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From the * AFSSA Nancy, Unité de Recherches
sur la Rage et les Maladies Emergentes, Malzéville, France; and the
Department of Medical Chemistry, University of
Szeged, Szeged, Hungary.
| Correspondence to: F. Boué, AFSSA Nancy, Domaine de Pixerecourt, BP 9, F-54220 Malzéville, France (e-mail: f.boue{at}afssa.fr). |
| Received for publication July 2, 2004; accepted for publication December 8, 2004. |
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Abstract |
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Key words: Sperm antigen, fox, tissue specificity, immunocontraception
Identification of fox (Vulpes vulpes) sperm proteins has been carried out to provide potential target proteins for a contraceptive vaccine (Bradley, 1994). Indeed, the European red fox is considered to be a pest in areas such as Australia (Cowan and Tyndale-Biscoe, 1997), and increase of fox population in Western Europe would dramatically increase the cost of the oral vaccination against rabies, likely with a less efficient result (Chautan et al, 2000). However, only a few fox sperm proteins have been identified and biochemically characterized: FSA-1 (Beaton et al, 1995), LDH-C4 (Bradley et al, 1996), and PH-20 (ten Have et al, 1998). Because none of these proteins could actually be used as a contraceptive antigen, it is necessary to identify more fox sperm proteins.
The immunologic approach has been used to identify sperm proteins (Haden et al, 2000). In our previous study on fox sperm antigens (Verdier et al, 2002b), sera containing antisperm antibodies were obtained after immunization of fox and rabbit with fox sperm proteins. These sera were used to select and localize by Western blotting 7 highly antigenic proteins on 2-dimensional (2D) gel electrophoresis of fox sperm proteins. It was supposed that most of these proteins were membrane proteins, because they were extracted with detergent; however, because of the experimental conditions used, it was unavoidable that some of them were not surface proteins.
The objective of this study was molecular characterization of a low-molecular isoantigen of fox spermatozoa, recognized by sera of rabbits and foxes immunized with fox sperm proteins. This antigen, named fSP8, has a molecular weight (MW) of 14.7 kd and an isoelectric point (IP) of 6.0 (Verdier et al, 2002b).
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Materials and Methods |
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Preparation of Spermatozoa, Extraction of Sperm Proteins, and Purification of fSP8 Using 2D Electrophoresis
Sperm samples were obtained from 50 wild foxes (Vulpes vulpes).
They had been caught for rabies monitoring during the reproduction period in
the Northeast of France (January to March). Epididymides were dissected and
the sperm was collected from the cauda epididymis by retrograde flushing from
the vas deferens toward the proximal cauda with phosphate-buffered saline
(PBS: 140 mM NaCl, 16 mM KCl, 6.7 mM Na2HPO4, 1.4 mM
KH2PO4, 1 mM MgCl2, pH 7.3). Spermatozoa
samples were pooled, washed twice with PBS by centrifugation for 5 minutes at
800 g, and pellets of 108 spermatozoa were stored at -80°C
until analyzed (maximum 9 months). For the 2D electrophoresis, each pellet was
solubilized in 200 µL of a solution of 9.5 M urea, 2% Igepal, 2% pH3-10
Ampholines (Amersham Bioscience, Orsay, France), and 5% ß-mercaptoethanol
in bi-distillated water.
The 2D gel electrophoresis was based on the procedure of O'Farrell (O'Farrell, 1975). Sperm proteins were run on a first-dimensional 4% urea gel pre-equilibrated with 9.5 M urea and pH3-10 Ampholines. The evaluation of the IP as a function of the migration distance was carried out as described previously (Verdier et al, 2002a,b). The second-dimension electrophoresis was performed in a 12% acrylamide gel at 12°C under reducing conditions according to Laemmli (1970). Gels were then stained with Coomassie Blue according to Wu and Welsh (1996). fSP8 spots are well separated on 2D gels and could be unambiguously cored out (Verdier et al, 2002b). This protein was selected and identified after Western blotting of the 2D electrophoresis purified protein with fox and rabbit immunoreactive sera (Verdier et al, 2002b). Spots of fSP8 carefully cored from the gel were used for deglycosylation assay, microsequencing, and mass spectrometry analysis.
Deglycosylation Assay and Lectin Labeling
Spots of protein fSP8, purified on 14 2D electrophoresis gels, were
electroeluted from the gel and pooled. They were deglycosylated using a
commercial kit (enzymatic deglycosylation kit, Biorad, Marnes la Coquette,
France), as recommended by the manufacturer. The MW of the deglycosylated
protein was determined on a Tris-Trycine gel (Biorad) and was silver nitrate
stained (Morrissey, 1981).
Eight spots of the protein fSP8 electroblotted onto nitrocellulose membrane (Towbin et al, 1979) were lightly stained with Coomassie Blue, and the relevant band was excised and cut in 8 strips. They were saturated for 1 hour in 10% bovine serum albumin (BSA) in Tris-buffered saline (TBS: 0.15 M NaCl, 0.05 M Tris-HCl, pH 7.5), washed 5 minutes in PBS, and incubated for 16 hours with 50 µg/mL of the following biotynilated lectins, selected according to the wide spectra of their binding sugars: Concanavalin A, Dolichos biflorus, Phaseolus vulgaris PHA-E, Phytolacca americana, Pisum sativum, Tetragonolobus purpureas, Ulex europaeus UEA I, and Wisteria floribund in TBS-BSA. After 3 washings of 5 minutes apiece in 0.05% Tween20 in TBS, the presence of lectin is stained with a commercial kit (Pierce, Rockford, Ill) using avidin-biotinylated-peroxidase and 3,3'diaminobenzidine-Zinc, according to the manufacturer's instructions. Total sperm fox proteins were used as positive control.
Microsequencing of fSP8
Twenty spots of the protein P8 were carefully cored from 2D gels, pooled,
purified by sodiuom dodecyl sulfate polyacrylamide gel electrophoresis, and
electroblotted onto polyvinylidenedifloride membrane. The membrane was stained
with Coomassie Blue R-250 and the relevant band excised and subjected to
N-terminal amino acid sequence analysis
(Edman, 1956) on an Applied
Biosystem gas phase sequencer (model 476) equipped with a phenylthiodantoin
analyzer (model 120A). The sequencing program was run as recommended by the
manufacturer. Similarities of the amino acid (AA) sequence obtained to known
proteins were then searched into the full National Center for Biotechnology
Information nonredundant protein database.
Generation of a fSP8 cDNA Probe
For testis collection, two fertile foxes were obtained from the Norwegian
Fur Breeders Association (Eidsvoll, Norway). They were housed individually in
outdoor cages in compliance with the Canadian Council on Animal Care
guidelines in our experimental farm (agreement A 54747). Testes were taken
during reproduction season and immediately frozen at -80°C. Tissue was
homogenized in liquid nitrogen, then total ribonucleic acid (RNA) was
extracted using Trizol (Invitrogen Life Technologies, Renfrewshire, Scotland)
and messenger RNA (mRNA) was purified with Oligotex kit (Qiagen, Courtaboeuf,
France) according to the manufacturer's instructions.
Reverse transcription (RT) was performed by Rapid Amplification of cDNA extremities (RACE) polymerase chain reaction (PCR) using a commercial kit (Invitrogen Life Technologies), according to the manufacturer's instructions. A quantity of 50 ng of mRNA was added to 500 nM of an Adaptator Primer and heated 10 minutes at 70°C. Then, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 10 mM dithiothreitol, and 500 µM of each oligonucleotide were added, the mix was incubated 5 minutes at 42°C, and 200 U of Superscript II reverse transcriptase was added. The tube was then incubated for 50 minutes at 42°C, 15 minutes at 70°C, and 1 minute at 4°C. After addition of 2 units of RNase H, the mix was incubated for 20 minutes at 37°C.
The PCR was performed using a primer deduced from the Edman sequence (gTNCCNACNgAYgAYgARCARg) synthesized by Genome Express (Paris, France) and one Abreged Universal Adaptator Primer (ggCCACgCgTCgACTAgTAC), in a mix of 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1 mM MgCl2, 2 µM of each primer, 0.2 mM dNTP, and 1 U of Taq Platinium DNA polymerase. Thermal cycling was done in a mastercycler gradient thermal cycler (Eppendorf, Hamburg, Germany), using a program of 1 cycle at 94°C for 5 minutes, 35 cycles of 94°C for 1 minute, 65°C for 30 seconds, 72°C for 1 minute, and a last cycle at 72°C for 10 minutes. PCR products were separated by electrophoresis on 2% agarose gel stained with ethidium bromide. A 350-bp DNA fragment was extracted from the gel with Genelute column (Sigma) and cloned in pUC18/SmaI vector (Amersham Bioscience) according to the manufacturer's instructions. Plasmids were purified with a Perfectprep plasmid mini kit (Eppendorf), screened, and sequenced on a Perkin-Elmer Biosytem DNA sequencer using Dye terminator Chemistry (ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit, Applied Biosystem, Courteboeuf, France) with M13 forward primer (AgCggATAACAATTTCACACAgg) and M13 reverse primer (CCCAgTCACgACgTTgTAAAACg). Plasmids purified from positive clones were digested with the enzymes XbaI and PstI.
Construction and Screening of Fox Testis cDNA Library
Fox testis cDNA library was realized using ZAP Express cDNA synthesis kit
and ZAP Express cDNA Gigapack III gold cloning kit (Stratagene, Amsterdam, The
Netherlands), according to the manufacturer's instructions. After plating and
growing of the phages, they were blotted in duplicate onto a nitrocellulose(+)
membrane (Amersham Biosciences). Membranes were treated for 2 minutes in 0.2 M
NaOH, 1.5 M NaCl; for 5 minutes in 0.5 M Tris-HCl, pH 8, 1.5 M NaCl; and for 1
minute in 0.2 M Tris-HCl, pH 7.5, saline sodium citrate buffer (SSC) 2x,
and DNA was fixed by cross linking using ultraviolet irradiations. The library
was screened by Southern blot. Labeling of DNA probe with digoxigenin and
detection were realized using digoxygenine High Prime DNA Labeling and
Detection Starter Kit II (Roche Diagnostics, Meylan, France), according to the
manufacturer's instructions. Hybridization was performed for 16 hours at
42°C. Then, 1.5 x 105 phages were plated for the first
screening. Positive clones were purified by isolation of the plaque and two
additional screening procedures. After the last screening, positive clones
were converted into plasmid using helper phage, as described in the
manufacturer's protocol. Plasmids pBK-CMV, containing the target inserts, were
sequenced by gene walking, as described. The first primers used were T3
forward (AATTAACCCTCACTAAAggg) for sense sequence and T7 reverse
(gTAATACgACTCACTATAgggC) for antisense sequence. The other primers (forward
and reverse) were synthesized to cover all the sequences.
Mass Spectrometry
Spots of protein fSP8 were excised from 10 Coomassie Blue–stained 2D
electrophoresis gels and analyzed by mass spectrometry
(Shevchenko et al, 1996). The
gel plugs were diced into small pieces and de-stained by washing twice with
water and acetonitrile. Protein was reduced in gel with 10 mM dithiothreitol
in 100 mM NH4HCO3 for 30 minutes at 56°C and
alkylated with 55 mM iodoacetamine in 100 mM NH4HCO3 for
45 minutes in the dark at room temperature. The alkylation solution was
removed, and the gel pieces were washed once with 100 mM
NH4HCO3, dehydrated with acetonitrile, and dried in a
vacuum centrifuge. The gel pieces were swollen in digestion buffer containing
50 mM NH4HCO3, 5 mM CaCl2, and 12.5 ng/µL
trypsin for 30 minutes on ice. The digestion buffer was exchanged with 50 mM
NH4HCO3 per 5 mM CaCl2, and samples were
digested overnight at 37°C. Peptides were extracted from gel pieces in 5%
vol/vol formic acid for 1 hour, followed by 3 extractions with 50:50
acetonitrile per 5% vol/vol formic acid for 1 hour. The combined extracts were
reduced to 20 µL in a vacuum centrifuge, and the peptides were purified on
a C18 ZipTip (Millipore Corporation, Bedford, Mass) according to the
manufacturer's instructions. Samples were mixed at a 1:1 ration with a
saturated aqueous solution of 2,5-dihydroxybenzoic acid matrix.
Mass spectrometric analyses were carried out on a Bruker Reflex IV matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Bruker-Daltonics, Bremen, Germany) equipped with a nitrogen laser (337 nm). Spectra were collected in positive ion reflector mode with delayer extraction. The ion acceleration voltage was 20 kV. The spectra were calibrated externally using peptide standards, but if necessary, they were internally recalibrated on trypsin autodigestion products (m/z 842.50 and 2211.10). The monoisotopic masses for all peptide ion signals in the acquired spectra were determined and used for comparison with theoretical spectra of the fSP8 cDNA sequence.
RT-PCR Analysis of fSP8 Organ Specificity
Organs samples from 1 male and 1 female fox (12 months old) were dissected
and frozen at -80°C. Tissues were separately homogenized, and total RNA
was isolated. For the nonsexually differentiated organs, including brain,
heart, liver, kidney, spleen, lung, duodenum, and muscle, RNA extracted from
the male and the female were mixed together. RNA from sexually differentiated
organs including epididymis, testis, vagina, uterus, and ovary were
independently isolated.
Total RNA was extracted using Trizol (Invitrogen Life Technologies), and mRNA was purified with Oligotex kit (Qiagen) according to the manufacturer's instructions. RT was performed using Maloney Murine Leukemia Virus reverse transcriptase and oligodT primers (Applera, Paris, France). The PCR was performed using hot start Taq platinum DNA polymerase (Invitrogen Life Technologies). Thermal cycling was done in mastercycler gradient thermal cycler (Eppendorf), using a program of 1 cycle at 94°C for 5 minutes; 30 cycles of 94°C for 1 minute, melting temperature for 30 seconds; and 72°C for 1 minute; and a last cycle at 72°C for 10 minutes. PCR products were separated by electrophoresis on 2% agarose gel stained with ethidium bromide. All samples were analyzed by 2 independent RT-PCR experiments. To amplify the part of the fSP8 sequence common with other cytochrome c oxidase Vb (COCVb) sequences, a gAgggAggTCATgATggCT forward primer designated from the well-conserved part of fSP8 was used with the TCAgTgggCCAACTggTgg reverse primer and a melting temperature of 55°C. To amplify the fSP8-specific sequence, the ATgAAgAgAggCTCAgCTgC forward primer was designated from the fSP8-specific sequence and used with the same reverse primer and a melting temperature of 66°C.
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Results |
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Microsequencing of fSP8
To obtain structural information on the identity of fSP8, microsequencing
of 20 spots of this protein was undertaken. A peptide sequence of 14 AA was
obtained (Table 1). Database
search using sequence information identified more than or equal to 85% of
identity with the bovin cytochrome c oxidase polypeptide Vb (P00428).
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Cloning and Sequence Analysis of fSP8
To isolate the cDNA encoding fSP8, a RACE PCR was first used. A degenerated
primer was deduced from the fSP8 NH2-terminal sequence. By using
this primer, a 350-bp piece of cDNA was amplified from testicular RNA. This
piece was used to screen, by Southern blot, a cDNA library from fox testis.
The nucleotide sequence of the full-length cDNA (AJ421970) consisted of 879 bp
with an inframe start codon at nucleotides 400–402
(Figure 1). The cDNA contained
a 384-bp open-reading frame (ORF) with untranslated regions of 399 bp at the
5' end and of 96 bp at the 3' end. The ORF encoded a protein of
128 AAs with a predicted molecular weight of 14.0 kd and an IP of 8.3. The
peptide obtained by microsequencing the protein spot was recovered in the
predicted AA sequence of the molecule
(Figure 1, underlined).
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Analysis of the predicted AA sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated the presence of one potential N-linked glycosylation sites (96–99), two potential kinase C phosphorylation sites (AA 26–28 and 83–85), one potential AMPc- and GMPc-dependant kinase phosphorylation site (2–5), and three possible protein casein kinase phosphorylation sites (38–41, 44–47, and 72–75). Moreover, fSP8 presents a homology with the zinc binding site of the subunit Vb of the cytochrome c oxidase (99–121).
Sequence analysis according to Bendtsen et al (2004) predicts no apparent signal peptide sequence nor any cleavage site sequences. The instability index calculated according to Guruprasad et al (1990) was computed to be 42.32, classifying the protein as unstable.
Comparison of the deduced fSP8 sequence to the full National Center for Biotechnology Information nonredundant protein database revealed that the fox sperm protein fSP8 had in its C-terminal part the closest AA similarity to the Vb polypeptide of the cytochrome c oxidase from bovin (P00428; Figure 2), mouse (P19536), and human (P10606). The 30 AA at the NH2-terminal extremity of fSP8 do not show similarity with the COCVb and COCVb precursor from human (gi|17017988; Figure 3), rat (gi|627986), and mouse (gi|109770).
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Mass Spectrometry
To confirm that the protein originally identified and cored from the gel
was cloned, and to validate that the ORF deduced from the cDNA sequence, AA
determination was performed by tandem mass spectrometry on peptides generated
by overnight trypsin digestion at 37°C of the protein spot within pieces
of the gel. The extracted peptides were concentrated and then analyzed by mass
spectrometry. Six generated peaks of strong intensity, corresponding to 42% of
the protein, were close to the theoretical results deduced from the fSP8 AA
sequence without and with posttranscriptional modifications (oxidation,
acetylation, myristilation, and guanidylation)
(Table 2).
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Organ Specificity Expression of fSP8
To assess the expression of the fSP8 by different organs, RT-PCR was used
with mRNA isolated from various tissues by using a primer corresponding to the
specific fSP8 sequence. Another RT-PCR was performed with a primer pair
corresponding to the ubiquitous sequence of the COCVb.
The mRNA coding for fSP8 sequence was specifically expressed in the testis (Figure 4A). The band stained corresponded to the predicted size of 384 bp. The ubiquitous sequence of 248 bp corresponding to the COCVb was expressed at a high level in all of the tissue tested, including brain, heart, liver, kidney, spleen, lung, duodenum, muscle epididymis, testis, vagina, uterus, and ovary (Figure 4B), indicating the good quality of the mRNA used.
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Discussion |
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fSP8 presents a homology with the bovine COCVb, as shown both by the NH2-terminal sequence and the full-length cDNA sequence. COCVb is a protein localized on the internal membrane of the mitochondria, but it also interacts with cytoplasmic proteins such as the androgen receptor (Beauchemin et al, 2001). This complex has been described in most of the tissues (eg, brain, muscle, and liver) of several species (eg, Bos taurus, Mus musculus, Homo sapiens). It is localized in the inner mitochondrial membrane and is composed of cytochrome A and cytochrome B, 2 copper atoms, and 13 different protein subunits, 3 of which are encoded by the mitochondrial DNA. By transferring electrons from reduced cytochrome c to oxygen to form water, it generates an electrochemical gradient across the mitochondrial inner membrane that drives the synthesis of adenosine triphosphate. The COCVb is the most conserved among the nuclear-encoded subunits of the cytochrome c oxidase. It contains 3 conserved cysteines in its C-terminus that are believed to bind zinc (Rizzuto et al, 1991) and that are conserved in the sequence of fSP8. Among the 13 subunits of the cytochrome c oxidase, 2 have been described in the fox (Vulpes vulpes): the subunit I (AF028206) and the subunit II (AF028230). Unlike fSP8, these 2 subunits are encoded by a mitochondrial gene and do not present any similarity to COCVb. It has been established in rats and mice that COCVb is expressed in various tissues, including spermatocytes, spermatogonia, spermatids, epididymial sperm, and Sertoli cells (Klissenbauer et al, 2002).
The 30 AA located at the NH2 extremity of fSP8 shares no significant homology with any other protein, as shown by protein database screening. In the precursor sequence of the fSP8 homologs (COCVb of human, mouse, and rat), this region corresponds to a transit peptide containing a conserved 3-AA motif predicted for mitochondrial proteins undergoing a 2-step proteolytic processing, which may have a role in the transport of the protein to the cytoplasmic side of the inner membrane (Hendrick et al, 1989; Basu and Avadhani, 1993). The NH2-terminal sequence of fSP8 shares a poor homology with these transit peptides (Figure 3), and no apparent signal peptide sequence nor cleavage site sequences were predicted according to Bendtsen et al (2004). This indicates that fSP8 may undergo a particular processing, conferring to this fox protein specific properties including its NH2-terminal sequence, interaction with other proteins, and subcellular localization during its processing.
Several facts and hypotheses explain the antigenicity of fSP8. First, we have experimentally proved that in foxes, the NH2-terminal sequence of fSP8 is testis specific. As is the case with fSP8, a few other glycolytic enzymes have been shown to be composed of a sequence common to other tissues and a testis-specific sequence (Mori et al, 1993). We assume that this testis-specific sequence is responsible of the fSP8 antigenicity. The synthesis of spermatozoa proteins, including fSP8, occurs only at puberty. In their normal condition, testis-specific proteins are then isolated from the immune system by the hematotesticular barrier (Yanagimachi, 1994), but in our studies, in order to select sperm immunogenic proteins, fox spermatozoa extracts were subcutaneously injected into fox (Verdier et al, 2002b), allowing contact between fSP8 and the immune system. The ubiquitous sequence of the COCVb should then be recognized as "self" by the immune system, since the NH2-terminal sequence should be recognized as "nonself," inducing an immune response against fSP8.
Another fact to be considered to explain the high immune response against fSP8 is the probable high quantity of fSP8 expression in spermatozoa. It has been shown that the expression of the COCVb varies in different tissues, as mitochondria from tissues with higher oxygen consumption, such as the heart, kidney, and brain, contain higher levels of subunit Vb mRNA and protein than the liver (Vijayasarathy et al, 1998). We assume that this protein is expressed in a high level in spermatozoa as a result of the correlation between the COCVb activity and sperm mobility (Soderquist et al, 1991). In our previous studies (Verdier et al, 2002b), we should have injected into fox significant quantities of mitochondrial proteins, including fSP8, to facilitate the immune response against them. Indeed, mitochondria are situated just below the plasmic membrane of the middle piece of spermatozoa (Yanagimachi, 1994), which should have allowed a large quantity of mitochondrial proteins, including fSP8, to be extracted with detergent and then to be injected into foxes. Similar contamination of membrane fraction by the COC has been proved in other works on human spermatozoa (Mack et al, 1986).
Identification of sperm antigens can lead to the development of antisperm vaccines, which are attractive to control fertility in wild species such as fox. For this use, the antigenicity and the organ specificity are of a crucial importance. Further investigation on the fSP8 accessibility to antibodies should now be assessed.
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Acknowledgments |
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Footnotes |
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References |
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