annealing (1 min, 55 °C), and elongation (1 min, 72 °C), followed by a final extension step (10 min, 72 °C). The product was purified by agarose gel electrophoresis and cloned into pCRII TA-vector (Invitrogen) according to the procedure described in the instruction manual. DNA sequencing was performed using AmpliTaq polymerase-specific primers and an Applied Biosystems automated DNA sequencer (ABI 377).

Expression of Recombinant GSTs-- Specific sense and antisense oligonucleotide primers were designed for PCR amplification and cloning of cDNAs encoding rodent and human GST subunits as follows: hGSTM3-sense, 30 mer, 5'-AGC CCG TCC ATA TGT CGT GCG AGT CGT CTA-3'; hGSTM3-antisense, 30 mer, 5'-TGC AAG TCT GGA TCC TGA TCA GCA TAC AGG-3'; rGSTM5-sense, 25 mer, 5'-AAG ATC CCC CCA TAT GTC GTG CTC C-3', rGSTM5-antisense, 24-mer, 5'-AGA GCA GCG GGA TCC AGC TCA GCA-3'; mGSTM5-sense, 28-mer, 5'-GCC AAG ATC GCC CCA TAT GTG ATC CAA G-3'; mGSTM5-antisense, 28-mer, 5'-CTC AGC AGC AGC GGG ATC CGG CTC AGC A-3'. All sense and antisense oligonucleotides contained NdeI or BamHI restriction sites (underlined) for cloning. The PCR reactions were performed in the presence of corresponding sets of sense-antisense primers and cDNA templates under conditions stated in the previous section ("RT-PCR Analysis"). HTGT-6, a hGSTM3-cDNA probe (15), was used as a template for hGSTM3 amplification.

The PCR products were purified using Qiagen-PCR purification kit, digested with NdeI and BamHI, then ligated into the NdeI/BamHI restriction site of a pET3a expression vector. Escherichia coli BL21 (DE3) competent cells (Novagen) were transformed by ligation mixtures and selected on ampicillin resistance media. Bacterial clones that expressed recombinant GSTs were detected by an immunoscreening procedure using anti-hGSTM3-specific antiserum under conditions described above for the immunoblot procedures. Plasmid DNAs were purified and sequenced in both directions using T7 promoter and T7 terminator oligonucleotide primers.

E. coli BL21(DE3) clones carrying recombinant plasmids were used for expression of recombinant GSTs. In brief, the overnight culture of bacterial cells was diluted at a ratio of 1:100 with fresh LB medium and grown in a rotary shaker until an A540 = 0.1-0.2 was attained. The synthesis of the recombinant GST was induced by addition of 1 mM isopropyl-beta -D-thiogalactopyranoside (Life Technologies, Inc.) and then the incubation was prolonged for 6-8 h at 37 °C. Longer incubation times for expression of these particular GSTs usually resulted in the formation of oxidized protein products. The bacteria were collected by centrifugation for 15 min at 3000 × g, washed with TBS, resuspended in the loading buffer used for GST purification, and disrupted by sonication. The soluble fraction obtained following centrifugation (30 min, 20,000 × g) was applied for protein purification by GSH affinity chromatography as mentioned above for tissue GSTs. To obtain GSH-free proteins, recombinant GSTs were dialyzed extensively against 25 mM Tris-HCl, pH 7.5, containing 0.1 mM EDTA and 1 mM dithiothreitol, until no catalytic activity with CDNB was observed in the absence of exogenous GSH. The purity of GSTs was confirmed by polyacrylamide gel electrophoresis and by reversed-phase HPLC analysis as described above.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Characterization of Rat Testicular GST Subunits-- For identification of a putative rat homologue of the human hGSTM3 subunit, rat testicular GSTs were purified and subunits resolved by reversed-phase HPLC (Fig. 1A). Peptide sequence-specific antibodies raised against the unique C-terminal sequence of the hGSTM3 subunit (15) were used to survey the protein-containing HPLC eluate by immunoblot analysis. The subunit tentatively identified as rGSTM52 on the basis of its similar retention time as the hGSTM3 subunit (27.1 min) was the only component that reacted with the highly specific hGSTM3 antiserum (Fig. 1C).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1.   Profile of rat testicular GSTs. A, HPLC pattern of GSTs isolated from rat testis by GSH affinity chromatography. The components have been tentatively identified (M1 = rGSTM1 subunit, etc.) on the basis of their relative retention times, electrophoretic mobilities shown in B, and their molecular masses determined by ESI-MS shown above the HPLC results. B. Coomassie Blue-stained SDS-polyacrylamide gel electrophoretic pattern of each HPLC fraction and of total testicular GSTs (T). Each component is labeled according to the assignments in A. C. Immunoblot of the gel shown in B using anti-hGSTM3 peptide sequence-specific antisera (see "Experimental Procedures").

To characterize further the rGSTM5 component and to substantiate rat testicular subunit assignments, the GSTs were analyzed by on-line electrospray ionization-mass spectrometry (ESI-MS). The nine major peaks obtained in order of elution were: rGSTM1, rGSTM2, rGSTM6, rGSTP1, rGSTA3, rGSTM3, rGSTM5, rGSTA1, and rGSTA4 subunits as determined by SDS-polyacrylamide gel electrophoresis (Fig. 1B) and ESI-MS (Fig. 1). The subunit masses determined by ESI-MS are shown in Fig. 1. Some molecular masses were in agreement with those calculated from the deduced sequences for rat subunits, and the molecular masses obtained for rGSTA1 and A4 were 42 Da greater than those expected from the deduced sequences, indicating that the N termini were acetylated. Complete amino acid sequences for the testicular rGSTM5 and rGSTM6 subunits have not been reported previously. The larger mass of the rGSTM5 subunit (Fig. 2) and its predominance in testicular tissue are also characteristic of the human hGSTM3 subunit. Among the Mu-class GSTs hGSTM3 is unique in that it has both N and C termini extended by several amino acid residues (15).


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2.   Electrospray ionization-mass spectrum of the rat rGSTM5 subunit. The putative rGSTM5 component was resolved by HPLC methods as described in the legend to Fig. 1. ESI-MS data were obtained according to procedures described under "Experimental Procedures." A reconstructed spectrum with molecular masses of the major and minor (glutathionylated) components is shown in the inset.

Tissue Distribution of rGSTM5-- The rGSTM5 subunit comprises approximately 30% of the GST content of adult rat testis. This subunit type is also relatively abundant in rat brain where it represents over 10% of the GST composition. However in terms of absolute amounts, testis is decidedly the richest source of rGSTM5 subunits with levels of over 4.0 µg/mg of cytosolic protein, as determined by quantitative HPLC of subunit content. The levels of this subunit in adult rat testis are thus usually about 30-fold greater than the levels in brain (0.14-0.18 µg/mg of cytosolic protein). Very low amounts of the rGSTM5 subunit were present in other rat tissues including liver, kidney, heart, and lung.

Primary Structure of rGSTM5-- The rGSTM5 subunit was first purified by reversed phase HPLC, incubated with iodo[14C]acetic acid and digested to completion with Lys-C protease. Peptide fragments were resolved by HPLC, radiolabeled peptides identified, and sequences of selected peptides determined (Fig. 3A). A sequence alignment of rGSTM5 peptides determined here, and those reported previously for rat rGSTM1 (a representative rat Mu-class subunit) and the human GSTM3 subunit are shown in Fig. 3B.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3.   A, peptide map of the rGSTM5 subunit. Lys-C endoproteinase digestions of carboxymethylated rGSTM5 subunits were carried out according to the procedure outlined under "Experimental Procedures." An aliquot of a digest was resolved by reversed-phase HPLC on a Vydac C18 column (4.6 × 250 mm) using a linear gradient of acetonitrile (5-55% in 0.1% trifluoroacetic acid for 50 min) at a flow rate of 1 ml/min. The indicated peptides (1-9) were isolated and prepared for sequence analysis; sequences including the putative lysine residues at the cleavage sites are shown in the inset. The X denotes an unidentified residue. 14C-Radiolabeled peptides, which are presumed to contain cysteine residues labeled with iodo[14C]acetate, are indicated by an asterisk. B, sequence alignment of rGSTM5 peptides with rat rGSTM1 and human hGSTM3 subunits. Peptides obtained by Lys-C protease digestion of the rGSTM5 (rM5) subunit are aligned with deduced sequences of rGSTM1 (rM1) and hGSTM3 (hM3) subunits (from Refs. 34, 35, and 15, respectively). Amino acid identities to GSTM1 are indicated by the dashes. Putative lysine residues at the protease cleavage sites are included in the rGSTM5 subunit sequence. Residues that differ from those of rGSTM1 subunits, but are identical in hGSTM3 and rGSTM5 subunits, are marked by an asterisk.

Although containing several amino acid differences, peptides obtained for the rGSTM5 subunit exhibited far greater sequence identity to the human hGSTM3 form as opposed to other rat Mu-class subunits (Fig. 3B). Analysis of the peptide map of rGSTM5 yielded some unexpected results. For instance, a small radiolabeled peptide refractory to Edman degradation was obtained; this could be the putative N-terminal peptide that is blocked. Moreover, a large peptide (number 9: SMVLGYWDIRGLAXAIRMLLEF) had an almost identical sequence as that near the N terminus of the hGSTM3 subunit (Fig. 3B). Cyanogen bromide cleavage of GST11-11 (Yo or rGSTM5) (17) yielded an identical sequence between the putative CNBr cleavage site methionine residues of this particular peptide 9, and Hsieh et al. (22) recently found this peptide after protease digestion of a rat testicular GST. Those authors were apparently unaware that the N-terminal sequence of this subunit extends beyond that of other Mu-class subunits and assumed that this peptide was near the N terminus of the GST with the first methionine at position 2. However, from the mass of the rGSTM5 subunit (Fig. 2), it is likely that it contains extended N and C termini, so the origin of peptide 9 (Fig. 3A) remained unclear. In addition, the sequence of a tetrapeptide near the C terminus terminated at a lysine residue (WGNK, peptide 1; Fig. 3A), so that the rGSTM5 cannot contain the Lys-Pro linkage found in the hGSTM3 subunit (see Fig. 3B), which is refractory to the Lys-C cleavage.

Purification of the Mouse mGSTM5 Subunit-- A cDNA clone for a mouse sperm fibrous sheath GST (mGSTM5) was recently identified (20). The mGSTM5 subunit has not been purified hitherto, although a testis-specific GST designated Ft by Lee (23) may correspond to this form. Mouse testis GSTs were therefore resolved by reversed-phase HPLC (Fig. 4). The subunit eluting at 26 min (similar to the retention time of rGSTM5 and hGSTM3 subunits) was the only mouse GST that cross-reacted with hGSTM3 peptide sequence-specific antisera and was found by ESI-MS to have a molecular mass of 26,546 Da (Fig. 4, inset). It is interesting to note that Mitchell et al. (24) did not detect this higher molecular weight subunit of testis in an HPLC-MS survey of mouse tissue GSTs purified using S-hexyl-GSH affinity matrices. This mass is greater than that of other GSTs and agrees with the molecular mass calculated from the deduced primary structure of the mouse fibrous sheath GST (20). The ESI-MS result also confirmed that the N and C termini are indeed extended for the translated mouse testicular protein product.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.   Mouse testicular GSTs. Resolution of mouse testis GST subunits by HPLC according to procedures described in the legend to Fig. 1. The GSTs purified by GSH affinity chromatography were applied to a Vydac C4 reversed-phase HPLC column (4.6 × 150 mm) and eluted as described under "Experimental Procedures." The arrow indicates the putative mGSTM5 subunit that was analyzed by the ESI-MS shown in the inset.

Molecular Cloning of rGSTM5-- On the basis of these findings, it was important to determine the complete amino acid sequence of the rat rGSTM5 subunit. Total RNAs were therefore obtained from fresh rat and mouse testicular tissue, and primers designed on the basis of known hGSTM3 and mGSTM5 sequences were used for RT-PCR (see "Experimental Procedures"). The cDNAs of both mouse mGSTM5 and rat rGSTM5 were obtained using the RT-PCR methods and their sequences determined. The sequence found for the mGSTM5 cDNA was identical to that published previously (20). Fig. 5A shows the sequence of the rGSTM5 open reading frame and the deduced amino acid sequence of rGSTM5 is compared with those of hGSTM3 and mGSTM5 subunits in Fig. 5B. It is noteworthy that the rGSTM5 subunit has 97% sequence identity to the deduced amino acid sequence for mGSTM5 (20) and 88% identity to the sequence of the hGSTM3 subunit (15). These sequence homologies among different species are far greater than homologies of these type subunits with other Mu-class GST subunits, even within the same species. Although Hsieh et al. (22) reported that this subunit had a blocked N terminus that is not acetylated, the molecular mass of rGSTM5 was precisely that predicted from the deduced sequence reported here (Fig. 5B) plus an acetylated (42 Da) N-terminal serine residue.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 5.   A, nucleotide and deduced amino acid sequences of rGSTM5. Total RNA from rat testis was subjected to RT-PCR using the primers described under "Experimental Procedures." The PCR product was purified by agarose gel electrophoresis, cloned into a pCRII TA vector, and its sequence determined by use of an ABI 377 automated sequencer. The putative ATG translation initiation codon is labeled position 1, and the termination TGA codon is labeled with a dash. B, alignment of human hGSTM3 (hM3), mouse mGSTM5 (mM5), and rat rGSTM5 (rM5) subunit sequences. The deduced hM3 and mM5 sequences are from Campbell et al. (15) and Fulcher et al. (20), respectively. Sequence identities are indicated by the dashes. The X at position 2 of mM5 reflects a missing codon for this species, which was assigned to residue 5 by Fulcher et al. (20) (MSSKS-M). However, the alignment shown here is more plausible based on its greater homology with the deduced sequence for the rat rGSTM5 subunit.

The protein sequence deduced from the cDNA also accounts for some of the unexpected results of the peptide mapping experiments (Fig. 3). For instance, the C-terminal tripeptide was released after digestion because rGSTM5 contains a Lys-Ser linkage (KSIC) rather than the Lys-Pro (KPVC) linkage at the C terminus of the hGSTM3 subunit. The lysine residue at position 4 near the N terminus provides a cleavage site to yield the large peptide starting with serine 5 (peptide 9 of Fig. 3A).

Expression and Catalytic Properties of rGSTM5-5, mGSTM5-5, and hGSTM3-3-- To allow study of the catalytic and other properties of GSTs with rGSTM5 and subunits, the proteins were expressed in E. coli (see "Experimental Procedures"). After purification by GSH affinity chromatography, the proteins were characterized by ESI-MS. For the rGSTM5 subunit, a major component with a molecular mass of 26,498 Da, and a minor form with a mass of 26,676 Da were detected, which reflect the expressed protein with and without the nascent N-formylmethionine residue. Likewise the hGSTM3-3 and mGSTM5-5 forms were expressed with and without the nascent N-formylmethionine residue.

Apparent pKa values calculated from plots of log(kcat) versus pH were identical for all three GSTs (6.20 ± 0.05), with maximal velocities obtained at pH 7.0 ± 0.2. The KmGSH values were constant for these isoenzymes over a pH range of 5.8-8.0, so that the plots log [kcat/KmGSH] versus pH also attained maximal values at pH 7.0. In contrast, the values of KmCDNB were pH-dependent and decreased until the optimal pH for enzymatic reaction was reached. Apparent pKa values of 6.6 ± 0.1 determined from each plot of log[kcat/KmCDNB] versus pH, however, were similar for all three enzymes.

Kinetic parameters determined at pH 6.2 (apparent pKa) with GSH and CDNB as substrates for the homodimeric recombinant isoenzymes followed steady-state kinetic analyses consistent with a random sequential or ordered Bi Bi bisubstrate reactions (see Table II), rather than with other bisubstrate reaction mechanisms. The likelihood of an ordered Bi Bi mechanism of catalysis was suggested by product inhibition studies, where the GS-DNB conjugate was found to be a competitive inhibitor with respect to GSH (Ki = 32 ± 16 µM for all enzymes tested) but a mixed-type inhibitor toward CDNB (Ki = 2.6 ± 0.3 mM for hGSTM3-3 and 1.2 ± 0.1 mM for the rodent enzymes). The catalytic efficiencies (kcat/Km values) for GSTs were not greater than 3-4 × 106 M-1 min-1 (Table II) less than those of most other GSTs and much lower than those expected for diffusion-controlled reactions.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Kinetic constants for recombinant Mu-class GSTs
CDNB was used as a substrate.

The kinetic constants of rGSTM5-5 and mGSTM5-5 were virtually identical, which is not surprising in view of the extensive amino acid sequence homologies of these two protein subunits. The hGSTM3-3 enzyme was slightly less active with CDNB (Table II). KmCDNB values in general were relatively high (about 1.1 mM for rodent enzymes and 2.8 mM for the human GST); by contrast, the KmGSH values were much lower (see Table II).

Certain GSH conjugates were effective competitive inhibitors, with respect of GSH. Their Ki values depended on the side chain substituent with hydrophobic GSH conjugates having increased affinities toward GSTs relative to that of GSH (Table III). The Ki for S-methylglutathione was close to the KmGSH value for each of the enzymes (Tables II and III). As a rule, Ki values decreased as the alkyl hydrophobic side chain length increased (S-methyl > S-butyl > S-hexyl > S-octyl (Table III). Aromatic analogues of GSH conjugates such as S-methyl-2-naphthyl- and S-(p-nitrobenzyl)glutathione were also effective competitive inhibitors vis à vis GSH as a substrate. Surprisingly, polar and more hydrophilic GSH conjugates such as S-(1,2-dicarboxyethyl)glutathione and glutathionesulfonic acid were much more effective inhibitors for these enzymes (see Table III), as compared with other types of GSTs. It is noteworthy that recoveries of this class of GST from commonly used S-hexyl-GSH affinity matrices are poor and probably accounts for underestimates of their content in some studies (25). It is likely that the free amino group of GSH, not present in the S-hexyl-GSH-agarose matrices, is required for high affinity binding.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Inhibitors of recombinant GST Mu class enzymes
Inhibition constants were obtained for the reaction with CDNB as a substrate.

rGSTM5 Subunit Assembly Patterns-- To determine if rGSTM5 subunits exist as, or can form heterodimers with, other Mu-class GST subunits, rat testicular cytosolic proteins as well as purified GSTs from rat testis were first resolved by isoelectric focusing methods (Fig. 6A). The isoelectric focusing gels were blotted with the hGSTM3-specific antibodies and with a Yb-"common" antiserum that cross-reacts with other Mu-class GSTs (26). The immunoblots in Fig. 6B show that a single major component with a pI near 6.2 did react with the hGSTM3-specific antiserum. It was noteworthy that no heterodimer formation between rGSTM5 and the common rGSTM1 and M2 subunits was detected. The Yb-common antibodies reacted with multiple components likely arising from homo- and heterodimeric combinations of other Mu-class subunits (Fig. 6B).


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 6.   Isoelectric focusing of rat testicular GSTs. A, a Coomassie Blue-stained isoelectric focusing gel, pH 3-10, of GSTs (5 µg) purified from rat testis (G) and a cytosolic extract (100 µg of total protein) (C) of rat testis. B, immunoblot of rat testicular cytosolic extracts probed with hGSTM3 (M3) peptide sequence-specific antisera (see text). The same blot was stripped and reprobed with Yb-common antisera that cross-reacts with most rat Mu-class GSTs.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Among rat Mu-class GSTs the rGSTM5 subunit has exceptional properties. Because the rGSTM5 subunit is found primarily in testis and is labile under oxidative conditions (17), it has not been studied extensively heretofore. On the basis of several specific criteria, including their unique structures, catalytic properties, and tissue distribution in the respective species in which they are found, rGSTM5, as well as hGSTM3 and mGSTM5, subunits should be grouped together in a separate subdivision of mammalian Mu-GSTs. A definitive cross-species interrelationship for this type of Mu-class GST subunit has thus been established. There is also evidence for an interspecies Alpha-class GST subfamily, consisting of mGSTA4, rGSTA8, and hGSTA4, which are structurally related to each other (5). From an evolutionary standpoint these Mu-class genes are the most divergent.

Aspects of primary structure, including the N- and C-terminal extensions and overall sequence homologies, suggest that rGSTM5, mGSTM5, and hGSTM3 subunits from these three different species are more closely related to one another than they are to other Mu-class GSTs even within the same species. These forms contain 7-8 cysteine residues per subunit as opposed to 3 for most other mammalian Mu-class GSTs. In addition, the protein undergoes S-thiolation to form a mixed disulfide or monoglutathionylated rGSTM5 subunit (see ESI-MS data in Fig. 2). The high cysteine contents could account for their lability during and after purification. The common catalytic properties of the rGSTM5-5, hGSTM3-3, and mGSTM5-5 enzymes summarized in Tables II and III also distinguish these forms from other GSTs of the Mu-class. It is suggest that all of these enzymes exhibit ordered Bi Bi catalytic mechanisms in contrast to other GSTs where random sequential Bi Bi mechanisms have been proposed (27-30).

Although rat, mouse, and human GSTs of this class have C-terminal cysteine residues, and the rat and mouse forms (rGSTM5 and mGSTM5) have over 97% sequence identity, it is noteworthy that 2 of the 3 additional residues at the C terminus are different in each species studied (Fig. 5). The three-dimensional structures of these forms have not been solved, and thus the functions of those particular 3-residue C-terminal extensions are presently unknown. For other Mu-class GSTs, however, the C-terminal domain folds back near the GSH sites near the N terminus and on the hydrophobic binding pocket (30, 31). In preliminary studies,3 deletion of those 3-residues from the hGSTM3 subunit did not affect catalytic properties of the mutated enzyme. Possibly the different C-terminal tripeptide sequences could serve in as yet undetermined species-specific recognition functions for these subunits.

Despite their lesser catalytic efficiencies, their Km values for GSH are lower than, or comparable with, those of other GSTs (Table II), and this class of GST adhere to, and can be quantitatively recovered from, epoxy-GSH affinity columns. Most of the residues associated with GSH binding (G-site) by Mu-class GSTs (31), including Tyr10, Trp11, Leu16, Arg46, Trp49, Lys53, NLP (62-64), QS (75, 76), and Asp109, are conserved in rGSTM5, mGSTM5 and hGSTM3 subunits (because they contain N-terminal extensions, numbering of residues are 4 amino acids greater than comparable residues of other Mu-GSTs). In addition, the H-site probably accommodates more polar substances compared with other Mu-class GSTs, in view of the effective inhibition by the polar GSH conjugates (Table III).4

Significantly, rGSTM5 isolated from rat testis is not found as a heterodimer with other common Mu-class subunits (rGSTM1, rGSTM2), even though the other forms exist as heterodimers and as homodimers among themselves. In fact, the existence of heterodimers has been a major criterion for classification of GST subunit families. It is not yet known if structural features at the intersubunit interface contact sites preclude the assembly of rGSTM5 subunits with other Mu-GST subunits. A crucial feature of Mu-class dimer formation involves the insertion of a phenylalanine residue (Phe60, which corresponds to Phe56 of other Mu-GSTs) of one subunit into a hydrophobic pocket between helices 4 and 5 of the second subunit (31). However, Phe60 and the relevant hydrophobic side chains of helices 4 and 5 (Ile102, Gln106, Gln140, Phe141, and Phe144) are conserved in the rGSTM5, mGSTM5, and hGSTM3 subunits. Moreover, the prominent residues involved in electrostatic interactions at the dimer interface, particularly the arginine salt bridge (Arg85) and the side chains of Arg85, Gln75, Arg81, Asp101, and Glu104 (31), are also conserved. Conceivably then, the overall orientation of these domains may not be conducive for interaction with other Mu-type subunits. It is more likely, however, that heterodimers do not assemble because of different cellular or subcellular locations of the rGSTM5 subunits and the other forms.

A striking feature about this class of GSTs is their tissue distribution. In all three (mouse, rat, human) species studied these forms were found primarily in testis and to a lesser extent in brain (12-15). Other tissues are virtually devoid of this GST isoform (12). The molecular basis for this decided tissue-specific expression is unknown. That rodent and human germ cells are particularly rich in this GST (20, 32) suggests that this class of GST may have specific or unique functions in males in reproductive processes. These GSTs could serve in protecting germ cells from reactive electrophilic compounds and hydroperoxides, but based on their unique structures and other properties, other unspecified functions not usually ascribed to GSTs, cannot be ruled out.

    ACKNOWLEDGEMENTS

We thank Edward Nieves for assistance with the ESI-MS experiments and Ruth Angeletti and the staff of the laboratory for macromolecular analysis of the Albert Einstein College of Medicine for their assistance. The technical help of John Stevenson is acknowledged. We particularly appreciate the careful review of the manuscript, advice, and encouragement of Dr. John D. Hayes.

    FOOTNOTES

* This work was supported by Grant CA-42448 and Cancer Center Core 5-P30-CA1330 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U86635.

Dagger Supported by genetics and biochemistry training grant T32 GM-07128.

§ To whom correspondence should be addressed. Tel.: 718-430-2276; Fax: 718-430-8565; E-mail: irving{at}aecom.yu.edu.

1 The abbreviations used are: GST, glutathione S-transferase; CDNB, 1-chloro-2,4-dinitrobenzene; HPLC, high performance liquid chromatography; GS-DNB, S-(2,4-dinitrophenyl)glutathione; RT-PCR, reverse transcription polymerase chain reaction; ESI-MS, electrospray ionization mass spectrometry.

2 Nomenclature for rat GSTs that is consistent with the subunit class-based system for human GSTs (1), and similar to that used in a recent review by Hayes and Pulford (5), has been adopted in this study. Table I may be used for comparison to the "Y" system frequently employed for rat GSTs.

3 Y. V. Patskovsky and I. Listowsky, unpublished observations.

4 Y. V. Patskovsky, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Mannervik, B., Awasthi, Y. C., Board, P. G., Hayes, J. D., Di Ilio, C., Ketterer, B., Listowsky, I., Morgenstern, R., Muramatsu, M., Pearson, W. R., Pickett, C. B., Sato, K., Widersten, M., and Wolf, C. R. (1992) Biochem. J. 282, 305-306[Medline] [Order article via Infotrieve]
  2. Listowsky, I. (1993) in Hepatic Anion Transport and Bile Secretion: Physiology and Pathophysiology (Tavolini, N. N., and Berk, P. D., eds), pp. 397-405, Raven Press, New York
  3. Rushmore, T. H., and Pickett, C. B. (1993) J. Biol. Chem. 268, 11475-11478[Free Full Text]
  4. Awasthi, Y. C., Sharma, R., and Singhal, S. S. (1994) Int. J. Biochem. 26, 195-308[Medline] [Order article via Infotrieve]
  5. Hayes, J. D., and Pulford, D. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 445-600[Abstract]
  6. Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M. K., Warholm, M., and Jornvall, H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7202-7206[Abstract]
  7. Jakoby, W. B., and Ziegler, D. M. (1990) J. Biol. Chem. 265, 20715-20718[Free Full Text]
  8. Armstrong, R. N. (1994) Adv. Enzmol. Rel. Areas Mol. Biol. 69, 1-44
  9. Ketterer, B., and Christodoulides, L. G. (1994) Adv. Pharmacol. 27, 37-69[Medline] [Order article via Infotrieve]
  10. Kamisaka, K., Listowsky, I., Gatmaitan, Z., and Arias, I. M. (1975) Biochemistry 14, 2175-2180[Medline] [Order article via Infotrieve]
  11. Listowsky, I., Abramovitz, M., Homma, H., and Niitsu, Y. (1988) Drug Metab. Rev. 19, 305-318[Medline] [Order article via Infotrieve]
  12. Rowe, J. D., Nieves, E., and Listowsky, I. (1997) Biochem. J. 325, 481-486[Medline] [Order article via Infotrieve]
  13. Takahashi, Y., Campbell, E. A., Hirata, Y., Takayama, T., and Listowsky, I. (1993) J. Biol. Chem. 268, 8893-8898[Abstract/Free Full Text]
  14. Johnson, J. A., Finn, K. A., and Siegel, F. L. (1992) Biochem. J. 282, 279-289[Medline] [Order article via Infotrieve]
  15. Campbell, E., Takahashi, Y., Abramovitz, M., Peretz, M., and Listowsky, I. (1990) J. Biol. Chem. 265, 9188-9193[Abstract/Free Full Text]
  16. Hayes, J. D. (1988) Biochem. J. 255, 913-922[Medline] [Order article via Infotrieve]
  17. Kispert, A., Meyer, D. J., Lalor, E., Coles, B., and Ketterer, B. (1989) Biochem. J. 260, 789-793[Medline] [Order article via Infotrieve]
  18. Jensson, H., Alin, P., and Mannervik, B. (1985) Methods Enzymol. 113, 504-507[Medline] [Order article via Infotrieve]
  19. Cornish-Bowden, A. (1995) Fundamentals of Enzyme Kinetics, pp. 143-147, Portland Press Ltd., London
  20. Fulcher, K. D., Welch, J. E., Klapper, D. G., O'Brien, D. A., and Eddy, E. M. (1995) Mol. Reprod. Dev. 42, 415-424[Medline] [Order article via Infotrieve]
  21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. Hsieh, C.-H., Tsai, S. P., Yeh, H.-I., Sheu, T.-C., and Tam, M. F. (1997) Biochem. J. 323, 503-510[Medline] [Order article via Infotrieve]
  23. Lee, C. Y. (1982) Mol. Cell. Biochem. 49, 161-168[Medline] [Order article via Infotrieve]
  24. Koehler, R. T., Villar, H. O., Bauer, K. E., and Higgins, D. L. (1997) Proteins Struct. Funct. Genet. 28, 202-216[CrossRef][Medline] [Order article via Infotrieve]
  25. Mitchell, A. E., Morin, D., Lakritz, J., and Jones, D. A. (1997) Biochem. J. 325, 207-216[Medline] [Order article via Infotrieve]
  26. Abramovitz, M., Ishigaki, S., Felix, A. M., and Listowsky, I. (1988) J. Biol. Chem. 263, 17627-17631[Abstract/Free Full Text]
  27. Jakobson, I., Warholm, M., and Mannervik, B. (1979) Biochem. J. 177, 861-868[Medline] [Order article via Infotrieve]
  28. Ivanetich, K. M., and Goold, R. D. (1989) Biochim. Biophys. Acta 998, 7-13[Medline] [Order article via Infotrieve]
  29. Ivanetich, K. M., Goold, R. D., and Sikakana, C. N. (1990) Biochem. Pharmacol. 39, 999-2004
  30. Ji, X., Zhang, P., Armstrong, R. N., and Gilliland, G. L. (1992) Biochemistry 31, 10169-10184[Medline] [Order article via Infotrieve]
  31. Armstrong, R. N. (1997) Chem. Res. Toxicol. 10, 2-18[CrossRef][Medline] [Order article via Infotrieve]
  32. Jakoby, W. B., Ketterer, B., and Mannervik, B. (1984) Biochem. Pharmacol. 33, 2539-2540[Medline] [Order article via Infotrieve]
  33. Comstock, K. E., Widersten, M., Hao, X.-Y., Henner, W. D., and Mannervik, B. (1994) Arch. Biochem. Biophys. 311, 487-495[CrossRef][Medline] [Order article via Infotrieve]
  34. Townsend, A. J., Goldsmith, M. E., Pickett, C. B., and Cowan, K. H. (1989) J. Biol. Chem. 264, 21582-21590[Abstract/Free Full Text]
  35. Lai, H. C., Qian, B., and Tu, C. P. D. (1989) Arch. Biochem. Biophys. 273, 423-432[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.