©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heterologous Expression and Enzymatic Properties of a Selenium-independent Glutathione Peroxidase from the Parasitic Nematode Brugia pahangi(*)

(Received for publication, April 24, 1995)

Liang Tang (1) (2) Kleoniki Gounaris (1) Caroline Griffiths (3) Murray E. Selkirk (1) (2)(§)

From the  (1)Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, London, United Kingdom ( (2)Wellcome Centre for Parasitic Infections) and the (3)Wellcome Research Laboratories, Beckenham, Kent, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A full-length cDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera frugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). The recombinant protein was glycosylated and secreted from the cells in tetrameric form. The purified protein showed glutathione peroxidase activity with a range of organic hydroperoxides, including L-alpha-phosphatidylcholine hydroperoxide, but no significant activity against hydrogen peroxide. Glutathione was the only thiol tested that served as a substrate for the enzyme, which showed no activity with the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). No glutathione-conjugating activity was detected against a range of electrophilic compounds that are common substrates for glutathione S-transferases. The apparent (pseudo) K for glutathione was determined as 4.9 mM at a fixed concentration of linolenic acid hydroperoxide (3 µM). The enzyme showed low affinity for hydroperoxide substrates (apparent K for linolenic acid hydroperoxide and L-alpha-phosphatidylcholine hydroperoxide of 3.8 and 9.7 mM, respectively at a fixed glutathione concentration of 3 mM).


INTRODUCTION

Glutathione peroxidases (GPxs) (^1)catalyze the reduction of hydrogen peroxide (H(2)O(2)) and organic hydroperoxides by glutathione (GSH), thus limiting oxidative damage to biological tissues (1) . The best studied of this class of enzymes has a cytosolic location and exists as a homotetramer with a subunit mass of 21 kDa(2) , and a variant with similar mass and tetrameric association but restricted tissue distribution has been identified from mammalian sources(3) . Secreted GPxs have been isolated from mammalian plasma and also exist in a tetrameric state(4, 5) . Doubt about their physiological role as efficient anti-oxidant enzymes has been expressed due to the low extracellular concentration of GSH and the absence of an efficient system for regenerating GSH from its oxidized form (GSSG), although recent studies have suggested that these enzymes could utilize the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) as an alternative electron donor in vivo(6) . Cytosolic GPxs can reduce H(2)O(2) and fatty acid hydroperoxides but not phospholipid hydroperoxides(7) , whereas recent data demonstrate that the plasma enzymes show activity against the latter substrates and cholesterol 7alpha hydroperoxide(8, 9) . Another member of the GPx family, termed phospholipid hydroperoxide glutathione peroxidase (phospholipid hydroperoxide GPx), readily reduces phospholipid and cholesterol hydroperoxides(10, 11, 12, 13) . Phospholipid hydroperoxide GPx is a monomeric protein of 19 kDa with a distinct but related primary structure to classical GPx(14) . The ability of this enzyme to act on phospholipid and cholesterol hydroperoxides in situ suggests that it plays an important role in the limitation of lipid peroxidation in biological membranes. Phospholipid hydroperoxides are poor substrates for cytosolic GPx unless first acted on by phospholipase A(2) to release sn-2 fatty acyl hydroperoxides(7) , and it has been reported that the plasma GPx largely lacks the membrane interfacial properties of the phospholipid hydroperoxide GPx(8) .

All of the enzymes described above contain a selenium atom within a catalytically active selenocysteine residue. Detailed kinetic measurements of bovine GPx (15) and the determination of the three-dimensional structure of the enzyme (16, 17) have led to a proposed mechanism of enzyme action, which has recently been revised (18) . The kinetic pattern has been described as a ping-pong mechanism involving an oxidation-reduction cycle of selenium at the active site of the enzyme, although the intermediates in the catalytic cycle have not been fully elucidated(15, 17, 18) . The involvement of selenocysteine in catalysis has been confirmed by carboxymethylation ( (19) and (20) ; Floh, cited in (12) ) or elimination of the residue(21) , which leads to inactivation of the enzyme.

Although selenocysteine-independent GPx activity has been reported in the literature, the majority of these cases are due to structurally unrelated glutathione-S-transferases that reduce fatty acid hydroperoxides by an entirely different mechanism. Recently, genes encoding homologs of GPx in which the codon directing incorporation of selenocysteine was observed to be substituted by a cysteine codon have been isolated from Escherichia coli(22) , plants(23, 24) , and mammals, the last isolated mainly from epididymal tissues(25, 26, 27) . Analysis of the diversity of glutathione peroxidases based on primary sequence places these selenium-independent homologs in a separate clade together with the plasma enzymes(18) , although no relevant peroxidase activity has yet been reported for the cysteine-containing homologs. We recently reported the isolation of cDNAs encoding a cysteine-containing (i.e. selenium-independent) GPx homolog from the parasitic nematode Brugia pahangi(28) . This glycoprotein, termed gp29, has been localized to the cuticular matrix of the parasite (29, 30) and is therefore a secreted protein, as are all of the selenium-independent homologs reported from mammals(25, 26, 27) . Purification of the native enzyme for functional analysis is impossible due to limitations on parasite material. We therefore expressed gp29 in insect cells via a baculovirus vector in order to examine its putative enzyme activity, and we report here on the properties of this selenium-independent GPx.


EXPERIMENTAL PROCEDURES

Generation of Recombinant Baculovirus and Expression of gp29 in Insect Cells

A full-length cDNA encoding gp29 was generated by polymerase chain reaction from cDNA 29.1 (28) using the 5` primer 5`-CTATCTAGAATGTCCGCACAACT-3` for the sense strand (methionine start codon in boldface type) and the 3` primer 5`-CTATCTAGATTAAATTTACGTTCCAGTTCATC-3` for the antisense strand (stop codon in boldface type). Both primers contained an XbaI restriction site (underlined), which facilitated subcloning of the amplified fragment into the baculovirus transfer vector pVL1393 (Invitrogen). Orientation of the insert was checked by restriction mapping, and the construct was transferred into Autographa californica nuclear polyhedrosis virus (AcNPV) by recombination according to the manufacturer's instructions (Baculogold, Pharmingen). Spodoptera frugiperda (Sf) cells were infected and recombinant plaques confirmed by polymerase chain reaction with oligonucleotides homologous to AcNPV sequences flanking the gp29 insert. Sf cells were grown as described (31) in suspension or monolayer cultures in TC-100 medium supplemented with 10% fetal calf serum, 1% antibiotic and antimycotic solution (Life Technologies, Inc.; catalog number 15240-021) and 2 mML-glutamine at 27 °C. Cultures infected with viral constructs coding for gp29 or wild type AcNPV were further grown in and adapted to the same culture medium without fetal calf serum. Culture supernatants (20 µg) and cell pellets (50 µg) were harvested at days 0, 1, 2, 3, and 5 post-infection and analyzed by 15% SDS-polyacrylamide gel electrophoresis and Western blotting with a rabbit polyclonal antiserum to gp29 from B. pahangi(29) , either prior to or following digestion with serial dilutions of peptide:N-glycosidase F (N-glycanase; Boehringer-Mannheim) as described previously(32) . Sf cells infected with the recombinant gp29 viral construct were pulsed with 100 µCi ml [S]methionine for 15 min and chased with TC-100 medium for 15 min, 30 min, 1 h, 4 h, and 24 h. Culture supernatants were collected and concentrated or immunoprecipitated with the rabbit anti-gp29 serum as described previously (29) prior to resolution by SDS-polyacrylamide gel electrophoresis and subsequent autoradiography.

Purification of Recombinant gp29

The culture supernatant from infected Sf cells was collected and centrifuged at 25,000 g for 1 h at 4 °C to remove the cells. Proteins in the supernatant were precipitated with (NH(4))(2)SO(4) at concentrations between 30 and 65% and redissolved in 50 mM potassium phosphate buffer, pH 7.5. The soluble protein sample was desalted to remove excess (NH(4))(2)SO(4) before further purification by cation exchange chromatography (Hiload 16/10 S-Sepharose, Pharmacia Biotech Inc.). The ion exchange column was pre-equilibrated with buffer A (20 mM bis-Tris, pH 6.0). After the sample was loaded the column was washed with 50 ml of buffer A and eluted with a linear gradient (0-100%) of buffer B (2 M NaCl in buffer A). Fractions containing gp29 were pooled and further separated by gel permeation chromatography (Hiload 16/60 Superdex 200, prep grade, Pharmacia) at 4 °C with 50 mM potassium phosphate buffer, pH 7.5, containing 150 mM NaCl. Protein molecular weight markers (MW-GF-200, Sigma) were utilized to create a calibration curve for the calculation of protein mass. Purification to homogeneity was effected by concanavalin A-agarose affinity chromatography as described previously(32) . 50 ml of protein sample from gel permeation chromatography was incubated with 10 ml of ConA-agarose (Sigma) overnight at 4 °C. The ConA-agarose gel mixture was then packed into a polypropylene Econo-Column (Bio-Rad) and washed with 30 ml of potassium phosphate buffer (50 mM, pH 7.5) containing 1.2 M NaCl, 50 mM potassium phosphate, pH 7.5. The bound protein was eluted with 0.2 M methyl alpha-D-mannopyranoside in potassium phosphate buffer. The purity of gp29 at each stage was evaluated by SDS-polyacrylamide gel electrophoresis and silver staining.

Preparation of Hydroperoxide Substrates

Fatty acid hydroperoxides and phospholipid hydroperoxides were prepared as described previously(12, 33) . Fatty acids (-linolenic acid and linoleic acid) and L-alpha-phosphatidylcholine were dissolved in 0.1 M sodium borate buffer, pH 9.0, at a final concentration of 0.5 mM, and sodium deoxycholate was added to the L-alpha-phosphatidylcholine reaction mixture at 3 mM. The reaction was started by adding 1.14 µg ml and 40 µg ml of lipoxygenase, respectively, to the reaction mixtures of fatty acids and phospholipids and incubated at room temperature for 30 min. The formation of hydroperoxides was monitored spectrophotometrically at 234 nm, and concentrations were calculated using a molar extinction coefficient of 25,000 M cm(33) . Hydroperoxides were purified by reverse-phase chromatography on Sep-Pak C18 cartridges (Millipore Corp.) and eluted with 5 ml of 100% methanol as described previously(12) . Methanol was removed by evaporation, and hydroperoxides were stored at -70 °C for further use.

Enzymatic Properties of gp29

The peroxidase activity of gp29 with different hydroperoxide and thiol substrates was examined, in addition to glutathione S-transferase activity. GPx from bovine erythrocytes (Sigma catalog number G6137) and glutathione-S-transferase from bovine liver (Sigma catalog number G4385) were used as positive controls under our assay conditions. GPx activity was determined by the glutathione reductase-coupled assay(34) . The reaction mixture contained 50 mM potassium phosphate buffer, pH 7.5, 0.4 mM EDTA, 1 mM sodium azide, 0.13 mM beta-NADPH, 1.2 units/ml GSH reductase, and 3 mM GSH. Purified gp29 (100 pmol) was added to 1 ml of reaction mixture and incubated for 5 min at 37 °C. An equivalent quantity (12 µg) of protein from the culture medium of Sf cells infected with wild type baculovirus was used as a control. The reaction was initiated by adding 3 µM hydroperoxides and monitored at 340 nm for 2 min. Backgrounds of the reaction from the control sample (i.e. resulting from autoxidation of GSH) were subtracted from assay values. In reactions with L-alpha-phosphatidylcholine hydroperoxide as substrate, hydroperoxide free Triton X-100 (Sigma) was added to the reaction mixture at 0.1% (v/v) as described previously(13) . Enzyme activity was calculated using a molar extinction coefficient of 6,220 M cm. The apparent (pseudo) K of gp29 for GSH and hydroperoxide substrates was calculated from a double-reciprocal plot of 1/v against 1/[substrate], with linolenic acid set at 3 µM in the first instance and GSH set at 3 mM in the second instance.

The optimal pH profile of gp29 was determined by using 3 µM cumene hydroperoxide as substrate, and the pH range of potassium phosphate buffer was 6.0-9.5. The specificity of gp29 for different thiols was investigated with the coupled assay as described above and the DTNB method(35) . For the DTNB assay, 1 ml of 50 mM potassium phosphate buffer, pH 7.5, containing 0.1 mM EDTA, 3 µM -linolenic acid hydroperoxide, and 0.5 mM respective thiol was incubated with 100 pmol of gp29 or 12 µg of protein from the culture supernatant of cells infected with wild type AcNPV, at 37 °C for 30 min. 50 µl of the reaction mixture was taken and added to a cuvette containing 1.5 mM 5,5`-dithiobis(nitrobenzoic acid. Residual sulfhydryl compounds were determined by measuring the optical density at 412 nm using a molar extinction coefficient of 13,000 M cm.

The ability of thioredoxin to act as an electron donor to gp29 was investigated via the thioredoxin system as described previously(6, 36) . Thus, 100 pmol of gp29 was incubated with 50 pmol of thioredoxin reductase (American Diagnostica Inc., catalog number 702) and 36 µg of thioredoxin reductase (Sigma catalog number 3658) in 1 ml of 50 mM potassium phosphate, pH 7.5, 1 mM EDTA, and 128 µM NADPH. The reaction was initiated by addition of hydroperoxides at 3 µM (hydrogen peroxide, cumene hydroperoxide, -linolenic acid hydroperoxide, and L-alpha-phosphatidylcholine hydroperoxide), and absorbance was monitored at 340 nm. Positive controls for the activity of thioredoxin and thioredoxin reductase were performed by monitoring the reduction of insulin (Sigma, catalog number 5500) via the same system. Oxidation of NADPH was linear with insulin concentrations between 4 and 40 nmol. ml.

Glutathione S-transferase (i.e. glutathione-conjugating) activity of gp29 was measured as described previously(37) . The reaction mixture contained 100 mM potassium phosphate, pH 7.0, 5 mM GSH, 100 pmol of gp29 or 12 µg of control proteins, and electrophilic compounds (1 mM 1-chloro-2,4-dinitrobenzene, 1 mMp-nitrobenzyl chloride, 0.2 mM 4-nitropyridine-N-oxide, and 5 mM 1,2-epoxy-3-[p-nitrophenoxy]propane). The reactions were monitored spectrophotometrically at the optimal wavelength of each substrate. The specific glutathione-S-transferase activities for each substrate were calculated with the molar extinction coefficients reported by Habig et al.(37) .


RESULTS

Expression, Purification, and Structural Properties of Recombinant gp29-The full-length cDNA encoding gp29 contained an N-terminal signal peptide that had previously been shown to direct translocation of the protein into microsomal membrane preparations in vitro(28) . We therefore assessed whether gp29 expressed by AcNPV was secreted from the Sf cells by pulse-chase labeling with [S]methionine. Fig. 1illustrates that a protein of 29 kDa was secreted from the cells containing the gp29 construct. This protein appeared in the culture supernatant 1 h after pulsing infected cells, accumulated in a progressive manner, and was identified as gp29 by immunoprecipitation with an antibody (29) specific for the native parasite protein (Fig. 1, lane6). Although a single protein of 29 kDa was detected in culture supernatants, cell extracts contained different forms of gp29 ranging in mass from 25 to 32 kDa (Fig. 2A). Digestion with N-glycanase indicated that these represented processing intermediates (Fig. 2B) and that the mature secreted protein contained two N-linked oligosaccharide chains on a peptide backbone of 25 kDa (Fig. 2C), identical to the properties of the native parasite protein(32) .


Figure 1: Secretion of gp29 from Sf cells. Sf cells infected with the AcNPV/gp29 construct were pulsed with [S]methionine for 15 min and chased with cold TC-100 medium for 15 min (lane1), 30 min (lane2), 1 h (lane3), 4 h (lane4), and 24 h (lane5). Culture media were concentrated and resolved on a 7-25% gradient SDS-polyacrylamide gel. The culture medium from the 4-h chase was immunoprecipitated with a polyclonal antibody to gp29 (29) prior to resolution (lane6). The relative mass of marker proteins is shown in kDa.




Figure 2: N-linked glycosylation of gp29 in Sf cells. Processing intermediates of gp29 in Sf cells and the mature secreted protein were detected by Western blotting with cell extracts (50 µg) and culture media (20 µg) 2 days postinfection with AcNPV/gp29. PanelA, lane1 shows differentially processed forms of gp29 in cell extracts, and their relative mass is indicated in kDa; lane2 shows that a single protein species of 29 kDa is secreted into culture medium. PanelsB and C show digestion of cell extracts and culture medium, respectively, with increasing concentrations of peptide:N-glycosidase F (N-glycanase) prior to SDS-polyacrylamide gel electrophoresis and Western blotting. Concentrations of N-glycanase utilized in both cases were as follows: 1 milliunit ml (lane1), 10 milliunits ml (lane2), 100 milliunits ml (lane3, and 1 unit ml (lane4). Glycosylated (G) and deglycosylated (DG) gp29 resolved with relative masses of 29 and 25 kDa, respectively.



Gp29 was purified to homogeneity from culture supernatants by sequential cycles of (NH(4))(2)SO(4) precipitation, ion exchange, gel permeation and concanavalin A affinity chromatography (Fig. 3). The purification was monitored by Western blotting with specific antibody to the native protein and assay for peroxidase activity with cumene hydroperoxide as substrate. Fractions collected from gel permeation chromatography showed peaks in GPx activity and immunological reactivity with the specific antibody at approximately 120 kDa, indicating that the mature recombinant protein was tetrameric, again consistent with the properties documented for the native parasite protein(30, 38) .


Figure 3: Purification of recombinant gp29. gp29 was purified from culture medium by sequential rounds of ammonium sulfate precipitation, cation exchange, gel permeation, and ConA-agarose affinity chromatography as described under ``Experimental Procedures.'' Samples were resolved on a 15% SDS-polyacrylamide gel and visualized by silver staining. Lane1, profile following ammonium sulfate precipitation; lane2, profile following cation exchange and gel permeation chromatography; lane3, the purified protein following ConA-agarose affinity chromatography. The relative mass of marker proteins is shown in kDa, and gp29 is marked with an arrow.



Enzymatic Activity of gp29

Purified gp29 showed GPx activity with a range of hydroperoxides but was susceptible to inactivation at high concentrations, as previously reported for mutants of GPx in which selenocysteine was replaced by cysteine(39) . Enzyme inactivation was avoided by employing a GSH:ROOH ratio greater than 100. Linolenic acid hydroperoxide was used as a standard substrate, and under our assay conditions (3 µM linolenic acid hydroperoxide and 3 mM GSH), the reaction velocity was a linear function of enzyme concentration up to 0.13 µM (Fig. 4A). A double-reciprocal plot with linolenic acid fixed at 3 µM (Fig. 4B) allowed the apparent K for GSH to be determined at 4.9 mM. This is similar to that reported for the plasma GPx with t-butyl hydroperoxide as substrate(4) , 4 times higher than that calculated for the phospholipid hydroperoxide GPx with linoleic acid(11) , and 7 times higher than that calculated for the cytosolic GPx with H(2)O(2)(15) .


Figure 4: GPx activity of gp29. PanelA, the rate of oxidation of GSH was measured with a fixed concentration of linolenic acid hydroperoxide (3 µM) and a range of concentrations of gp29 (0-210 pmol in a final volume of 1 ml). PanelB, double-reciprocal plot of GPx activity. GPx activity (v is expressed as nmol of NADPH oxidized min ) was measured with different concentrations of GSH (0.5-8 mM) as described under ``Experimental Procedures.''



The pH profile of peroxidase activity is shown in Fig. 5. The pattern and the pH range of activity was similar to that reported previously for bovine GPx (40) and a mutant enzyme in which the active site selenocysteine was substituted by cysteine(39) . A sharp increase in enzyme activity was observed above pH 7.0, and maximal activity was observed at pH 8.5, consistent with S as the active form. At higher pH conditions we observed a significant increase in non-enzymatic oxidation of GSH.


Figure 5: Effect of pH on GPx activity of gp29. GPx activity was assayed at a range of conditions (pH 6.0-9.5) with 100 pmol of gp29, 3 mM GSH, and 30 µM cumene hydroperoxide in a final volume of 1 ml at 37 °C.



The relative activity of gp29 with different substrates was investigated, and the results are summarized in Table 1. For the hydroperoxide substrates tested, gp29 showed maximal activity with linolenic acid hydroperoxide and lower activity against smaller substrates (cumene hydroperoxide and t-butyl hydroperoxide). Negligible activity was observed with hydrogen peroxide. gp29 also demonstrated significant activity with phosphatidylcholine hydroperoxide. No activity was demonstrable with any of the thiols tested other than GSH, and substitution of GSH and GSH reductase with thioredoxin and thioredoxin reductase also resulted in no demonstrable peroxidase activity, in contrast to that documented for the human plasma GPx(6) . No GSH-conjugating activity was detectable with a range of substrates commonly utilized by glutathione S-transferase (Table 1).



The apparent K(m) values of gp29 for linolenic acid hydroperoxide and phospholipid hydroperoxide (L-alpha-phosphatidylcholine hydroperoxide) were determined. A double-reciprocal plot of 1/v against 1/[ROOH] gave a linear plot for both hydroperoxide substrates (Fig. 6). The apparent K and V(max) of gp29 was calculated as 3.8 mM and 1.0 nmol min for linolenic acid hydroperoxide and 9.7 mM and 0.9 nmol min for L-alpha-phosphatidylcholine hydroperoxide, respectively, in reactions with a GSH concentration of 3 mM. A comparison of these K(m) values with those calculated from data reported for mammalian GPx and phospholipid hydroperoxide GPx at the same GSH concentration is shown in Table 2, illustrating the greatly reduced affinity of gp29 for the hydroperoxide substrates.


Figure 6: Double-reciprocal plot of GPx activity on phosphatidylcholine hydroperoxide and linolenic acid hydroperoxide. GPx activity was assayed with 100 pmol of gp29, 3 mM GSH, and 3-18 µML-alpha-phosphatidylcholine hydroperoxide or 1.5-18 µM -linolenic acid hydroperoxide in a final volume of 1 ml at pH 8.0, 37 °C. v is expressed as µmol of GSH oxidized min, and [ROOH] is expressed as mM.






DISCUSSION

The recombinant form of gp29 expressed in insect cells is similar in many respects to the native parasite protein in that it is secreted in tetrameric form and bears two N-linked oligosaccharide chains. Both proteins bind concanavalin A, suggesting that the oligosaccharides are of typical high mannose composition in both cases. Genes encoding homologs of gp29 have now been sequenced from four different species of filarial nematode (41) , (^2)and in all cases the UGA codon that specifies incorporation of selenocysteine at the active site of the enzyme (position 52 in the bovine cytosolic GPx) is substituted either by UGC or UGU (i.e. codons specifying insertion of cysteine). Genomic Southern analysis indicates that gp29 is encoded by a single copy gene in Brugia malayi, and thus these organisms do not appear to possess an additional, selenocysteine-containing GPx(41) .

The enzymatic reaction of GPx has been described as a ping-pong mechanism that involves initial reduction of the hydroperoxide substrate and a subsequent two-step oxidation of glutathione, and the selenocysteine residue appears to be crucial for redox catalysis by virtue of the low pK and high nucleophilicity of the selenol group (15) . Feeding a selenium-deficient diet to rats causes a drop in aortic GPx activity, assayed with hydrogen peroxide as substrate, to undetectable levels after 6 weeks(42) . The role of selenocysteine in activity of the bovine erythrocyte GPx has been examined by site-directed mutagenesis, in which the selenocysteine residue was replaced by either cysteine or serine. The cysteine mutant alone showed GPx activity, although this was reduced approximately 1000-fold when compared with that of the natural enzyme(39) . Analagous reductions in the activities of other selenoenzymes such as E. coli formate dehydrogenase (43) and type 1 iodothyronine deiodinase (44) have been reported for cysteine mutants.

Gp29 thus shows GPx activity, although the specific activity is considerably lower than that reported for the selenocysteine-containing GPxs. The apparent K of gp29 for GSH does not differ dramatically from values reported for mammalian selenoenzymes, but the rate-limiting step in the reaction would appear to be oxidation of the enzyme by hydroperoxides, presumably due to the low nucleophilicity of the sulfhydryl group of the active site cysteine relative to selenocysteine(45) . We could not detect significant catalytic activity against H(2)O(2), and high concentrations of all hydroperoxides tested inactivated the enzyme. Similar observations were reported for the cysteine mutant of bovine GPx(39) , and the authors suggested that this might be due to overoxidation of the sulfhydryl group of the active site cysteine from a sulfenic state to a sulfinic acid that could be sensitive to beta elimination, leading to inactivation of the enzyme.

To date, at least 24 amino acid sequences have been identified as homologs of GPx. The diversity of these proteins has recently been reviewed, and a dendrogram of the GPx superfamily has been constructed based on partial sequences that are unequivocally homologous(18) . This analysis allowed three distinct molecular clades to be defined: 1) the cytosolic enzymes (cGPx) with a side branch corresponding to an enzyme isolated from the human gastrointestinal tract (giGPx), 2) the phospholipid hydroperoxide GPx family, and 3) the plasma enzymes and homologs in which the active site selenocysteine has been substituted by cysteine.

The similarity between the filarial GPx (gp29) homologs and the mammalian epididymal homologs, which are also predicted to be secreted proteins, is particularly striking when one specifically considers residues in and around the active site. Both classes of proteins contain cysteine in the place of selenocysteine, and of the five basic amino acids suggested to orient binding of glutathione, only one of these residues (corresponding to Arg in the bovine cGPx sequence) is conserved. Of the others, Arg is deleted in both cases, Lys is substituted by an acidic residue (glutamate in the case of the filarial proteins and aspartate in the case of the epididymal homologs), Arg is substituted by histidine in both cases, and Arg is substituted by an uncharged residue. For the phospholipid hydroperoxide GPx family, none of the five charged residues are conserved, and a model of the active site has failed to highlight any isosteric and similarly charged substitute residues that could be envisaged to bind GSH(18) . The authors have therefore questioned whether GSH is the real physiological substrate for phospholipid hydroperoxide GPx, although these enzymes are active glutathione peroxidases and no donor substrate has yet been found that reacts faster than GSH(18) . Further substitutions in the active site region common to the filarial and epididymal homologs in relation to the cGPxs include replacement of Glu by valine, Ser by threonine, Leu by tyrosine, Asp by glutamine, Asn by proline, and Thr by valine.

Although the enzymatic properties of the epididymal homologs have not yet been reported, it has been suggested that they may function to protect spermatazoal membranes, unusually rich in unsaturated fatty acids, from lipid peroxidation(27) . Rat testis contains a high level of phospholipid hydroperoxide GPx, which appears to be partially cytosolic and partially linked to nuclei and mitochondria(46, 47) . The secreted epididymal GPx could complement these activities via concentration in seminal fluid. Gp29 is the major soluble glycoprotein secreted into the cuticular matrix of filarial nematode parasites, and in an analogous manner it may serve to protect parasite surface membranes from oxidative damage. The deletions and substitutions referred to above create a much more hydrophobic active site, which may be particularly amenable to lipid hydroperoxide substrates, consistent with the substrate specificity of gp29.

Reservations with respect to the anti-oxidant capacity of mammalian secreted GPxs have been expressed in view of the low concentration of GSH in blood plasma and the lack of any efficient extracellular system for regenerating GSH from GSSG(48) . A recent study has demonstrated that the thioredoxin and glutaredoxin systems act as efficient electron donors to the human plasma GPx(6) , and thus the former system could participate in the activity of secreted GPxs in vivo, particularly when one considers that both thioredoxin and thioredoxin reductase have been identified as plasma membrane-associated proteins on mammalian cells(49, 50) . Our current data suggest that the thioredoxin system does not act as an efficient electron donor to the parasite enzyme, however, and it is not yet known whether this will prove to be the case for other selenium-independent GPxs.

Filarial nematodes are bound by a cuticular matrix, delineated by a proximal hypodermal membrane and a distal epicuticular membrane. Immunoelectron microscopic studies suggest that gp29 is concentrated at the hypodermal membrane but is constantly turned over via secretion through the cuticle(29) , and thus the precise site of biological activity remains undefined. It is possible that cofactors present in the cuticle influence enzyme activity, but assays of whole parasite extracts have yielded results very similar to those reported here for gp29 expressed in insect cells. It is also unlikely that glutathione serves as a natural substrate, since the major structural components of the cuticle are collagens cross-linked by disulfide bonds (51) , and secretion of glutathione into this environment would be expected to have deleterious consequences due to formation of mixed thiols. The utilization of an extrinsic substrate acquired from mammalian plasma is therefore an attractive proposition, although no activity could be detected with thioredoxin in our present study, highlighting a divergence with the activity of the selenocysteine-containing plasma GPx. The apparently disadvantageous substitution of the active site selenocysteine residue by cysteine is puzzling, but the similarities in the active site of gp29 and epididymal GPx homologs (remarkable given that the comparison is made between mammalian and invertebrate sources) suggest that the enzymes may have similar features in terms of substrate specificity and kinetic properties as well as biological functions. Thus, although we have demonstrated GPx activity for gp29 against fatty acid hydroperoxides and phospholipid hydroperoxides, there may exist alternative roles for the selenocysteine-independent GPx homologs in vivo.


FOOTNOTES

*
This investigation received financial support from the Onchocerciasis Control Programme in West Africa-UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases Macrofil Chemotherapy Project. We also acknowledge support from the Medical Research Council and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44171 594 5214; Fax: 44171 225 0960.

^1
The abbreviations used are: GPx, glutathione peroxidases; AcNPV, Autographa californica nuclear polyhedrosis virus; Sf cells, Spodoptera frugiperda cells.

^2
C. A. Tripp, personal communication.


ACKNOWLEDGEMENTS

We thank Prof. F. Ursini and Prof. L. Floh for allowing us to read their manuscript ((18) ) prior to publication and Dr. A. E. G. Cass for constructive criticism.


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