Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14

Gene 519 (2013) 271–278

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Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14 Edith Serrano a, Rosaura Pérez-Pé a, Lucía Calleja a, Natalia Guillén a, Adriana Casao a, Ramón Hurtado-Guerrero b, c, Teresa Muiño-Blanco a, José A. Cebrián-Pérez a,⁎ a b c

Departamento de Bioquímica y Biología Molecular y Celular, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón (IUCA), Facultad de Veterinaria, Spain Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, Spain Fundación ARAID, Paseo María Agustín 36, Zaragoza, Spain

a r t i c l e

i n f o

Article history: Accepted 7 February 2013 Available online 24 February 2013 Keywords: Protein cloning Gamete biology Cryoprotectant proteins Ram spermatozoa

a b s t r a c t In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22 kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76–85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Spermatozoa have limited biosynthetic activity (Amann et al., 1993; Hammerstedt et al., 1987). Hence, their functionality is mainly controlled by external factors acting through cell surface and plasma membrane components. The acquisition of fertilizing ability requires extensive sperm plasma membrane remodeling during epididymal transit and in the female reproductive tract, by processes generally referred to as epididymal maturation and capacitation respectively (Austin, 1985; Yanagimachi, 1994). The adsorption of proteins from the epididymal (see Dacheux et al., 2005; Marengo, 2008 for review) and seminal (Desnoyers et al., 1992; Souza et al., 2008) fluids results Abbreviations: BSP, bovine seminal plasma; cDNA, complementary to RNA; CRAC, cholesterol recognition/interaction amino acid consensus; FN2, fibronectin type 2; PCR, polymerase chain reaction; RSVP, ram seminal vesicle protein; SP, seminal plasma. ⁎ Corresponding author at: de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, C/ Miguel Servet, 177, 50013-Zaragoza, Spain. Tel.: + 34 976761637; fax: + 34 976761612. E-mail address: [email protected] (J.A. Cebrián-Pérez). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.02.016

in a functional, mature sperm with an adequate plasma membrane for specific interactions with the oocyte. Seminal plasma (SP) of mammals is composed of secretions from several glands of the male reproductive tract (Mann et al., 1981) and contains various proteins, some of which are able to bind to the sperm plasma membrane. In certain species, most of these proteins are secretory products of the seminal vesicle, an accessory reproductive gland in most male mammals (Aumuller et al., 1988; Chandonnet et al., 1990; Dostalova et al., 1995). The seminal vesicle secretion accumulates in the lumen of this reproductive gland after puberty. This fluid constitutes a relevant portion of seminal plasma at ejaculation in several species, and influences the metabolism, motility, and surface properties of spermatozoa (Manco et al., 1988; Peitz, 1988). SP provides metabolic support as an energy source for the sperm cells and is a key modulator of sperm functionality (reviewed by Caballero et al., 2012; Muiño-Blanco et al., 2008) in terms of viability (Ashworth et al., 1994; Maxwell et al., 1996), motility (Baas et al., 1983; Bernardini et al., 2011; Graham, 1994) and resistance against the damaging effects of cold shock (Berger et al., 1985; Leahy et al.,

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2009; Mogielnicka-Brzozowska et al., 2011; Pursel et al., 1973). Furthermore, a sperm surface receptor for a SP protein complex that increases sperm motility and viability has been found in rabbit spermatozoa (Minelli et al., 2001a, 2001b). The addition of SP also resulted in improved subsequent fertility following artificial insemination in ram (Maxwell et al., 1999), boar (Rozeboom et al., 2000) and stallion (Alghamdi et al., 2004). It has been suggested that some of the coating proteins are essential for maintaining the stability of the membrane up to the process of capacitation (decapacitation factors) (Cross, 1996; Fraser et al., 1996; Manjunath et al., 1994, 2002a; Ollero et al., 1994). Therefore, they must be removed, modified or masked before the spermatozoa undergo the acrosome reaction (Fraser et al., 1996; Yanagimachi, 1994), an essential requisite to successful fertilization (Desnoyers et al., 1992; Fraser et al., 1996; Roberts et al., 2003; Yanagimachi, 1994). Based on these data, it has been suggested that the main role of seminal plasma is to maintain the spermatozoa in a decapacitated state. The fact that the effect of SP on spermatozoa is influenced by several factors such as the source of SP (donor, season and SP fraction; Strzezek et al., 2005) and the subsequent processing of the spermatozoa (reviewed in Leahy et al., 2011) must be taken into account. It is well known that low temperatures alter the function of spermatozoa (Watson, 1995). Cold shock results in the destabilization of sperm membranes and impairment of sperm function. The particularly high sensitivity of ram spermatozoa to cold-shock stress, compared with other species such as bull, rabbit and man (Fiser et al., 1986; Holt, 2000; Holt et al., 1984) has been reported. The reduced longevity and fertilizing ability of cryopreserved ram spermatozoa has been suggested to result from the premature sperm capacitated state due to cold-shock (Ashworth et al., 1994; Bailey et al., 2000; Perez et al., 1996). We have already shown that ram SP proteins can prevent (Pérez-Pé et al., 2001b, 2001c, 2002) and repair (Barrios et al., 2000; García-López et al., 1996; Ollero et al., 1997) the cold-shock sperm membrane damage. Furthermore, a specific protein fraction has been isolated from ram SP and identified as responsible for these protective effects (Barrios et al., 2000) with a similar protein profile (Barrios et al., 2005) to those identified by other authors in ram SP of different breeds (Bergeron et al., 2005; Bernardini et al., 2011; Jobim et al., 2005; Maxwell et al., 2007). We have also proved that three ram SP proteins of approximately 14, 20 and 22 kDa are mainly responsible for this protective ability (Barrios et al., 2005). They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22 (Fernandez-Juan et al., 2006). These proteins contain two fibronectin type II domains (FN2 domain), first described in the proteins collectively called bovine seminal plasma proteins (BSP, A1/A2, A3, 30 kDa) (Fan et al., 2006; Manjunath, 1984), which are secreted by the seminal vesicles (Topfer-Petersen et al., 1995). Homologous proteins have been identified in the SP of different mammalian species (Calvete et al., 1997; Lefebvre et al., 2007; Manjunath et al., 2009; Plante et al., 2012a). The FN2 domain is a collagen-binding domain that binds to extracellular matrix and cytoskeleton components to stabilize them and determine the shape of the cell and cytoskeleton organization (Pankov et al., 2002). The FN2 domains confer many binding properties on the BSP such as binding to glycosaminoglycans (Therien et al., 2005), choline phospholipids (Desnoyers et al., 1992), high and low-density lipoproteins (Manjunath et al., 1989, 2002a) and gelatin (Manjunath et al., 1987b). Seasonal differences in the ability of ram SP proteins to prevent cryoinjury have been shown (Pérez-Pé et al., 2001a) as well as in the content of specific proteins able to protect sperm against stressful events (Cardozo et al., 2006; Marti et al., 2007). Therefore, the isolation of these beneficial proteins from SP is of great interest but is necessarily limited, particularly during the non-breeding season. Thus, the availability of these proteins through cloning and in vitro expression could be useful for the formulation of improved diluents for preserving ram spermatozoa during freezing or storage. Semen extender

with added SP proteins might protect ram semen against cold shock during cryopreservation, allowing the storage of spermatozoa for extended periods and the improvement of fertilization results with frozen semen. Furthermore, although there is a wide array of proteins containing FN2 domains, very few of these have been successfully expressed in a recombinant system. Likewise, their soluble expression has not been frequently achieved, and hence recombinant proteins obtained from insoluble inclusion bodies have been mostly used (Banyai et al., 1990; Jani et al., 2005; Lefebvre et al., 2009; Plante et al., 2012a, 2012b; Tordai et al., 1999). Therefore, the development of an efficient strategy to produce soluble recombinant FN2 domain-containing proteins is required. Moreover, knowledge of the RSVP genes and protein structure is essential for understanding the exact function of these proteins. Therefore, the objective of this study is to characterize and express the RSVP14 gene and to understand the underlying structural features of this protein responsible for its membrane stabilizing properties.

2. Materials and methods 2.1. RNA isolation and reverse transcriptase-polymerase chain reaction Tissue samples from seminal vesicles were collected from a freshly slaughtered Rasa Aragonesa male ram and immediately frozen in liquid nitrogen. Total RNA was extracted by the guanidine thiocyanate/phenol extraction method (Chomczynski et al., 1987, 2006) by homogenization in 1 mL of TRI reagent (Sigma-Aldrich) per 200 mg of tissue. RNA concentration was measured in a NanoDrop ND-100 Spectrophotometer (Wilmington, DE, USA). 500 ng of total RNA was reverse transcribed using poly (dT) primers and the SuperScript III RT enzyme (Invitrogen, CA, USA).

2.2. Amplification and sequencing of cDNA encoding RSVP14 Degenerate PCR primers (Table 1) for RSVP14 were designed according to the primary sequence of the protein (Barrios et al., 2005) in the 5′ region. Primer RSVP14-5′ was based on amino acid residues 1–6. The 3′ primer was Oligo (dT)20. PCR was performed with 2 mL of cDNA. Using these primers, 35 PCR cycles were carried out on reverse-transcribed RNA from ram seminal vesicles. Cycling conditions consisted of 45 s at 94 °C, 1 min and 30 s at 57 °C, and 3 min at 72 °C. A 1 min denaturation step at 94 °C preceded cycling; at the end, a final 10 min extension at 72 °C was performed. PCR products were separated on 1% agarose gel in 1 × Tris–borate–EDTA (TBE) buffer containing 0.5 μL/mL ethidium bromide (Sigma-Aldrich, Madrid, Spain) and were visualized under ultraviolet (UV) light. Molecular size was estimated by using Step Ladder 50 bp (Sigma-Aldrich, Madrid, Spain). PCR products were purified using the GENECLEAN Turbo Nucleic Acid Purification kit (Q-BIO gene, Morgan Irvine, CA, USA) according to the manufacturer's instructions. PCR fragments covering the complete RSVP14 cDNA were sequenced. This was performed on both strands with RSVP14-5′/Oligo (dT)20 primers. DNA was sequenced and analyzed on an ABI Prism 3700 sequencer (Applied Biosystems, Foster City, CA, USA).

Table 1 Oligonucleotide sequences used for PCR amplifications and sequencinga. RSVP14-5′

5′-GATGATGAIC TIACICG-3′

14Sgfl-Fw 14Pmel-Rv 14StarG-Fw 14StarG-Rv

5′-ATCGGCGATC GCCATGGGCC GCAGCTGGGG CT-3′ 5′-ATCGGTTTAA ACCTAGCAAT ACTTCCAAGC TC-3′ 5′-AGCGGCTCTT CAATGGCGCC GCAGCTGGGG AT-3′ 5′-AGCGGCTCTT CTCCCGCAAT ACTTCCAAGC TCCATC-3′

a

Oligonucleotide primers were synthesized by Invitrogen. I indicates inosine.

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2.3. Cloning of cDNA sequences into expression vectors Oligonucleotide primers for the amplification of the cDNA fragment encoding RSVP14 were designed (Table 1) based on the previously obtained cDNA sequence. In order to clone into the vector for the expression in insect cell extract, the recognition sites for restriction enzymes SgfI and PmeI were added to primers 14Sgfl-Fw and 14Pmel-Rv. The signal peptide was included in the amplification as indicated by the manufacturer's instructions. PCR was performed with Pfu DNA Polymerase (Fermentas, Ontario, Canada). Cycling conditions consisted of a 1 min initial denaturation at 94 °C, 30 cycles at 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min, and a 10 min final extension step at 72 °C. The amplified PCR products were analyzed on 1% agarose gel and sequenced in an ABI Prism 3730 (Applied Biosystems, Foster City, CA, USA). Following incubation with the restriction enzymes SgfI and PmeI, both the PCR-amplified product and the pF25A ICE T7 Flexi Vector (Promega, Madison, Wisconsin, USA) were run in a 1% agarose gel, gel-purified using a GeneJet gel extraction kit (Fermentas, Ontario, Canada) and ligated overnight using T4 DNA ligase. This ligation mixture was used to transform Escherichia coli strain JM109 competent cells. The bacterial colonies containing recombinant plasmids were selected on LB agar medium, with 100 mg/L ampicillin. There was no need to carry out blue/white colony screening since these plasmids contain a lethal barnase gene, which is replaced by the insert, and thus only the correctly transformed bacteria can grow. pPICZα-A plasmids (Invitrogen, Oregon, USA) were used for expressing RSVP14 in Pichia pastoris. RSVP14 cDNA was synthesized to optimize its expression in Pichia, and the sites for XhoI and SacII were added (GenScript, Piscataway, NJ, USA). The optimization parameters were determined according to the OptimumGene™ algorithm (GenScript, NJ, USA) taking into account the codon usage bias, GC content, mRNA secondary structures and RNA instability motifs. The sequence codifying the signal peptide was substituted by the Kozak and the Shine-Dalgarno sequences, to increase the efficiency of translational initiation. Both the cDNA and the plasmid were digested, purified and then ligated overnight. DH5α cells were transformed and selected in low salt LB agar with 25 mg/L zeocin. To express RSVP14 in bacteria using the StarGate system (IBA, Göttingen, Germany), primers 14StarG-Fw and 14StarG-Rv were designed according to the manufacturer's instructions. PCR conditions were the same as those mentioned above. The PCR product was gel-purified, sequenced, and ligated with the vector pENTRY-IBA51 which adds a His6 tag. DH5α cells were transformed and selected in LB agar plates with 50 mg/L kanamycin and 50 mg/L X-gal for white/ blue selection. Selected JM109 and DH5α colonies were incubated overnight at 37 °C with shaking in 3 mL LB medium (low salt LB for pPICZα-A). Plasmids were extracted using the PureYield plasmid miniprep system (Promega Biotech, Madrid, Spain) and sequenced. The confirmed recombinant strains were frozen at − 80 °C in 15% glycerol. 2.4. Protein expression in P. pastoris pPICZα-A plasmids were linearized with SacI and transformed in Pichia strain X-33 by electroporation. A positive control plasmid that codified Gas2 protein of Saccharomyces cerevisiae was also transformed and processed simultaneously. The yeasts were selected in YPDS plates (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol, 2% agar) with 100 mg/L zeocin for 3 days at 30 °C. Three colonies were incubated at 30 °C overnight in BMGY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, 1.34% yeast nitrogen base (YNB), 4 × 10−5% biotin, 1% glycerol) and centrifuged at 3000 ×g for 5 min. The pellet was resuspended in 1 mL of BMMY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, 1.34% YNB, 4 × 10 − 5% biotin, 1% methanol) to induce expression. The culture

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was placed in falcon of 50 mL and incubated at 30 °C with shaking for 4 days. Methanol was added to a final concentration of 0.5% every 24 h to maintain expression. The cells were centrifuged at 12,000 ×g for 2 min and the supernatant was stored at − 80 °C. 2.5. Protein expression in insect cell extract The protein was expressed in a cell-free protein synthesis system prepared from insect extract (TNT T7 Insect Cell Extract Protein Expression System, Promega Biotech, Madrid, Spain). Four μg of recombinant plasmid was added to 40 μL of TNT T7 Master mix and incubated at 30 °C for 4 h. Total protein concentration was calculated with a Micro BCA Protein assay (Thermo Scientifics, Rockford, IL, USA). The expression reaction was frozen at −20 °C. 2.6. Protein expression in E. coli Plasmids were transformed into OrigamiB (DE3) pLysS competent cells. The transformed bacteria were grown overnight on LB-agar plates containing 100 mg/L ampicillin. Selected colonies were grown in 5 mL LB media with 100 mg/L ampicillin to an OD600 of 0.6–0.8. Expression of RSVP14 was induced by adding isopropyl-b-D-thiogalactoside (IPTG) to final concentrations of 0.5 and 1 mM. Inductions were performed at 37 °C for 8 h and 16 °C for 16 h. One mL culture samples were collected before and after the IPTG induction. From the samples, E. coli cells were harvested by centrifugation at 6800 ×g for 2 min at 4 °C and resuspended in 500 μL of lysis buffer (20 mM TRIS, 500 mM NaCl, 2 mM imidazole, pH7.5). The cells were lysed by sonication and centrifuged at 14,000 ×g for 10 min at 4 °C. The pellet was resuspended in 500 μL of lysis buffer. 2.7. Antibody against RSVP14 An anti RSVP14 polyclonal antibody was raised by rabbit immunization against a synthetic peptide based on the NH2 terminal sequence of RSVP14 in Freund's complete adjuvant. After 15 days, the rabbits were re-immunized with the same peptide in Freund's incomplete adjuvant. The antiserum was obtained 15 days after the second immunization by centrifuging 10–20 mL of blood at 3000 ×g for 30 min, and purification by protein G affinity chromatography in a HiTrap Protein G column (Pharmacia-Biotech, Buckinghamshire, UK) following manufacturer's instructions. 2.8. SDS-polyacrylamide gel electrophoresis and western blotting SDS-PAGE was performed in 16% polyacrylamide gel using a Mini protean III system (Bio-Rad, Hercules, CA, USA). Ten μL of each expression reaction was mixed with 5 μL of loading buffer (0.045 M Tris/HCl, 0.8 mM EDTA, 3% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.004% bromophenol blue). Electrophoresis was performed for 90 min at 130 V at 4 °C. A mixture of pre-stained protein standards with molecular weights ranging from 10 to 250 kDa (Bio-Rad, Hercules, CA, USA) was used as a marker. For Coomassie staining, SDS-PAGE gels were submerged for 2 h in staining solution (0.1% Coomassie blue G, 45% methanol and 10% acetic acid) and de-stained overnight (30% methanol and 10% acetic acid). Images were captured with an Odyssey Clx equipment (Li-Cor Biosciences, Lincoln, NE, USA). The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using a Trans-blot Turbo (Bio-Rad, Hercules, CA, USA). The transference was performed for 10 min at 2'5 A-25 V and the membrane was air dried for 15 min. Non-specific sites on the membrane were blocked for 1 h with 5% bovine serum albumin (BSA) in phosphate buffer saline (PBS, 136 mM NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, pH 7.4). RSVP14 was detected by incubating overnight at 4 °C with the rabbit generated antibodies diluted 1:60,000 in PBS with 1% BSA and 1% Tween. After exhaustive washing, the membranes were

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incubated with a secondary anti-rabbit DyLight 680 conjugated (Thermo Scientific, Madrid, Spain) diluted 1:15,000 for 1 h at room temperature. After washing, the membrane was scanned using the Odyssey Clx equipment (Li-Cor Biosciences, Lincoln, NE, USA). As negative controls, we used TNT T7 Master mix without recombinant plasmids, bacterial and yeast samples before induction, to rule out non-specific binding to the transferred proteins. 2.9. Sequence alignment and characterization Sequence homologies were identified using the NCBI BLAST algorithm. The acquired sequences were aligned using Clustal X (version 2.0). Characterization of RSVP14 was carried out using ExPASy software developed by the Swiss Institute of Bioinformatics. 3. Results 3.1. Characterization of RSVP14 cDNA The analyzed cDNA sequence (Fig. 1) comprises 603 bp containing an open reading frame of 390 bp, which begins with a methionine codon at position 19 and ends with a TAG termination codon at position 408. The 3′ untranslated region contains a polyadenylation signal (AATAAA). The GenBank accession number is FM211030 (sequence submitted by the authors). This cDNA encodes a 129 amino acid protein with a molecular mass of 14 kDa and a predicted isoelectric point of 5.05. The deduced protein displayed a 25-amino acid signal peptide. We found that the RSVP14 sequence contains one potential O-linked glycosylation site at position 30. Likewise, seven phosphorylation sites

could be predicted, one of which is tyrosine (position 50), four are serine (positions 34, 35, 38 and 82) and two are threonine (positions 30 and 49). The results of this study also showed that two fibronectin type-II collagen-binding domains are located at positions 44–81 (CVFPFTYKDKRHFDCTFHGSIFPWCSLDADYVGRWKFC) and 89–129 (CVFPFI FGGKKHETCTKIGSIFGAWCSLSPNYDQDGAWKYC). Our results also prompted us to predict four disulphide bonds between amino acids 44–68, 58–81, 89–114 and 103–129. 3.2. Analysis of the RSVP14 sequence homology A GenBank database search using BLAST alignments revealed that the RSVP14 nucleotide sequence shares a similarity with the BSP protein family including mainly bull (NM_001001145, 85%) and stallion (NM_001081933, 76%). Protein alignment (Fig. 2) also showed a similarity with bull SP proteins (72%), and BSP-like proteins from boar (52%) and stallion (50%). The similarity was mainly observed in the signal peptide and the two FN-2 collagen-binding domains, being the common structure in this protein family. 3.3. Amplification and cloning of RSVP14 coding cDNA Ligation of pPICZα-A plasmids and the synthesized cDNA for expressing RSVP14 in P. pastoris was analyzed by restriction enzyme digestion with XhoI and SacII. An insert of 400 bp along with a vector band was obtained (Fig. 3A). An approximately 400 bp amplified DNA fragment was obtained from the previously obtained cDNA, with both 14Sgfl-Fw/14Pmel-Rv primers and 14StarG-Fw/14StarG-Rv (Figs. 3B, C). The DNA was purified, cloned in pF25A ICE T7 and pENTRY-IBA51, and transformed in E. coli strains JM109 and DH5α, respectively. Five colonies of each were selected for plasmid isolation. The PF25A clones showed an insert of 400 bp along with a vector band after digestion with Sgfl and Pmel (Fig. 3B). The pENTRY restriction analysis was carried out with HindIII and XbaI (Fig. 3C). The cloning of the protein gene was confirmed by nucleotide sequencing. 3.4. Protein expression in P. pastoris The methylotrophic yeast P. pastoris has become one of the leading eukaryotic expression systems for general laboratory use due to its many advantages over other systems. It is able to form disulphide bonds and glycosylated proteins and, at the same time, has a high productivity and is simple to manipulate. We tried expressing RSVP14 in P. pastoris using pPICZα-A plasmids engineered to produce His-tagged secreted protein. After inducing RSVP14 expression with methanol, the cells were harvested by centrifugation and the medium was checked for proteins by SDS-PAGE. We did not detect RSVP14 by either Coomassie staining (Fig. 4) or western blot assay despite its higher sensitivity (data not shown). 3.5. Protein expression in insect cell extract

Fig. 1. Complementary cDNA and the deduced amino acid sequence of RSVP14. The signal peptide is in italics and the stop codon is indicated with an asterisk. The two FN-2 domains are underlined. The eight cysteine residues that can form disulphide bonds are double underlined. The potential O-linked glycosylation site is shaded and the phosphorylation sites are in open boxes. The polyadenylation site is dashed–underlined. The CRAC domain is between vertical bars.

Insect cells are able to carry out complex post translational modifications and are therefore quite suitable for making soluble protein of mammalian origin (Agathos, 1991). The baculoviral system is, however, complex and time consuming; thus, we first tried the expression of RSVP14 in a cell-free protein synthesis system prepared from insect cells. The purified pF25A plasmid was used to express the protein by a coupled transcription/translation reaction. The resulting protein was detected by SDS-PAGE and western blotting. Immunoblotting results revealed a protein band of ∼ 14 kDa, which is not present in the expression medium without plasmid used as a negative control (Fig. 5).

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Fig. 2. Multiple sequence alignment of RSVP14, BSP and other related seminal plasma proteins by ClustalW. Invariant conserved residues are marked with an asterisk. Signal peptides are dashed–boxed. FN-2 domains are boxed.

3.6. Protein expression in E. coli Recombinant proteins in E. coli are usually expressed in fusion with an affinity tag to facilitate the purification by affinity chromatography. The polyhistidine (His)-tag is commonly used because it does not usually interfere with protein activity, and it allows easy purification using nickel-affinity chromatography (Porath et al., 1975). His6tagged RSVP14 was expressed in OrigamiB (DE3) pLysS E. coli cells. When the cells were induced at 16 °C for 16 h there was no protein expression (data not shown). However, following induction at 37 °C for 8 h, the recombinant protein was detected after cell lysis, both in the pellet and the supernatant (Fig. 6). There were no significant variations between induction with 0.5 mM or 1 mM IPTG (data not shown). 4. Discussion As the molecular composition of SP is very complex and variable among species and individual males (reviewed by Strzezek et al., 2005), the structure of SP proteins and their specific effects on spermatozoa may also differ among themselves. To provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa, in this study we have sought to examine the ram cDNA sequence encoding for one SP protein (RSVP14) shown to be able to protect ram spermatozoa against stressful events (Barrios et al., 2000, 2005; Fernandez-Juan et al., 2006), and to look for homologies with related proteins. Additionally, a first approach has been made to the recombinant protein production.

Fig. 3. Agarose gels of (A) restriction enzyme analysis of pPICZα-A recombinant plasmid with XhoI and SacII enzymes (lane 2): vector (a), insert gene RSVP14 (b). RSVP14 PCR-amplified sample using (B) 14Sgfl-Fw and 14Pmel-Rv primers (lane 2); restriction enzyme analysis of pF25A recombinant plasmid with Pmel and SgfI enzymes (lane 3): vector (a), insert gene RSVP14 (b). (C) Using 14StarG-Fw and 14StarG-Rv primers (lane 2); restriction enzyme analysis of pENTRY recombinant plasmid with HindIII and XbaI enzymes (lane 3): vector (a), insert gene RSVP14 (b). DNA ladders (lanes 1); the arrows indicate 500 bp.

The cDNA sequence obtained for RSVP encodes a 129 amino acid polypeptide with the expected molecular mass of 14 kDa (Barrios et al., 2000, 2005) and presents a 25-amino acid signal peptide. In BSP proteins, the signal peptide targets the proteins for secretion (Scheit et al., 1988), and BSP-like proteins of other species are also secreted proteins (Boisvert et al., 2004; Plucienniczak et al., 1999). We have already shown by immunohistochemical analysis that RSVP14 is expressed in the cytoplasm of the secretory epithelial cells, and that the apical end of some secretory cells forms protrusions which dilate and pinch off after selective accumulation of the protein (Fernandez-Juan et al., 2006). Consequently, it can be inferred that RSVP14 is also a secreted protein, and the presence of the signal peptide confirms this. The results obtained also verified that the RSVP sequence contains one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. Our data are consistent with those in previous reports in which we showed, using 2D-electrophoresis, that RSVP14 is composed of four protein spots that are glycosylated (Cardozo et al., 2008) and phosphorylated on tyrosine, serine and threonine residues (Barrios et al., 2005). Despite important differences among species, the role of mammalian SP proteins in the main events that lead to fertilization may be highly conserved (Calvete et al., 2007). A glycosylation site at position 30 has also been evidenced in BSP proteins, one of the major protein families of mammalian SP (Manjunath, 1984). BSP-A1, BSP-A2, BSP-30 kDa

Fig. 4. SDS-PAGE of protein expression in Pichia pastoris stained with Coomassie: RSVP14 (lane 1) and positive control of Gas2 protein (lane 2).

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Fig. 5. Western blot analysis of RSVP14 recombinant protein expression in insect cell extracts (lane 1). TNT T7 ICE Master Mix as negative control (lane 2).

and other BSP-like proteins such as equine SP-1 and porcine pB1 (Calvete et al., 1997; Manjunath et al., 1987a) share this feature. Furthermore, it is worth noting that sperm capacitation and the acrosome reaction are regulated by intracellular signaling pathways and associated with protein phosphorylation (Aitken et al., 1995; O'Flaherty et al., 2006; Visconti et al., 2002). Protein tyrosine, serine and threonine phosphorylation has been shown to be associated to ram sperm capacitation (Colas et al., 2008, 2010; Grasa et al., 2006; Luna C., et al., 2012; Pérez-Pé et al., 2002), and a partial loss and redistribution of phosphorylated proteins towards new domains involved in later stages of fertilization take place during capacitation and the acrosome reaction (Barrios et al., 2005; Grasa et al., 2009). These changes may have a physiological significance in consolidating certain phosphorylated proteins to specific sperm regions involved in acrosomal exocytosis and zona pellucida recognition, binding and penetration, as already suggested (Grasa et al., 2009). The seven possible phosphorylation sites found in this study would suggest an important role for RSVP14 in the processes of sperm capacitation and acrosome reaction, and they may be relevant in the interaction between sperm and oocyte. Our results also showed that RSVP contains two FN-2 domains. The presence of two tandemly repeated FN-2 domains is the signature characteristic of the BSP protein family and related proteins of different species (Boisvert et al., 2004; Calvete et al., 1997; Fan et al., 2006; Lefebvre et al., 2007; Manjunath et al., 2009; Plante et al., 2012a). This is relevant and denotes the significance of these FN-2 domains in sperm functionality. By means of these domains, RSVP could contribute to the protein structure surrounding the spermatozoon in a similar way to fibronectin. It could bind to the sperm

Fig. 6. Protein expression in E. coli before (0 h) and after induction with 1 mM IPTG at 37 °C for 8 h. P, pellet; SN, supernatant.

surface at ejaculation, stabilizing membrane phospholipids and cytoskeleton, maintaining the spermatozoa in a decapacitated state (decapacitating factors), and later participating in membrane modification during capacitation, as already suggested for BSP (Greube et al., 2001; Manjunath et al., 2002a; Therien et al., 2001) and ram SP proteins (Barrios et al., 2005). This might be the explanation for the protective effect of these proteins against cold-shock injury to the membranes of ram spermatozoa. We also deduced that RSVP14 contains four disulphide bonds. This structure of eight cysteine residues forming intramolecular disulphide bridges is present in the BSP protein family, and other similar proteins of porcine and murine, while nine cysteine residues have been reported in human BSPH1 (Lefebvre et al., 2007). Furthermore, and related with the possible implication of RSVP14 in the capacitation processes, our results revealed a cholesterol recognition/interaction amino acid consensus (CRAC) domain in positions 70–77 of RSVP14. A CRAC domain has also been found in the equine protein SP-1 and human BSPH1, while BSP-A3 and BSP-30 kDa have three and BSP-A1 and BSP-A2 have four (Scolari et al., 2010). These findings indicate a connection between RSVP14, BSP and other related proteins with cholesterol. The significance of cholesterol in the activation of certain signaling pathways involved in fertilization processes has been widely evidenced (see Flesch et al., 2000 for review). The release of cholesterol from the sperm membrane leads to an increase in membrane fluidity, which is essential for the concomitant acrosome reaction in human (Cross, 2003) and goat (Iborra et al., 2000) spermatozoa, and regulates the fertilization potential (Travis et al., 2002; Watanabe et al., 2011). Likewise, the relationship between cholesterol and capacitation has been established in several species (Manjunath et al., 2002b; Moreau et al., 1998), and it has been demonstrated that changes in the cholesterol membrane content activate protein tyrosine phosphorylation (Bailey, 2010; Osheroff et al., 1999; Travis et al., 2002; Visconti et al., 1995a, 1995b, 1999a, 1999b). These data reinforce the significance of the CRAC domain of RSVP14 and its role in capacitation. Our results also revealed that RSVP14 shares 76–85% identity with other mammalian SP proteins, particularly with the BSP protein family. This indicates a high degree of conservation in the mammalian SP protein genes during evolution, which is consistent with the fact that SP proteins have a common role in the molecular events leading to fertilization (Calvete et al., 2007). Given the seasonal variability in the content of the beneficial ram SP proteins, the possibilities of purifying the native RSVP14 from SP are very limited. Therefore, we explored several possibilities of its in vitro expression. Although we did not succeed with the approach using P. pastoris, we achieved sound results using insect cell extracts, which revealed a band of approximately 14 kDa molecular weight (MW), as already expected for RSVP14. The presence of other unspecific minor bands of higher MW may be due to the fact that the antibody was raised in rabbit against a synthetic peptide and is rather nonspecific. Although different procedures have been essayed to express proteins containing FN-2 domains in E. coli, this is the first time to our knowledge that a cell-free protein synthesis system prepared from insect cell extract has been used for the expression of FN2 domain-containing proteins. This approach might be useful for the research on this kind of proteins, although optimization of the system is required for obtaining significant quantities of protein. The production of soluble recombinant eukaryotic proteins in E. coli is a difficult process due to several problems such as the formation of inclusion bodies, toxicity, misfolding and unglycosylation. However, the many advantages of this production method make it the obvious choice when attempting protein mass production because it is fast and cost effective. The recovery of FN2 domain-containing proteins from inclusion bodies has been achieved using guanidine (Banyai et al., 1990; Tordai et al., 1999), urea (Plante et al., 2012a, 2012b) or fused with thioredoxin (Lefebvre et al., 2009). In this study, we used the polyhistidine (His)-tag which is commonly used because it does not usually interfere with protein activity and it

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allows easy purification using nickel-affinity chromatography (Porath et al., 1975). Under physiological conditions, the E. coli cytoplasm is maintained in a reduced state that disfavors the formation of stable disulphide bonds in proteins. However, mutants such as the strain used in this study have been genetically modified to favor disulphide bond formation in the cytoplasm, thus increasing the possibilities of recovering correctly folded protein in the supernatant after cell lysis (Bessette et al., 1999). This is important because proteins recovered in the pellet usually form inclusion bodies and are inactive, aggregated and insoluble (Fischer et al., 1993). In this study the recombinant protein was detected after cell lysis not only in the pellet but also in the supernatant, which will facilitate the purification. In conclusion, the RSVP14 gene was successfully cloned and expressed in E. coli OrigamiB (DE3) pLysS. Deduced characterization of RSVP14 revealed biochemical and functional similarities with BSP and other mammalian related proteins, which suggests that they might play a similar role in sperm capacitation and functionality. The presence of FN-2 and CRAC domains supports a dual role of RSVP14 that could act as a decapacitating factor at ejaculation (avoiding premature capacitation) and later, in the female tract, participate in membrane modifications during capacitation. Further work is needed to assess whether the recombinant protein has the same protective effect as the native RSVP14, and to know the mechanism by which this protein participates in ram sperm capacitation. Acknowledgments Supported by grants CICYT-FEDER AGL 2010-18975 and DGA A-26. E. Serrano was financed by the IUCA fellowship 149-01. The authors thank ANGRA for supplying the sires. References Agathos, S.N., 1991. Production scale insect cell culture. Biotechnol. Adv. 9, 51–68. Aitken, R., et al., 1995. Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J. Cell Sci. 108, 2017–2025. Alghamdi, A.S., et al., 2004. Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri. Reproduction 127, 593–600. Amann, R., et al., 1993. The epididymis and sperm maturation—a perspective. Reprod. Fertil. Dev. 5, 361–381. Ashworth, P.J., et al., 1994. Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma. Reprod. Fertil. Dev. 6, 173–180. Aumuller, G., et al., 1988. Binding of a major secretory protein from bull seminal vesicles to bovine spermatozoa. Cell Tissue Res. 252, 377–384. Austin, C.R., 1985. Sperm maturation in the male and female genital tracts. In: Metz, C.B., Monroy, A. (Eds.), Biol. Fertil. Academic Press, New York, pp. 121–155. Baas, J.W., et al., 1983. Factors in seminal plasma of bulls that affect the viability and motility of spermatozoa. J. Reprod. Fertil. 68, 275–280. Bailey, J.L., 2010. Factors regulating sperm capacitation. Syst. Biol. Reprod. Med. 56, 334–348. Bailey, J.L., et al., 2000. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J. Androl. 21, 1–7. Banyai, L., et al., 1990. The collagen-binding site of type-II units of bovine seminal fluid protein PDC-109 and fibronectin. Eur. J. Biochem. 193, 801–806. Barrios, B., et al., 2000. Seminal plasma proteins revert the cold-shock damage on ram sperm membrane. Biol. Reprod. 63, 1531–1537. Barrios, B., et al., 2005. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock. J. Androl. 26, 539–549. Berger, T., et al., 1985. Effect of male accessory gland secretions on sensitivity of porcine sperm acrosomes to cold shock, initiation of motility and loss of cytoplasmic droplets. J. Anim. Sci. 60, 1295–1302. Bergeron, A., et al., 2005. Isolation and characterization of the major proteins of ram seminal plasma. Mol. Reprod. Dev. 71, 461–470. Bernardini, A., et al., 2011. Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage. Theriogenology 76, 436–447. Bessette, P.H., et al., 1999. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc. Natl. Acad. Sci. U. S. A. 96, 13703–13708. Boisvert, M., et al., 2004. Isolation and characterization of gelatin-binding bison seminal vesicle secretory proteins. Biol. Reprod. 70, 656–661. Caballero, I., et al., 2012. Seminal plasma proteins as modulators of the sperm function and their application in sperm biotechnologies. Reprod. Domest. Anim. 47, 12–21.

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