From Equipe "Protéases et Vectorisation," INSERM EMI-U-0010 Université François Rabelais, Faculté de Médecine, 2 bis Boulevard Tonnellé, 37032 Tours cedex, France
Received for publication, June 2, 1999, and in revised form, January 18, 2001
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ABSTRACT |
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Rat FAD-dependent sulfhydryl oxidase
was purified; partial sequencing indicated that it was homologous to
human quiescin Q6. A cDNA (GenBankTM accession
no. AF285078) was cloned from rat seminal vesicles, and active
recombinant sulfhydryl oxidase was expressed in Chinese hamster ovary
epithelial cells. This 2472-nucleotide cDNA has an open reading
frame of 1710 base pairs, encoding a protein of 570 amino acids
including a 32-amino acid leader sequence and two potential sites for
N-glycosylation. One of them is used and the 64,000 Mr purified protein was transformed to 61,000 by the action of endoglycosidase F. Northern blotting and reverse transcription-polymerase chain reaction analyses showed that there were
small amounts of sulfhydryl oxidase in the rat testis, prostate, lung,
heart, kidney, spleen, and liver, and that the gene was highly
expressed in seminal vesicles and epididymis. Rat sulfhydryl oxidase
cDNA corresponds to the human cell growth inhibiting factor cDNA, which could be a differently spliced form of quiescin Q6. Comparing sulfhydryl oxidase sequences with those of human quiescin Q6
and mammalian and Caenorhabditis elegans quiescin
Q6-related genes established the existence of a new family of
FAD-dependent sulfhydryl oxidase/quiescin Q6-related genes
containing protein-disulfide isomerase-type thioredoxin and yeast ERV1 domains.
Sulfhydryl oxidases are enzymes that catalyze the reaction 2R-SH + O2 Sulfhydryl oxidase activity in the mammalian male genital tract was
first described in hamster epididymal fluid (9) and in seminal vesicle
fluids from rats and hamsters (9, 10). Rat sulfhydryl oxidase was
purified from seminal vesicles. It is a monomeric enzyme with an
apparent Mr of 66,000, a
pHi of 7.45, and 1 mol of noncovalently bound
FAD/mol of enzyme (14). The rat enzyme accepts a variety of small
sulfhydryl substrates including glutathione, cysteine, dithiothreitol
(DTT)1, and 2-mercaptoethanol
and can also markedly enhance the rate of renaturation of fully reduced
ribonuclease (14). Several possible functions have been proposed for
sulfhydryl oxidase in the rat male genital tract. These include the
generation of disulfide bonds in the proteins of seminal plasma or
spermatozoa, the preservation of spermatozoan membrane integrity,
antimicrobial activity (through the release of
H2O2), and the protection of spermatozoa
against the harmful effects of thiol after ejaculation (14). It has been shown recently that flavin-dependent sulfhydryl
oxidase from chicken egg white contains one redox-active cystine
bridge, and accepts a total of 4 electrons per active site (15). This
oxidase has a high catalytic activity toward reduced peptides and
proteins including insulin A and B chains, lysozyme, ovalbumin,
riboflavin-binding protein, and RNase (16).
Flavin-dependent chicken egg white sulfhydryl oxidase and
protein disulfide isomerase can also cooperate in vitro in
the generation and rearrangement of native disulfide pairings (16).
Neither protein nor nucleotide sequences were available for
FAD-dependent sulfhydryl oxidases until the partial
sequencing of chicken egg white sulfhydryl oxidase (17) (published
after the original submission of this work) and rat sulfhydryl oxidase (this work). These sequences were similar to those of four mammalian cDNAs. The first of these, encoding human bone-derived growth factor-1 (BPGF-1; GenBankTM accession no. L42379) was cloned from an
osteosarcoma cell line. The second, GEC-3 (GenBankTM accession no.
U82982), was the product of a gene whose expression is hormone-dependent in the uterine tissue of the guinea pig
(Cavia porcellus). The third, encoding the cell growth
inhibiting factor (CGIF; GenBankTM accession no. E12644) was cloned
from human lung fibroblast. The last is the product of quiescin
Q6 (GenBankTM accession no. U972760) (18), a gene that is
specifically expressed, together with collagen We have now cloned and sequenced rat seminal vesicle
FAD-dependent sulfhydryl oxidase cDNA. We believe that
this enzyme, together with quiescin Q6 and quiescin Q6-related genes,
constitutes a new family of FAD-dependent sulfhydryl
oxidase-related proteins. They contain protein-disulfide
isomerase/thioredoxin and ERV1 domains, which may assist in the
folding of a number of newly synthesized or secreted proteins.
Materials--
DTT, 5-5' dithiobis-2-nitrobenzoic acid (DTNB),
glutathione and papain were purchased from Roche Molecular
Biochemicals (Meylan, France). The papain (EC 3.4.22.2) was
repurified on a Mono-S column (Amersham Pharmacia Biotech) to
remove the glycyl-endopeptidase (EC 3.4.22.25) which usually
contaminates the commercial enzyme (20). Z-Phe-Arg-NH-Mec (Bachem,
Bubendorf, Switzerland) was prepared as a 10 mM stock
solution in 10% dimethylformamide. E-64 was obtained from the Peptide
Research Institute (Osaka, Japan). H2O2 was
supplied by Sigma. All other reagents were of analytical grade.
Catalase was prepared from frozen rat liver.
Purification and Sequencing of Sulfhydryl Oxidase from Rat
Seminal Vesicle Fluid--
Seminal vesicle fluid from Wistar rats was
collected directly (21). It was diluted in four volumes of 0.01 M potassium phosphate buffer, pH 6.8, and homogenized in an
Ultraturrax. The fraction of the centrifuged homogenate precipitated by
40-70% (NH4)2SO4 was redissolved and dialyzed
against 0.05 M Tris-HCl, pH 8.0, 0.15 M NaCl
and then chromatographed on carboxymethylated papain-Sepharose to
eliminate rat cystatin C, a known inhibitor of lysosomal cysteine proteinase inhibitors (22). Rat cystatin C was detected
immunochemically in the proteins eluted with phosphate buffer pH 12.0. Most of the papain inhibitory activity was in the flow-through of the column. The unbound material was equilibrated with 1.5 M
(NH4)2SO4 in 0.01 M potassium
phosphate buffer, pH 6.8, and loaded on to a phenyl-Sepharose column
(1.1 × 7.0 cm) (Amersham Pharmacia Biotech). Proteins were eluted
with a gradient of (NH4)2SO4 decreasing from 1.5 to 0.0 M. The column was then washed with 30% ethylene
glycol and the inhibitory activity eluted with a gradient of urea
(0.0-6.0 M) in 0.01 M potassium phosphate
buffer, pH 6.8. The inhibiting fractions were dialyzed against 0.01 M potassium phosphate buffer, pH 6.0, and loaded onto a
hydroxyapatite column (2.1 × 7 cm) (Bio-Rad). Proteins were
eluted by a potassium phosphate, pH 6.0, gradient from 0.01 to 0.20 M. The purity of the fractions was checked by reverse-phase
chromatography using an Aquapore BU300 (C4) cartridge (2.1 × 30 mm; Applied Biosystems) and an acetonitrile gradient (0-60%) in
0.075% trifluoroacetic acid. The molecular mass of the eluted peak was
determined by MALDI-TOF mass spectrometry using a Brücker reflex
mass spectrometer. The amino acid sequence was determined on an Applied
Biosystems 477A pulsed liquid sequencer using the chemicals and
programs recommended by the manufacturer. Phenylthiohydantoin
derivatives were identified using an on-line Applied Biosystems model
120A analyzer. Rat sulfhydryl oxidase cleavage was carried out on the
reduced and pyridylethylated enzyme (23) after purifying by
reverse-phase chromatography on an Aquapore BU300 (C4) cartridge using
an acetonitrile gradient (0-60%) in 0.075% trifluoroacetic acid.
CNBr cleavage was performed as previously described (24). The resulting
material was either analyzed directly by reverse-phase chromatography
on Aquapore BU300 or submitted to endoproteinase Lys-C, used according
to the manufacturer's directions (Roche Molecular Biochemicals),
followed by reverse-phase chromatography.
Deglycosylation of Rat Sulfhydryl Oxidase--
Rat sulfhydryl
oxidase was deglycosylated using endoglycosidase
F/N-glycosidase F (Sigma) at concentrations of 0.08-2.5
units/ml. Fractions were incubated overnight in 20 mM
phosphate buffer, pH 6.5, 10 mM EDTA, 1% Triton X-100,
0.2% SDS in the presence of 1% 2-mercaptoethanol and then analyzed by
SDS-PAGE.
Papain Inhibiting Activity--
Purified papain was titrated
against E-64 (25). Papain (0.01 µM range) and sulfhydryl
oxidase (nanomolar range) were incubated for 30 min at 30 °C in 0.1 M phosphate buffer, pH 6.8, 1 mM EDTA, 0.1%
Brij, and 2 mM DTT in the presence or not of catalase
(about 50 nM). The residual enzyme activity was measured
with Z-Phe-Arg-NH-Mec (5 µM final) on a Dynatech
Microfluor reader or a F-2000 Hitachi spectrofluorimeter.
Sulfhydryl Oxidase Assays--
Dithiotreitol (2 mM)
was oxidized in 0.1 M Tris-HCl buffer, pH 8.0, at 25 °C.
Aliquots (7 µl) of the incubation mixture were removed and added to
650 µl of 0.54 mM DTNB in the same buffer, and the
absorbance at 412 nm was measured (26).
Total RNA Extraction, Poly(A)+ RNA Purification, and
Northern Blot--
Total RNA was extracted from Wistar rat tissues
using the TriReagent method (Euromedex, France) following the
manufacturer's instructions. Poly(A)+ RNA was isolated
using oligo(dT) (27). For Northern blot analysis, total RNA (20 µg)
or poly(A)+ RNA from rat seminal vesicles (1 µg) was
separated on 1% agarose gel, transferred to a nylon membrane, and
prehybridized for 1 h at 68 °C with the QuickHyb hybridization
solution (Stratagene). A 300-bp probe was generated from the
3'-untranslated region of rat SOx cDNA by PCR using the forward
primer (5'-AACATCGTCAGAGAC-3') and the reverse primer
(5'-AGCTGGGTAGGCCAGAGAA-3'). One hundred nanograms of this probe was
[ Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Analysis--
The first cDNA strand was synthesized from 5 µg of
total RNA prepared from various rat tissues using Moloney murine
leukemia virus reverse transcriptase (Promega) and
oligo(dT)17 primer following the manufacturer's
instructions. At the end of reverse transcription, the reaction mixture
(20 µl) was diluted to 1000 µl with 10 mM Tris, pH 7.5, 1 mM EDTA and 1 µl of this solution was used to amplify a
400-bp SOx fragment or a 800-bp GAPDH fragment (internal standard). The sequences of the 5' and 3' primers for SOx were 5'-ACTTGAGCGAGGTGGACAGTCAAG-3' and 5'-AGCACAGGCACTCGGGAA-3'; for GAPDH
they were 5'-AGTTCAACGGCACAGTCAAGGCTGAGAAT-3' and
5'-GAGGGGCCTCCACAGTCTTCTGAGTGGC-3'. Amplification was performed
in a Progene thermocycler (Techne) using Platinum®
Pfx DNA polymerase (Life Technologies, Inc.). After one
cycle at 94 °C for 2 min, 62 °C for 2 min, and 68 °C for 40 min for second strand synthesis, the thermocycling parameters were 30 cycles of amplification at 96 °C for 10 s, 59 °C for 30 s, and 68 °C for 90 s. Ten microliters (1/5 of PCR products)
were analyzed on 1% agarose gel. Expression of SOx mRNA was
quantified by densitometry and corrected using GAPDH as internal standard.
cDNA Cloning of Rat SOx, Sequencing, and Plasmid
Construction--
The 5' end of the rat SOx cDNA was generated as
follows. One microgram of poly(A)+ mRNA from rat
seminal vesicles was first reverse transcribed using a reverse primer
5'-GCGAAGAACTCCACCGCCCAG-3'. This primer was synthesized on a conserved
5' region of human quiescin Q6, human BPGF-1, and guinea pig GEC-3
cDNAs. After addition of a poly(C) tail to the first cDNA
strand with terminal deoxynucleotide transferase (28), cDNA
amplification was performed with the same reverse primer and an
oligo(dG) primer, using the Expand Long Template PCR system (Roche
Molecular Biochemicals). The thermocycling parameters were: one cycle
at each of 95 °C for 5 min, 55 °C for 2 min, and 68 °C for 40 min; and 20 cycles at 94 °C for 10 s, 63 °C for 30 s,
and 68 °C for 1 min. The 300-bp amplified product was purified by
agarose gel electrophoresis and cloned into a pGEM®T
vector (Promega). DNA constructions were transformed into XL2-Blue Epicurian Coli ultracompetent cells (Stratagene). The two cDNA strands were sequenced with a ThermosequenaseTM II dye terminator cycle
sequencing kit (Amersham Pharmacia Biotech) in an ABI PRISM 377 DNA
sequencer (PerkinElmer Life Sciences). Based on the sequence of this 5'
end fragment, a specific 5' primer, 5'-ACTTGAGCGAGGTGGACAGTCAAG-3', was
synthesized to generate the full-length rat sulfhydryl oxidase cDNA. The reverse transcription reaction was performed from 1 µg
of rat seminal vesicle poly(A)+ RNA with an
oligo(dT)17 primer for 1 h at 42 °C. Amplification was performed as described in Ref. 28 with 1/1000 of the reverse transcription product, in the presence of an oligo(dT) primer and the
specific 5'-primer. Cycling parameters were one cycle at 95 °C for 5 min, 58 °C for 2 min and 68 °C for 40 min; 30 cycles at 94 °C
for 10 s, 60 °C for 30 s and 68 °C for 4 min; and one final cycle with an elongation step at 68 °C for 15 min. The
2.5-kilobase pair product corresponding to the full-length rat SOx
cDNA was then cloned in pCR3.1 T/A cloning vector (Invitrogen) to
generate pCR3.1-rSOx plasmid; it was sequenced with forward and reverse rSOx-specific primers. The pcDNA/3.1/LacZ/V5-His-TOPO (pcDNA-3-LacZ) came from the Eukaryotic TOPO TA cloning kit (Invitrogen).
Cell Culture and Transfections--
The Hamster ovary epithelial
cell strain CHO was maintained in 10% fetal calf serum (Eurobio,
France) in Dulbecco's modified Eagle's medium (DMEM) (Eurobio)
supplemented with 100 µg/ml each penicillin and streptomycin
(Eurobio) and 2 mM L-glutamine (Eurobio). Transfection was performed using the electroporation method. Briefly, subconfluent CHO cells were trypsinized and washed twice in DMEM supplemented with 10% fetal calf serum. Cells (2.4 106
cells in 400 µl of culture medium) with 10 µg of pcDNA-3-LacZ or pCR3.1-rSOx plasmid were then subject to electroporation at 150 V
for 25 ms in a ECM399 electroporator (BTX, San Diego, CA). They were
then plated in 75-cm2 flasks. Forty-eight hours after
transfection, the cells were passaged and the medium was supplemented
with 1 mg/ml G418. After 2 weeks in this selective medium, resistant
colonies were selected, passaged for 48 h in DMEM supplemented
with 2% fetal calf serum, and analyzed for expression and enzymatic
activity of SOx recombinant protein or for activity of
Immunoprecipitation, Western Blot, and Activity of Recombinant
Enzyme--
One hundred microliters of stably transfected CHO 10×
concentrated supernatant were incubated in 500 µl of
immunoprecipitation buffer (20 mM Tris, pH 7.5, 0.25 M NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% SDS,
1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride) with 5 µl of pre-immune or immune rat
seminal vesicle SOx serum. After gentle shaking at 4 °C overnight,
50 µl of 50% (v/v) Protein A-Sepharose (Amersham Pharmacia Biotech)
was added for an additional 2 h. Protein A-Sepharose was then
washed four times in immunoprecipitation buffer, resuspended in Laemmli
sample buffer, and heated for 3 min at 100 °C (29).
Immunoprecipitation was analyzed by Western blot with immune rat
seminal vesicle sulfhydryl oxidase serum after electrophoresis on a
12% polyacrylamide gel and transfer to nitrocellulose membranes.
Immunoblots were revealed by chemiluminescence with the detection
system RPN 2106 (Amersham Pharmacia Biotech). A concentrated
supernatant (10×) of CHO transfected cells was used to measure
recombinant sulfhydryl oxidase activity, as described previously.
Activity of 75 ng of native protein measured in 10× concentrated DMEM
supplemented with fetal calf serum was used as a control.
Purification and Sequencing of Rat FAD-dependent
Seminal Vesicle Fluid Sulfhydryl Oxidase--
While purifying the
specific inhibitors of lysosomal cysteine proteinases from seminal
vesicle fluid, using papain as target enzyme, we found a hitherto
unreported inhibitory activity that was not bound to carboxymethylated
papain Sepharose (data not shown). This was unusual behavior for any
known specific inhibitor of the cystatin superfamily (30). The unbound
papain inhibitory activity was fractionated by hydrophobic
chromatography. The activity was not eluted by either a decreasing
gradient of ammonium sulfate or by 30% ethylene glycol, but it was
eluted by an increasing gradient of urea without detectable loss of
activity. The inhibitory activity was further fractionated on
hydroxyapatite gel using a phosphate buffer gradient. The activity
eluted with the yellow retained fractions. One peak was at 39 min by
reverse-phase chromatography on Aquapore BU-300 but there was no
inhibitory activity in the eluted peak. It corresponds to a protein
with a Mr of 64,000 by SDS-PAGE and of 64,624 by
mass spectrometry (Fig. 1). These
molecular properties were similar to those of the previously described
rat sulfhydryl oxidase (EC 1.8.3.2), an androgen-independent protein in
seminal vesicle fluid that catalyzes the oxidation of sulfhydryl groups
to disulfides with the reduction of oxygen to hydrogen peroxide (11,
14). Since papain activity must be analyzed under reducing conditions,
usually in 2 millimolar DTT, the free thiol content of the reaction
mixtures in the presence of inhibitory fractions was measured using
DTNB. DTT progressively disappeared from the medium as a function of
time until it had all been consumed. The parallel production of
H2O2 was demonstrated by the appearance of
luminescence in the presence of luminol and peroxidase (data not
shown), and this luminescence was totally abolished by incubation with
rat liver catalase. The indirect inhibition of papain by sulfhydryl
oxidase was partially reversible, because only a fraction of the
inactivated papain could be reactivated when placed under reducing
conditions (2 mM DTT) (Fig.
2). This inactivation is explained by the
direct effect of H2O2 on protease, because
papain remains active when sulfhydryl oxidase acts in the presence of catalase (Fig. 2).
Direct sequencing of the unreduced or of the pyridylethylated rat
sulfhydryl oxidase gave a sequence of 25 amino acids
(ARLSVLYS(F/S)ADPLTLLDADTVRGAV). The pyridylethylated protein was
therefore submitted to CNBr cleavage and reverse-phase chromatography
(Aquapore BU 300). One narrow peak, eluting at 30.4 min, gave the
sequence XXVGSPNAAVLXLXI. Finally,
endoproteinase Lys-C cleavage was performed directly on the CNBr
cleavage product. A reverse-phase chromatography peak at 33.5 min gave
the sequence RLIDALESHRDTWPPACPXL. These amino-terminal and
internal sequences of the rat sulfhydryl oxidase did not correspond to
any protein sequence in data banks and were therefore the first elements of sequence of FAD-dependent sulfhydryl oxidase.
However, they were similar to the amino acid sequences derived from two human cDNAs, BPGF-1 and quiescin Q6 (hQ6) and to the amino acid sequence derived from the guinea pig (C. porcellus) GEC-3
gene product. Bone-derived growth factor-1 was cloned from an
osteosarcoma cell line. Quiescin Q6 is a gene that is expressed in
confluent cultures of human lung fibroblasts (19). The expression of
the GEC-3 gene is hormone-dependent in the uterine tissue
of guinea pig. We therefore decided to clone the rat sulfhydryl oxidase cDNA
Molecular Cloning of Rat Seminal Vesicle FAD-dependent
Sulfhydryl Oxidase--
A two-step cloning strategy was developed to
obtain the complete cDNA of sulfhydryl oxidase from seminal
vesicles using a primer based on the homology between the
amino-terminal sequence of the rat enzyme and the deduced open reading
frame of human quiescin Q6, guinea pig GEC3, and human BPGF-1 cDNA
(as described under "Experimental Procedures"). The rat 2472-bp
sulfhydryl oxidase cDNA contains a polyadenylation signal after the
stop codon (Fig. 3) showing that it codes
for a full-length sulfhydryl oxidase. This cDNA has an open reading
frame of 1710 bp coding for 570 amino acids. The deduced protein
contains a 32-amino acid putative signal peptide, which leads to a
mature protein with a calculated Mr of 60,035, a
value distinct from that of the native enzyme, which is 64,624 by mass
spectrometry (Fig. 1). Compared with the deduced protein sequence, the
higher mass of the native form is probably due to the carbohydrate
content of the mature protein, which is glycosylated, as seen in Fig.
1. However, only one of these two putative N-glycosylation
sites seems to be used in vivo, as observed by the
modification of SOx electrophoretic mobility on SDS-PAGE after
incubation with increasing concentrations of endoglycosidase F (data
not shown). The deduced amino acid sequence of rat sulfhydryl oxidase
contains all the amino acid sequences of the three peptide fragments
obtained from the native seminal vesicle enzyme (Fig. 3). Moreover, the
NH2 amino acid sequence of native protein, which started by
the ARLSV sequence, confirms the presence of a 32-amino acid cleavable
signal peptide, which is released in the mature secreted sulfhydryl
oxidase protein.
Rat FAD-dependent Sulfhydryl Oxidase Defined a New Gene
Family Including Human Quiescin Q6--
A search in the GenBankTM data
base showed a strong homology between nucleotide sequences of rat
sulfhydryl oxidase cDNA and several cDNA sequences including
human quiescin Q6, human cell growth inhibiting factor, human
bone-derived growth factor-1, and guinea pig GEC-3. Alignment of
sulfhydryl oxidase, hQ6, and GEC-3 cDNAs nucleotide sequences
reveals 80% identity in their coding regions (data not shown). This is
confirmed by amino acid sequence alignment (Fig.
4), where rat sulfhydryl oxidase is
74.6% identical to human quiescin Q6 and 67.5% identical to GEC-3. On the other hand, the 3' nucleotide noncoding sequences are divergent. Human quiescin Q6 has a 728-bp longer 3' end than rat sulfhydryl oxidase, human CGIF, and guinea pig GEC-3 cDNAs (Fig.
5). However, there is a great similarity
in the 3' end of rat SOx and other cDNAs with conservation in the
sequence surrounding the polyadenylation motif (Fig. 5). Rat sulfhydryl
oxidase and human Q6 have the same open reading frame starting with a
putative signal sequence. Two conserved putative
N-glycosylation motifs are also found in these NH2 regions (Fig. 4). Rat SOx, hQ6, and guinea pig GEC-3
contain a thioredoxin domain (amino acids 38-127) characterized by a
WCGHC PDI-type motif inserted in the well conserved
SAWAVEFFASWCGHCIAFAPTWK sequence and an ERV1 domain (amino acids
384-515) containing the CRDCA motif in the conserved FFGCRDCANHFEQM
sequence (Fig. 4) (17, 18). Three genes related to human quiescin Q6,
CeQ6r1 (GenBankTM accession no. Z69637), CeQ6r2 (GenBankTM accession no. U80848), and CeQ6r3 (GenBankTM accession no. U39646) from
Caenorhabditis elegans containing these PDI-like and ERV1 domains are also members of this family (18). The homologies found
among these deduced protein sequences associated with the sulfhydryl
oxidase activity of rat protein favor the definition of a new
sulfhydryl oxidase/quiescin Q6 gene family characterized by the
original juxtaposition of thioredoxin and ERV1 domains.
Expression and Enzymatic Activity of Recombinant Rat Sulfhydryl
Oxidase in CHO Cells--
The rat sulfhydryl oxidase cDNA was
stably expressed in CHO epithelial cells. This cell line, which has no
endogenous sulfhydryl oxidase secretion (Fig.
6A), was transfected with the
pCR3.1-rSOx plasmid. After 2 weeks in G418 selective medium, resistant
colonies were selected and serially passaged. Four stably transfected
colonies were assayed for recombinant sulfhydryl oxidase expression.
Recombinant protein was immunoprecipitated from transfected cell
supernatants or whole cell extracts with immune rat sulfhydryl oxidase
serum and subjected to Western blot analysis. Immunoprecipitation
revealed a Mr 64,000 band in the supernatant of
rSOx-transfected CHO cells, which was lacking in the supernatant of
nontransfected cells and pcDNA3.1-LacZ-transfected cells (Fig.
6A). The electrophoretic mobility of the recombinant protein
was identical to that of native enzyme from seminal vesicles. Finally,
any expression of rat recombinant rat sulfhydryl oxidase protein was
observed in immunoprecipitation from transfected whole cell extracts
(data not shown); this confirms that sulfhydryl oxidase protein is
mainly secreted.
Oxidative activity of recombinant protein in the supernatant of stably
transfected CHO cells was measured using DTT as a substrate (Fig.
6C). Sulfhydryl oxidase activities correlated with the
expression level of recombinant protein, as shown in the Western blot
experiment (Fig. 6A). Activity due to recombinant protein in
clone 4 supernatant corresponded to the activity of 75 ng of pure
native enzyme measured in the same experimental conditions. These data
show that recombinant sulfhydryl oxidase secreted by stably transfected
CHO cells displays enzymatic activity similar to that of rat seminal
vesicle fluid enzyme.
Rat Sulfhydryl Oxidase mRNA Is Highly Expressed in the Male
Reproductive Tract--
To study the expression pattern of sulfhydryl
oxidase mRNA, a Northern blot experiment was performed on total RNA
derived from different rat tissues. As shown in Fig.
7A, the size of the messenger
was around 2600 nucleotides, a value in agreement with the 2472 bp of
the cloned cDNA. Only 4 h of autoradiography were necessary to
detect a strong signal in seminal vesicles and a weaker signal in
epididymis, showing a very high and high level of expression,
respectively, in those tissues. Exposure for a week is needed to detect
presence of sulfhydryl oxidase mRNA in other tissues (data not
shown). To confirm the Northern blot analysis and to create a more
sensitive detection, the RT-PCR experiment was conducted using specific
rat sulfhydryl oxidase and GAPDH (internal control) primers on total
RNA isolated from different rat tissues (Fig. 7, B and
C). These data showed a predominant sulfhydryl oxidase
mRNA expression in seminal vesicles and epididymis but also a lower
basal expression in prostate, kidney, testis, heart, liver, spleen, and
lung.
FAD-dependent sulfhydryl oxidases have previously been
identified and characterized by their enzymatic activities and their biochemical properties in several tissues and species including rat
seminal vesicles (9, 11, 14), hamster seminal vesicles (10), chicken
egg white (13), and human platelets (31). Although sulfhydryl oxidases
were thought to be members of the pyridine nucleotide disulfide
oxidoreductases (32), no data on the nucleotide or amino acid sequences
of these molecules were available in the literature. Purification and
partial sequencing of rat FAD-dependent seminal vesicle
fluid sulfhydryl oxidase and molecular cloning of the corresponding
cDNA, indicated a great similarity at the nucleotide and the amino
acid levels with human quiescin Q6 and guinea pig GEC-3 cDNA. All
three deduced protein sequences possessed a putative peptide signal,
two conserved potential N-glycosylation sites, and putative
thioredoxin and ERV1 domains. Such structural homologies among these
different molecules argue strongly in favor of a new gene family and
confirm the preliminary work of Hoober et al. (17) on egg
white sulfhydryl oxidase and quiescin Q6. This family also includes
CeQ6-related sequences of C. elegans and
Drosophila (18). Because the quiescin behavior (i.e. induction of gene expression that takes place when
cells enter the quiescent phase) seems thus far to be restricted to human quiescin Q6 cDNA (19, 33), this family should be named the
sulfhydryl oxidase/quiescin Q6 family until the enzymatic activity of
its members, and their behavior during the proliferative cycle, has
been fully determined.
Two quiescin Q6 transcripts of 2500 and 3200 nucleotides have
simultaneously been found in the WI38 human lung fibroblast cell line
(where only the longer form has been cloned) (18) and are also present
in several of the cell lines or tissues analyzed (33). Furthermore, two
human quiescin Q6-related cDNAs, bone-derived growth factor-1 and
cell growth inhibiting factor, have also been independently cloned;
they show 98% identity with human quiescin Q6, although a few
differences cause modifications in their putative open reading frames.
These differences could be due to PCR or sequencing errors as well as
to real gene modifications. The 3228-nucleotide BPGF-1 cDNA was
cloned from an osteosarcoma cell line and seems to correspond to
quiescin Q6 cDNA (3298 nucleotides). The 2500-nucleotide CGIF
cDNA was isolated from another lung fibroblast cell line (MRC-5),
and but it lacks a 728-nucleotide segment (1879-2606 of quiescin Q6)
in its 3'-noncoding sequence. CGIF cDNA could therefore represent
an alternative transcript of quiescin Q6. Rat sulfhydryl oxidase
cDNA (2472 nucleotides), which lacks the same 3'-noncoding
728-nucleotide segment, appears therefore to be the homologue of human
cell growth inhibiting factor cDNA.
Analysis of the expression pattern of rat sulfhydryl oxidase by
Northern blotting indicated that the gene is actively expressed in the
epididymis and seminal vesicles. This was confirmed by RT-PCR and agree
with the distribution of sulfhydryl oxidase activity in the rat and
guinea pig tissues, showing low activity in testis, more activity in
the epididymis, and the greatest activity in seminal vesicles, but no
activity in muscle, brain, kidney, liver, or lung tissue (9, 10). On
the other hand, RT-PCR and Northern blot revealed low expression of the
rat sulfhydryl oxidase gene in all the tissues analyzed. This
corresponds to the wide variety of human tissues expressing human
quiescin Q6, including the heart, placenta, lung, liver, skeletal
muscle, and pancreas (33). Therefore, apart from the high concentration
of sulfhydryl oxidase in the male reproductive tract of rodents,
sulfhydryl oxidase and quiescin Q6 appear to be ubiquitous.
Although this molecule is clearly a secretory protein, sulfhydryl
oxidase immunoreactivity has also been found in the matrix of the
mitochondria of certain human, rat, and hamster testicular cells at
specific stages of functional activation (34-36). In rat and hamster,
this immunoreactivity appears in pachytene spermatocytes at stage I
(34). In photoperiodically-induced testicular involution in the
Djungarian hamster, immunoreactivity reappeared during recrudescence,
when the first spermatogenic wave had reached the pachytene stage (37).
In mature human testis, moderate sulfhydryl oxidase immunoreactivity
has been found in Leydig cells and spermatogonia and in pachytene
spermatocytes (36). Sulfhydryl oxidase also seems to be associated with
hypospermatogenesis and impaired fertility (38). Sulfhydryl oxidase
appears therefore to be implicated in the functional or the activation
state of mitochondria, at least in the cells of the mammalian male
reproductive tract.
The ERV1 gene is essential for mitochondrial biogenesis and the
survival of Saccharomyces cerevisiae cells. ERV1 is found in
the cytosol and mitochondria of the yeast cell where it plays an
essential role in normal mitochondrial morphology and the stability of
these organelles. ERV1p has been recently shown to be a FAD-linked sulfhydryl oxidase (39). Its enzymatic activity is supported by the
CXXCA redox disulfide motif located in the COOH-terminal domain of the protein (39). The similarity of this motif to the CRDCA
motif present in rat sulfhydryl oxidase, in human quiescin Q6 and
guinea pig GEC-3, and to the CQECA motif of chicken egg white
sulfhydryl oxidase (17) confirms the functional importance of the ERV1
motif in sulfhydryl oxidase activity and in FAD binding (17). However,
a difference in FAD attachment strength could be seen because FAD is
removed from ERV1p by 6 M urea, whereas rat sulfhydryl
oxidase is stable without any loss of FAD or activity in 6 M urea. A difference in catalytic activity is also found because ERV1p is able to oxidize only protein thiols, whereas rat and
chicken sulfhydryl oxidases are able not only to oxidize protein thiols
but also low molecular weight thiols including glutathione.
Nevertheless, independently of enzyme substrate, a sulfhydryl oxidase
activity seems to be essential for cellular processes involving
mitochondria in lower and higher eukaryotes.
Although rat sulfhydryl oxidase has been shown to enhance
markedly the renaturation of fully reduced ribonuclease (14, 16) or the
reoxidation of different proteins
(16),2 which is a major
property of protein-disulfide isomerase (40), its biological role in
the secretions as well as in mitochondria is still unknown. The
increased expression of quiescin Q6 as cultured lung fibroblasts enter
quiescence, together with collagen The number of molecules possibly showing sulfhydryl oxidase activity is
increasing. It includes the ancient rat, hamster, and chicken egg white
sulfhydryl oxidases, and their partial sequencing has led to the
identification of other members of this new family (human quiescin Q6
and cell growth inhibiting factor, hamster GEC-3, Q6-related gene
products of C. elegans or Drosophila) for which
possible sulfhydryl oxidase activity has yet to be demonstrated. Their
structure includes a PDI-like thioredoxin and a ERV1 domain. Yeast
ERV1p has been recently described as a protein
FAD-dependent sulfhydryl oxidase; its homologue, human
augmenter of liver regeneration (ALRp) is a mammalian hepatic growth
factor that can functionally substitute for ERV1 in yeast (41) and is
also probably a sulfhydryl oxidase. Therefore sulfhydryl oxidases are
essential molecules in lower and higher eukaryotes implicated in
mitochondrial function as well as in the cellular growth regulation,
and could be important in pathological states such as cancer development.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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R-S-S-R + H2O2. There are at
least three families of these enzymes, each depending on different
cofactors for their catalytic activity. They are
iron-dependent sulfhydryl oxidases (1-4),
copper-containing enzymes (5-8), and FAD-dependent
enzymes. The last of these have been found in the male rat genital
tract (9-11), in fungi (Aspergillus) (12), and in chicken
egg white (13).
chains, decorin, and
complement C1r, as cultured human lung fibroblasts begin to leave the
proliferative cycle and enter quiescence (19). The quiescin Q6 gene has
evolved by the fusion of two ancient genes, those for thioredoxin and ERV1 (18). The protothioredoxin gene first evolved into thioredoxin and
protein-disulfide isomerase (PDI) genes; then the quiescin Q6 form
diverged from the PDI form (18).
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-32P]dCTP random primer-labeled and added to
prehybridization buffer. Hybridization was performed overnight at the
same temperature. Membranes were washed at 68 °C for 20 min in 2×
SSC, 0.1% SDS and twice for 30 min at 68 °C in 0.1× SSC, 0.5%
SDS. They were then exposed to Kodak AR x-ray film at
70 °C using
intensifying screens for 4 h to 7 days. RNA markers (Promega) were
used to determine the size of SOx transcript.
-galactosidase on lacZ-transfected cells.
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Fig. 1.
Molecular mass determination of purified and
deglycosylated rat seminal vesicle FAD-dependent sulfhydryl
oxidase. A, SDS-PAGE analysis of purified sulfhydryl
oxidase. Mr St, molecular weight markers; SOx,
purified sulfhydryl oxidase (1 µg); endo F ,
purified sulfhydryl oxidase (2 µg) in deglycosylation buffer for
16 h at 37 °C; endo F+, purified
sulfhydryl oxidase (2 µg) in presence of 0.4 unit/ml endoglycosidase
F/N-glycosidase F in deglycosylation buffer for 16 h at
37 °C. B, MALDI-TOF mass spectrometry of rat seminal
vesicle fluid sulfhydryl oxidase, after reverse-phase high pressure
liquid chromatography on Aquapore BU300 of hydroxyapatite fractions,
using a Brücker reflex mass spectrometer (RI, relative
intensity).
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Fig. 2.
Effect of rat sulfhydryl oxidase on papain
activity. a, activity of papain incubated with sulfhydryl
oxidase. Papain (0.01 µM) was incubated with sulfhydryl
oxidase in 0.1 M phosphate buffer, pH 6.8, 1 mM
EDTA, 0.1% Brij 35, and 2 mM DTT at 37 °C. Activity of
the papain (0.25 nM final) was analyzed at different times
with 5 µM Z-Phe-Arg-NH-Mec. Fluorescence is expressed as
arbitrary units (AU). b, activity of papain
incubated with sulfhydryl oxidase in presence of catalase.
c, concentration of H2O2 produced by
the action of sulfhydryl oxidase on DTT. H2O2
concentration was deduced from the free sulfhydryl concentration
determined using DTNB. The H2O2 produced was
calculated on the basis of 1/2 H2O2 per R-SH
consumed by sulfhydryl oxidase.
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Fig. 3.
Nucleotide and deduced amino acid sequences
of rat sulfhydryl oxidase. The rat sulfhydryl oxidase open reading
frame contains 1710 bp between ATG (bp 50-52) and TGA (1760-1762)
coding for a 570-amino acid protein. The predicted amino acid sequence
of rSOx contains a signal peptide (amino acids 1-32, according to rat
sulfhydryl oxidase numbering). Arrow indicates the first
residue of mature purified enzyme as determined by
NH2-terminal sequencing. Sequences corresponding to the
amino-terminal and internal amino acid sequences are
underlined. The poly(A) signal (bp 2418-2423) is underlined
too. Potential N-glycosylation sites are
boxed.
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Fig. 4.
Alignment of rat sulfhydryl oxidase amino
acid sequence with human quiescin Q6, human BPGF-1, and guinea pig
GEC-3 amino acid sequences. The residues conserved in all the four
proteins are designated by an asterisk. The amino acids
conserved among three of the sequences are shaded
gray. The CGHC PDI-type thioredoxin motif and the CRDC redox
active disulfide bridge are underlined. The two
N-glycosylation motifs are boxed. The sequences
were aligned using the Clustal W (1.5) multiple sequence alignment
program.
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Fig. 5.
Comparison of the structure of sulfhydryl
oxidase cDNA with human quiescin Q6, human cell growth inhibiting
factor, and guinea pig GEC-3 cDNAs. Graphical representation
of alignment between rat SOx, human hQ6, human CGIF, and guinea pig
GEC-3 cDNAs using BLASTN 2.0.14 protein data base search program.
Black areas correspond to more than 75% identity
and dashed areas to nonhomologous sequences.
Polyadenylation signal is absent from GEC-3 cDNA.
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Fig. 6.
Expression and enzymatic activity of
recombinant SOx protein in stably transfected CHO cells. A,
Western blot analysis of recombinant SOx using antibodies raised
against rat SOx. Purified native enzyme (15 ng) was used as a control
of electrophoretic mobility (lane 1).
Lanes 2-7 correspond to immunoprecipitated
products (see "Experimental Procedures") from 100 µl of 10×
concentrated supernatant of nontransfected CHO cells (lane
2), pcDNA-3-LacZ-transfected cells (lane
3), and pCR3.1-SOx-transfected cell clones 1-4
(lanes 4-7 respectively). B,
recombinant protein from clone 3 was immunoprecipitated using preimmune
serum and Western blot analysis was performed with immune serum as a
specificity control. C, Enzymatic activity of recombinant
SOx. Initial velocity of sulfhydryl oxidase reaction is expressed as
the relative rate of DTT oxidation using the activity of 75 ng of
native protein measured in 10× concentrated DMEM supplemented with
fetal calf serum as a reference. Samples 1-7 are
the same as those in A (conditions of reaction are detailed
under "Experimental Procedures"). Each value represents the mean of
three distinct determinations ± S.D.
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Fig. 7.
Expression of sulfhydryl oxidase in rat
tissues. A, Northern blot analysis of tissue expression
of SOx. SOx mRNA in rat tissues was analyzed using a probe
corresponding to the 3'-untranslated region of SOx. One microgram of
seminal vesicles poly(A)+ RNA and 20 µg of total RNA of
other tissues were loaded per lane (exposition 4 h). B,
RT-PCR analysis of SOx in tissues. RT-PCR was performed on 5 µg of
total RNA from different rat tissues using primers specific for rat SOx
and GAPDH (internal standard); 10 µl of RT-PCR product were separated
on 1% agarose gel. For clarity, and quantification, images from the
ethidium bromide stained agarose gels are inverted. C,
quantification of SOx mRNA in rat tissues. The amounts of SOx
mRNA expression level were corrected using GAPDH as internal
standard and are represented as the means of values expressed as
percentage of the maximum ratio (SOx/GAPDH in seminal vesicles)
considered as 100%.
DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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chains, decorin and complement
C1r (19), and the extracellular location of quiescin Q6 in quiescent
but not in proliferating cells (33), are indicative of a role for this
potential sulfhydryl oxidase in extracellular matrix metabolism as
cells begin to leave the proliferation state. These distinct locations
of members of sulfhydryl oxidase/quiescin Q6 family could reflect
different functions for these proteins. The identification of their
biological substrates is therefore of importance.
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ACKNOWLEDGEMENTS |
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We thank V. Schubnel Courjou for animal handling, M. Brillard-Bourdet for the sequence analysis, and M. Ferrer-Di Martino for the mass spectrometry determination. The English text was edited by Dr. Geoff Watts.
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FOOTNOTES |
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* This work was supported by a grant from the Ligue Nationale Contre le Cancer (Comité du Cher).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/ EBI Data Bank with accession number(s) AF285078.
These authors contributed equally to this work (A. E.-F. for
the sulfhydryl oxidase purification and characterization and B. B. for the cloning, sequencing, and expression of sulfhydryl oxidase cDNA).
§ To whom correspondence should be addressed: Laboratoire "Enzymologie et Chimie des Protéines," INSERM EMI-U0010, Faculté de Médecine, 2 bis Blvd. Tonnellé, 37032 Tours cedex, France. Tel.: 33-2-47-36-62-06; Fax: 33-2-47-36-60-46; E-mail: esnard@univ-tours.fr.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010933200
2 A. Esnard-Fève and F. Esnard, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: DTT, dithiothreitol; BPGF-1, human bone-derived growth factor-1; CGIF, cell growth inhibiting factor; DTNB, 5,5'-dithiobis-2-nitrobenzoic acid; E-64, [L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane]; ERV-1, essential for respiration and viability-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GEC-3, guinea pig endometrial cell-3; hQ6, human quiescin Q6; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; PDI, protein-disulfide isomerase; PAGE, polyacrylamide gel electrophoresis; SOx, sulfhydryl oxidase; Z-Phe-Arg-NH-Mec, carbobenzoxy-L-phenylalanyl-L-arginine-4-methylcoumarinyl-7-amide; Mec, 4-methyl-7-coumarylamine; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s).
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