(Received for publication, April 24, 1995)
From the
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-
Glutathione peroxidases (GPxs) ( 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.
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 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, 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) . 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 [
Figure 1:
Secretion of gp29 from Sf cells. Sf
cells infected with the AcNPV/gp29 construct were pulsed with
[
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
Gp29 was
purified to homogeneity from culture supernatants by sequential cycles
of (NH
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.
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
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
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
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-
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) , ( 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 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 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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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-
-phosphatidylcholine hydroperoxide of 3.8 and 9.7
mM, respectively at a fixed glutathione concentration of 3
mM).
)catalyze the
reduction of hydrogen peroxide (H
O
) 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
O
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 7
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
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) .
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
)
SO
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
)
SO
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
-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-
-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-
-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 -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-
-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.
-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
.
-linolenic acid hydroperoxide, and L-
-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
.
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) .
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.
(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.
)
SO
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) .
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
O
(15) .
) was measured with different
concentrations of GSH (0.5-8 mM) as described under
``Experimental Procedures.''
as the active form. At higher pH conditions we observed a
significant increase in non-enzymatic oxidation of GSH.
values of gp29
for linolenic acid hydroperoxide and phospholipid hydroperoxide (L-
-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
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-
-phosphatidylcholine hydroperoxide, respectively, in
reactions with a GSH concentration of 3 mM. A comparison of
these K
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.
-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.
)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) .
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
O
, 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
elimination, leading to
inactivation of the enzyme.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.