Mannan-binding proteins from boar seminal plasma
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Mannan-binding proteins from boar seminal plasma Mannan-binding proteins from boar seminal plasma Document Transcript

  • Journal of Reproductive Immunology 62 (2004) 167–182 Mannan-binding proteins from boar seminal plasma Petra Jel´nková a , Jiˇ´ Liberda b , Pavla Maˇ ásková a , ı rı n Helena Ryšlavá b , Vˇ ra Jonáková a,∗ , Marie Tichá b e aInstitute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 37 Praha 6, Czech Republic b Department of Biochemistry, Charles University, Albertov 2030, 128 40 Praha 2, Czech Republic Received in revised form 10 November 2003; accepted 5 January 2004 Abstract The interaction of boar seminal plasma proteins and sperm with yeast mannan was investigated by the enzyme-linked binding assay (ELBA) and specific detection of proteins after SDS electrophoresis and blotting using biotinylated derivative of the polysaccharide. Heparin-binding proteins (especially AQN 1 and DQH proteins) and their aggregated forms showed affinity to yeast mannan. Besides that, these proteins were shown to bind to oviductal epithelium. The mannan-binding activity of boar proteins and sperm was inhibited most efficiently by ovomucoid, ovalbumin and N-glycans released from ovalbumin, but not with d-glucose, d-mannose and their phosphates. On the other hand, yeast mannan inhibited both the interaction of boar seminal plasma and sperm with heparin and the binding of these proteins to porcine oviductal epithelium. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. Proteins adsorbed to the immobilized polysaccharide were analyzed by RP-HPLC, SDS electrophoresis and N-terminal amino acid sequencing. AQN and AWN spermad- hesins and DQH protein (names are derived from the N-terminal amino acid sequence) were identified as components of the isolated fraction. The results suggest an involvement of mannan-binding proteins in the formation of the sperm oviductal reservoir in pig. The ability of these proteins to interact both the complex d-mannose- containing saccharide structures and the heparin may also play an important role in sperm release from the oviductal reservoir or the capacitation process. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Boar seminal plasma proteins; Mannan-binding proteins Paper presented at the 4th Congress of the European Society for Reproductive & Developmental Immunology, Rhodes, Greece, June 2003. ∗ Corresponding author. Fax: +420-2-33331274. E-mail addresses: petraj@zeus.img.cas.cz, vjon@zeus.img.cas.cz (V. Jon´ kov´ ). a a 0165-0378/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2004.01.007
  • 168 P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a 1. Introduction Mammalian fertilization is a series of events that involve a highly coordinated sequence of interactions between molecules located on the surface of both gametes as well as with substances present in the natural environment of the gametes (Töpfer-Petersen, 1999). In- teractions of the lectin type play an important role in some steps of this process (Jonáková et al., 1995; Tichá et al., 1998). First, saccharide chains of zona pellucida glycoproteins are supposed to bind receptors present on the sperm surface. Although the saccharide ligands were first characterized in mice (Bleil and Wassarman, 1988) and pigs (Yurewicz et al., 1991; Yonezawa et al., 1995), the ligands of other mammals have not been fully recognized. Recently, Amari et al. (2001) described the essential role of the non-reducing terminal -d-mannosyl residues of N-linked saccharide chains of bovine zona pellucida glycoproteins in sperm–egg binding. The second type of protein–saccharide interactions seems to be involved in the forma- tion of the mammalian oviductal sperm reservoir (Töpfer-Petersen et al., 2002); proteins attached to the sperm surface recognize oviductal epithelium glycopeptides. By initiation of capacitation, these associated proteins are shed from the sperm surface. The saccharide specificity of the interaction of sperm surface molecules with epithelial cells appears to vary among species (Suarez, 2001). Non-capacitated bull sperm are trapped in the reservoir by binding to l-fucosyl residues in the oviductal epithelium (Lefebvre et al., 1997; Revah et al., 2000). l-Fucose-binding sites are lost during capacitation and, at that time, d-mannose bind- ing sites are revealed for interaction with the ovum (Revah et al., 2000; Ignotz et al., 2001). In the pig, the formation of the oviductal sperm reservoir appears to comprise d-mannose residues (Green et al., 2001). Detailed study on the structure of oligosaccharides involved in the carbohydrate-mediated adhesion has been described (Wagner et al., 2002). Recent studies have shown that seminal plasma proteins participate in the formation and rearrangement of the protein coating of the sperm surface, which changes its composi- tion in different steps of the fertilization process (Dostálová et al., 1994; Töpfer-Petersen, 1999; Petrunkina et al., 2000). The binding properties of the sperm surface (including saccharide-binding) are changed due to these events. In a previous communication (Liberda et al., 2002), we have shown the presence of mannan-binding proteins in bull seminal plasma. Mannan binding to seminal plasma pro- teins was inhibited by d-mannose and d-fructose, but not d-mannose-6-phosphate, d- glucose-6-phosphate, ovalbumin and ovomucoid. The aim of this study was to investi- gate the mannan-binding properties of boar seminal plasma proteins that significantly differ in their physico-chemical properties and binding activities of those isolated from bull sem- inal plasma. Yeast mannan immobilized to Sepharose was used for isolation of proteins participating in the studied interaction. 2. Materials and methods 2.1. Chemicals Divinyl sulfone and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were purchased from Fluka (Buchs, Switzerland); Sepharose 4B from Pharmacia (Uppsala, Sweden);
  • P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a 169 heparin, avidin-peroxidase, ABTS (2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), d-mannose- and d-glucose-6-phosphate and ovalbumin from Sigma (St. Louis, MO); Immobilon-P-membrane and 4-chloro-1-naphtol were products of Serva (Heidelberg, Ger- many); N-glycosidase F (PNGase F) (E.C.3.5.1.52) of Bio-Lab (New England, USA). Ovo- mucoid was a gift from Dr. K. Bezouška (Department of Biochemistry, Charles University, Praha, Czech Republic). SDS-PAGE standards-broad range were products of Bio-Rad (Her- cules, USA). Biotinylated polyacrylamide derivative of heparin was prepared as described by Liberda et al. (1997). Saccharomyces cerevisiae cell wall polysaccharide containing mannan and phospho- mannan polymers (Stewart et al., 1968) was isolated as was described by Haworth et al. (1937). 2.2. Materials 2.2.1. Porcine oviductal epithelium products Oviducts were collected from mature sows (five animals), the isthmic region of oviduct was cut longitudinally and oviductal epithelium was gently separated. The obtained material was centrifuged for 20 min at 600 × g to remove cellular debris, dissolved in PBS to protein concentration 1 mg/ml (based on A280 measurement) and frozen. 2.2.2. Boar seminal plasma and sperm Boar ejaculates were obtained from the Veterinary Research Institute, Brno, Czech Re- public. Ejaculates were centrifuged (600 × g, 20 min, 5 ◦ C) to separate plasma from sperm. Seminal plasma was directly lyophilized, sperms were suspended in phosphate-buffered saline (PBS, 20 mM phosphate, pH 7.4, 150 mM NaCl) and washed by centrifugation (two times with PBS), and sperm pellet was then lyophilized. 2.3. Preparation of ovalbumin N-glycans Ovalbumin (5 mg) dissolved in 0.1 M Tris–HCl buffer pH 7.5 (1 ml) was incubated with PNGase F (5 U) under a toluene layer at 37 ◦ C for 48 h. After incubation, protein was precipitated using ethanol and separated by centrifugation. Ethanol solution of N-glycans was concentrated using Speed-Vac. The saccharide content in ovalbumin after the PNGase treatment was determined according to Dubois et al. (1956). 2.4. Isolation of proteins from boar seminal plasma Heparin-binding (H+ ) and non-heparin-binding (H− ) proteins were obtained by affinity chromatography of boar seminal plasma on a heparin-polyacrylamide column (3 cm × 15 cm). Fifty milliliters of boar seminal plasma was diluted 1:1 with PBS and applied to the column (Tichá et al., 1994). H− proteins were washed out with PBS buffer, the adsorbed H+ proteins were eluted with 3 M NaCl. Gel chromatography: boar seminal plasma (5 ml) was applied to a Sephadex G–75 SF column (2.0 cm × 118.0 cm) equilibrated with 0.1 M Tris–HCl containing 0.15 M NaCl, View slide
  • 170 P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a pH 7.2 (flow rate 17.2 ml/h). Fractions, 4.3 ml, were screened for absorbance at 280 nm, pooled, desalted on Sephadex G-25 in 0.2% acetic acid and lyophilized. Biotinylation of H+ , H− and protein aggregates I–V obtained by gel chromatography was performed as described previously (Maˇ ásková et al., 2000). n 2.5. Mannan–Sepharose The immobilized mannan was prepared by coupling of yeast mannan to divinyl sulfone- activated Sepharose as described previously (Liberda et al., 2002). 2.6. Biotinylated derivative of mannan Biotinylated derivative of yeast mannan with coupled ethylenediamine was prepared by combination of two procedures described previously (Novotná et al., 1996; Liberda et al., 1997). The aqueous solution of yeast mannan (50 mg in 5 ml) was mixed with 1% periodic acid (1 ml) and ethylenediamine (20 l). The mixture was shaken for 2 h and the reaction stopped by addition of ethylene glycol (4 ml). After 15 min incubation at room temperature, sodium cyanoborohydride (80 mg) was added. The reaction mixture was dialyzed against distilled water and lyophilized. N-Hydroxy-succinimido-biotin (10 mg) dissolved in dimethylformamide (25 l) was added to the solution of mannan derivative (20 mg) in 0.1 M borate buffer, pH 8.5 (3 ml). The mixture was stirred for 30 min at room temperature and then 0.2 M ammonium chloride was added to adjust the pH to 6.0. After dialysis against distilled water, the solution was lyophilized. 2.7. Affinity chromatography on mannan–Sepharose Ten milligrams of H+ proteins were dissolved in 4 ml 1 mM HCl and 6 ml PBS and applied to a mannan–Sepharose column (2.5 cm × 11 cm) pre-equilibrated with PBS. Non-mannan-binding (Man− ) proteins were washed with PBS, flow rate 3.2 ml/20 min. Mannan-binding (Man+ ) proteins were first eluted with 3 M NaCl (Man1 + ) and then with 4 M urea (Man2 + ). Fractions 4.6 ml/10 min were collected. These were pooled, de- salted on Sephadex G-25 in 0.2% acetic acid and lyophilized. The same conditions were used for chromatography of fractions of non-heparin-binding proteins or boar seminal plasma. 2.8. Reversed phase high performance liquid chromatography (RP-HPLC) Protein samples (Man− , Man1 + , Man2 + ) were subjected to the inert Biocompatible Quaternary Gradient system of HPLC (Waters, Milford, U.S.A.). RP-HPLC was performed using a 218 TP 104 Vydac C18 column (4.6 mm × 250 mm, 10 m particle size). One mil- ligram of the sample in 1 ml of 0.05% trifluoroacetic acid (TFA) was applied and proteins eluted with a linear gradient of 20–50% acetonitrile (ACN) in 60 min. Fractions correspond- ing to protein peaks were collected and lyophilized. View slide
  • P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a 171 2.9. Electrophoresis and blotting Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of different fractions of boar seminal plasma proteins and ovalbumin was carried out on 15% slab gel (Laemmli, 1970). Non-reduced samples of seminal plasma proteins and reduced pro- tein standards were applied. The relative molecular masses of the separated proteins were estimated by comparison with relative molecular-mass protein standards run in parallel. Tris–glycine buffer (pH 9.6) with 20% (v/v) methanol was used for transfer of proteins separated by SDS-PAGE onto Immobilon-P-membrane or the nitrocellulose membrane for specific detection. Electroblotting was carried out for 1.5 h at 500 mA according to the arrangement described by Towbin et al. (1979). 2.10. N-terminal amino acid sequence determination N-terminal amino acid sequencing was performed in a Protein Sequencer LF 3600 D (Beckmann Instruments) following the Instruction Manual. Proteins isolated by affinity chromatography on immobilized mannan (fractions Man1 + and Man2 + ), by RP-HPLC (fractions 1–5) and separated by SDS electrophoresis were used for the analysis. After SDS electrophoresis, proteins were transferred onto the Immobilon-P-membrane, visualized by Coomassie Blue and subjected to N-terminal amino acid sequencing. Searches for amino acid similarities were carried out using the protein sequence deposits in the BLAST-BASIC e-mail Server Databank. 2.11. Binding studies 2.11.1. Enzyme-linked binding assay (ELBA) 2.11.1.1. Mannan- and heparin-binding activity. Microtiter plates were incubated for 1 h at room temperature with 100 l of bovine serum albumin (BSA) solution (1% in PBS). After extensive washing with PBS, the wells were treated with 100 l of glu- taraldehyde solution (1% in distilled water) for 1 h. After thorough washing with PBS, 100 l of the PBS solution of seminal plasma proteins (100 g/ml) or sperm suspen- sion (500 g/ml) were applied and incubated for 24 h at 4 ◦ C. After extensive washing with distilled water, wells were deactivated using 100 l of BSA solution (1% in PBS) for 1 h at room temperature. The solution of biotinylated ethylenediamine derivative of mannan or polyacrylamide derivative of heparin (100 g/ml) was applied to each well (100 l); wells were incubated for 2 h at 37 ◦ C and then washed with PBS. Afterwards, 100 l of avidin-peroxidase solution (0.25 g/ml) in PBS containing 1% BSA were added to each well and incubated at 37 ◦ C for 1 h. After washing, each well was incubated with 250 l of substrate ABTS solution (10 mg/ml in 0.05 M phos- phate-citrate buffer, pH 5.0, containing 0.012% sodium perborate). After 30 min incu- bation at 37 ◦ C, the reaction was stopped by adding 50 l of 1% SDS. Absorbance at 405 nm was measured using a Micro-plate reader. Three parallel measurements were performed. For inhibition studies, solutions of biotinylated ethylenediamine mannan or heparin-poly- acrylamide derivatives in PBS containing different concentrations of monosaccharides and
  • 172 P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a their phosphates (1.5–100 mM) or polysaccharides and glycoproteins (0.02–1 mg/ml) were applied. 2.11.1.2. Interaction of oviductal epithelium with biotinylated proteins. Microtiter plates were incubated for 1 h at room temperature with 100 l BSA solution (1% in PBS). After extensive washing with PBS, wells were activated with 100 l glutaraldehyde solution (1% in distilled water) for 1 h. After thorough washing with PBS, 100 l of the solution of porcine oviductal epithelium (1 mg/ml PBS) were applied and incubated for 24 h at 4 ◦ C. After extensive washing with distilled water, the wells were deactivated using 100 l of BSA solution (1% in PBS) for 1 h at room temperature. The solution of biotinylated seminal plasma proteins (100 g/ml in PBS) containing mannan (0–2 mg/ml) was applied to each well (100 l); wells were incubated for 2 h at 37 ◦ C and then washed with PBS. Afterwards, 100 l of avidin-peroxidase solution (0.25 g/ml) in PBS containing 1% BSA was added to each well and incubated at 37 ◦ C for 1 h. After washing, peroxidase was incubated with 250 l of substrate ABTS solution (10 mg/ml in 0.05 M phosphate-citrate buffer, pH 5.0, containing 0.012% sodium perborate). After 30 min incubation at 37 ◦ C, the reaction was stopped with 50 l of 1% SDS. Absorbance at 405 nm was measured using a Micro-plate reader. As a control, biotin-labeled BSA was used. 2.11.1.3. Specific detection of proteins blotted onto nitrocellulose membrane. Nitrocel- lulose membrane with transferred proteins was washed with PBS (3 × 10 min), deacti- vated with 3% BSA in PBS (overnight at 4 ◦ C) and washed with 0.02% Tween 20 in PBS (3×10 min). The membrane was then incubated with 0.01% biotinylated mannan derivative in PBS for 2 h. After washing in PBS with Tween (3×10 min), the membrane was incubated in the avidin-peroxidase solution in PBS (3.75 g in 10 ml) for 45 min. After washing with PBS, the membrane was developed in the following solution: 0.05% 4-chloro-1-naphtol, 0.001% (w/v) CoCl2 and 0.09% hydrogen peroxide in 0.01 M Tris–HCl, pH 7.4. After development in the dark, the reaction was stopped by washing with distilled water. Conditions used for the specific detection of ovalbumin blotted onto nitrocellulose mem- brane after SDS electrophoresis. The nitrocellulose membrane with transferred ovalbumin was deactivated with 0.5% gelatin (from cold water fish skin) in PBS for 1 h. Then, the mem- brane was incubated with biotinylated aggregated protein fractions II and III obtained by gel chromatography of boar seminal plasma (40 g/1 ml PBS) for 2 h. The further procedure was the same as in the case of detection with biotinylated mannan derivative. 3. Results 3.1. Interaction of mannan with boar seminal plasma proteins and sperm Two methods were used to study the ability of boar seminal plasma proteins and sperm to interact with complex saccharide structures of yeast mannan: (i) the enzyme-linked binding assay analogous to the ELISA test; in this case, proteins or sperm were immobilized to wells of a microtiter plate; (ii) detection of protein bands transferred onto nitrocellulose membrane after separation by SDS electrophoresis. For these binding studies, biotinylated
  • P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a 173 derivative of mannan with coupled ethylenediamine was prepared. Mannan was isolated from S. cerevisiae according to Haworth et al. (1937). The ELBA assay showed a high mannan-binding activity of heparin-binding proteins and sperm in comparison with that the non-heparin-binding proteins. The dependence of mannan-binding activity on the concentration of proteins and sperm is shown in Fig. 1a. After separation of boar seminal plasma by size exclusion chromatography on Sephadex G-75, five fractions (I–V) with relative molecular masses of >100 000, 55 000, 45 000, 30 000 and 5000–15 000 were obtained; the positive interaction with mannan was detected only in aggregates II and III (Fig. 1b), which have previously been described to be composed only of H+ proteins (Jonáková et al., 2000; Maˇ ásková et al., 2000). No interaction was n observed in the case of the main component of boar seminal plasma aggregate IV containing the PSP I/PSP II heterodimer belonging to H− proteins. The results of ELBA tests were confirmed by specific detection of electroblotted proteins onto nitrocellulose membrane after separation by SDS electrophoresis using the biotinylated derivative of mannan. Out of isolated seminal plasma proteins, spermadhesins of the AQN family and DQH protein exhibited the mannan-binding activity (Fig. 2). The ELBA assay was used further for inhibition studies. The mannan-binding activity of boar seminal proteins was inhibited by ovalbumin and ovomucoid (Table 1). To prove that the saccharide moiety of ovalbumin is preferentially involved in the inhibition, oligosac- charide chains (N-glycans) were released from ovalbumin by the N-glycosidase F (PNGase F) treatment and then separated. Under the conditions used, more than 90% of saccharides present in ovalbumin were released. To compare the inhibitory activity, the concentration range of separated glycans corresponded to the range of saccharide content of ovalbumin used for the testing. The ability of N-glycans to inhibit the mannan-binding activity of boar seminal plasma proteins was slightly higher than that of intact ovalbumin (Table 1). No sig- nificant inhibition was observed in the case of monosaccharides (d-glucose and d-mannose) or their phosphates (d-glucose-6-phosphate or d-mannose-6-phosphates) and heparin. Table 1 Inhibition of mannan-binding activity of the heparin-binding fraction of boar seminal plasma proteins Inhibitor Activitya (%) d-Mannose 100 d-Glucose 100 d-Galactose 75 N-Acetyl-d-glucosamine 100 N-Acetyl-d-galactosamine 100 d-Mannose-6-phosphate 100 d-Glucose-6-phosphate 98 Heparin 98 Ovalbumin 35 Ovomucoid 32 N-Glycans of ovalbumin 30 a Inhibition of the mannan-binding activity expressed in percentage of mannan-binding activity in the presence of an inhibitor (in concentration 3 mM in the case of monosaccharides or their derivatives or 100 g/ml for macromolecular substances) tested in the ELBA assay. The concentration of ovalbumin N-glycans was expressed as ovalbumin equivalents.
  • 174 P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a (a) 0.25 Seminal plasma H+ 0.2 H- Spermatozoa Absorbance at 405 nm 0.15 0.1 0.05 0 500 250 125 63 32 16 8 0 Protein concentration (µg/ml) (b) 0,3 Fraction I 0,25 Fraction II Fraction III Absorbance at 405 nm 0,2 Fraction IV Fraction V 0,15 0,1 0,05 0 500 250 125 63 32 16 8 0 Protein concentration ( µ g/m l) Fig. 1. Mannan-binding activity of boar seminal plasma proteins obtained by affinity chromatography on a heparin-polyacrylamide column (a) and of protein fractions (I–V) obtained by size exclusion chromatography on Sephadex G-75 (b). The binding activity determined by the enzyme-linked binding assay was found to be dose-dependent on the concentration of proteins and sperm. The value of absorbance at 405 nm corresponds to the formation of a product of peroxidase reaction; avidin-peroxidase was bound to the complex of protein with biotinylated derivative of mannan immobilized in the microtiter plate. H+ : heparin-binding proteins, H− : non-heparin-binding proteins. Solutions of proteins (8–500 g/ml), biotinylated ethylenediamine derivative of mannan (100 g/ml) and suspension of sperm (8–500 g/ml) were used. The binding activity was expressed as absorbance at 405 nm.
  • P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a 175 Fig. 2. Specific detection of AQN spermadhesins and DQH protein from boar seminal plasma with biotinylated ethylenediamine derivative of yeast mannan. Proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose membrane. Prestained molecular-mass standards were used – carbonic anhydrase (35 400), soybean trypsin inhibitor (29 000), lysozyme (21 700), and aprotinin (7300). The ability of boar seminal plasma proteins to interact with ovalbumin is shown in Fig. 3. Ovalbumin after SDS electrophoresis and blotting onto nitrocellulose membrane yielded positive reactions with biotinylated aggregates III and II (from the gel chromatographic separation) which are composed only of H+ proteins. Fig. 3. Binding of biotinylated aggregates II (a) and III (b) (obtained by gel chromatography of boar seminal plasma) with ovalbumin. Ovalbumin was separated by SDS-PAGE and electrotransferred to nitrocellulose membrane. Prestained molecular-mass standards were used: myosin (207 000), -galactosidase (120 000), BSA (92 000), egg albumin (55 900), carbonic anhydrase (35 400), soybean trypsin inhibitor (29 000), lysozyme (21 700), and aprotinin (7300).
  • 176 P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a 120 H+ H- 100 Seminal plasma Spermatozoa 80 Activity (%) 60 40 20 0 0 0,2 0,4 0,6 0,8 1 Mannan concentration (mg/ml) Fig. 4. Inhibition of heparin-binding activity of boar seminal plasma proteins and sperm by mannan. Heparin-binding activity was determined using ELBA. Inhibition expressed in percentage of heparin-binding activity in the presence of mannan. The heparin-binding activity in the absence of mannan was equal to 100%. Solutions of seminal plasma (100 g/ml) and heparin-binding proteins (H+ ) (100 g/ml), biotinylated polyacry- lamide derivative of heparin (100 g/ml), yeast mannan (0.03–1 mg/ml) and sperm suspension (500 g/ml) were used. Boar seminal plasma proteins and boar sperm that exhibit a high mannan-binding activ- ity (Fig. 1a) are characterized by their ability to interact with heparin. The heparin-binding activity of both the proteins and sperm was strongly inhibited by yeast mannan (Fig. 4). 3.2. Interaction of boar seminal plasma proteins with porcine oviductal epithelium The ability of boar seminal plasma proteins to interact with porcine oviductal epithelium was assayed by ELBA. Oviductal epithelium adsorbed to microtiter wells interacted with biotinylated H+ proteins and their aggregates in fractions II and III (from the gel chromato- graphic separation) (Fig. 5a). A low interaction was observed in the case of H− proteins and fraction IV, similarly as for the mannan-binding activity. The interaction of H+ proteins and their aggregated forms with oviductal epithelium was inhibited by yeast mannan. The dependence of this interaction on mannan concentration is shown in Fig. 5b.
  • P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a 177 (a) 1,0 0,8 Absorbance at 405 nm 0,6 0,4 0,2 0,0 H+ H- I II IIII IV V (b) 1 0.8 Absorbance at 405 nm H+ 0.6 fr.II - H 0.4 fr.IV 0.2 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Mannan concentration (mg/ml) Fig. 5. Interaction of boar seminal plasma proteins with oviductal epithelium (a) and inhibition of this interaction by yeast mannan (b). The interaction was studied using ELBA. The value of absorbance at 405 nm corresponds to the formation of a product of peroxidase reaction; avidin-peroxidase was bound to the complex of oviductal epithelium with biotinylated protein fractions immobilized in the microtiter plate in the absence (a) and presence (b) of mannan. Solutions of biotinylated H+ , H− and protein aggregates I–V (100 g/ml), oviductal epithelium (1 mg/ml) and mannan (0–2 mg/ml) were used. 3.3. Isolation and characterization of mannan-binding proteins isolated from boar seminal plasma Divinyl sulfone-activated Sepharose was used for the preparation of immobilized yeast mannan. H+ proteins of boar seminal plasma were separated on the mannan–Sepharose
  • 178 P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a Fig. 6. RP-HPLC of mannan-binding proteins (Man+ ) obtained by affinity chromatography on mannan–Sepharose (a) and SDS-PAGE of separated protein fractions (b). Proteins were separated on a 218TP104 Vydac C18 column in 0.05% TFA, with a linear gradient of 20–50% acetonitrile (60 min). Absorbance at 226 nm (—) and acetoni- trile gradient (- - -). Proteins in fractions 1–5. SDS-PAGE of protein fractions under non-reducing conditions. Molecular-mass standards – ovalbumin (45 000), carbonic anhydrase (31 000), soybean trypsin inhibitor (21 500), and lysozyme (14 400) were used. column into three fractions: Man− fraction, which did not interact with the immobi- lized ligand, and Man+ fraction adsorbed to the affinity column and eluted with 3 M NaCl (Man1 + ) and 4 M urea (Man2 + ). A small amount of Man1 + proteins was eluted with 3 M NaCl; the main portion of adsorbed proteins, Man2 + , was obtained using 4 M urea. No proteins present in the H− fraction interacted with immobilized mannan. Only a small amount of protein was obtained from the affinity chromatography of boar seminal plasma. Proteins adsorbed to the mannan–Sepharose column (Man1 + and Man2 + fractions) were separated by RP-HPLC (Fig. 6a), by SDS electrophoresis (Fig. 6b), blotted onto PVDF membrane and identified using N-terminal amino acid sequencing. The following proteins were found in the mannan-binding fraction eluted from the affinity column with 3 M NaCl and 4 M urea: DQH protein, differently glycosylated forms of AQN 2 and AQN 3, truncated forms of AWN 1 and AWN 2 spermadhesins (Table 2). The composition of Man1 + and Man2 + fractions did not significantly differ.
  • P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a 179 Table 2 Proteins identified in the mannan-binding fraction Fractiona Relative N-terminal amino acid sequence Identified protein molecular mass 1 13 000 DQHL . . . DQH protein 2 14 000 AQNKGSD . . . AQN 2 spermadhesin 3 12 000 AQNKGSD . . . AQN 3 spermadhesin 4 14 000 RRS . . . , RSXG . . . , SRSXGVL . . . Truncated forms of AWN 1 spermadhesin 5 15 000 N-terminus blocked AWN 2 spermadhesin a See Fig. 6. 4. Discussion The saccharide-binding ability of sperm-associated proteins has been described in dif- ferent species (Suarez et al., 1998; Töpfer-Petersen, 1999). Many of these proteins from seminal plasma originate in accessory sexual glands and associate with sperm during ejacu- lation. Lectin-type interactions with these proteins are an important phenomenon occurring in different phases of the fertilization process. Growing evidence suggests that such type of interaction participates in the deposition of sperm in the isthmic region of oviduct after its passage through uterus. The formation of the sperm reservoir in the oviduct is accom- plished due to the interaction of sperm surface proteins with the epithelial cells lining the duct (Suarez, 2001). It has been shown that the saccharide-binding specificity of protein receptors varies among species (Töpfer-Petersen et al., 2002). The formation of the oviduc- tal sperm reservoir in pig is the result of high-affinity sites for oligomannosyl residues and low affinity for binding of d-galactose (Wagner et al., 2002). Ovalbumin and mannopentose were found as the most potent inhibitors of sperm binding to the pig oviduct (Wagner et al., 2002). In a previous communication, we have described that yeast mannan inhibited the inter- action of bull seminal plasma proteins with bovine zona pellucida (Liberda et al., 2002). In the present communication, we have studied the mannan-binding proteins in boar seminal plasma. Contrary to bull seminal plasma proteins (Liberda et al., 2002), the interaction of boar proteins with mannan was inhibited most by ovalbumin, its separated N-glycans and ovomucoid, slightly by d-galactose, but not by d-mannose and d-glucose. Direct binding studies proved an interaction of heparin-binding proteins with ovalbumin. The inhibition studies are in good correlation with results of inhibition of sperm binding to pig oviductal epithelium (Wagner et al., 2002). In addition to that, we have shown that boar seminal plasma heparin-binding proteins interacted with porcine oviductal epithelium and this in- teraction was inhibited by mannan. Boar seminal plasma proteins with mannan-binding activity are most probably responsible for formation of the porcine sperm oviductal reser- voir and thus they might participate in modulation or regulation of sperm capacitation in the female oviduct. The second difference between the interaction of boar seminal plasma proteins with mannan and that of bull proteins was the inhibition of heparin-binding activity of these proteins by mannan. We have described (Liberda et al., 2001) that yeast mannan increased
  • 180 P. Jel´nkov´ et al. / Journal of Reproductive Immunology 62 (2004) 167–182 ı a the heparin-binding activity of bull seminal plasma proteins. In the case of boar proteins, mannan strongly inhibited the interaction with this acidic polysaccharide. This might be explained by different roles of proteins exhibiting the mannan-binding activity in the fertil- ization process of these two species. The results of both methods used for studying mannan-binding activity (ELBA (Fig. 1) and specific detection of proteins after electrophoretic separation (Fig. 2)) were in an agreement. Proteins interacting with mannan belong to the heparin-binding ones. Affin- ity chromatography of boar seminal plasma proteins on immobilized mannan–Sepharose confirmed the results of the binding studies. Mannan-binding proteins were isolated from the heparin-binding fraction; no proteins were retained from the non-heparin-binding fraction. The components of the mannan-binding fraction were identified according to relative molecular-mass determination and N-terminal amino acid sequencing: the DQH protein, differently glycosylated forms of AQN, AWN 1 and AWN 2 spermadhesins. All these proteins were found not only in seminal plasma, but also associated with ejaculated sper- matozoa (Jonáková et al., 1998; Veselský et al., 1999) and could be released from the sperm surface using acidic buffer (Jonáková et al., 1991; Dostálová et al., 1994). These proteins adsorbed to the sperm surface might participate in the specific interaction of sperm with the saccharide moiety of the epithelium. The role of AQN spermadhesins and not the AWN ones in the formation of the sperm oviductal reservoir has already been suggested (Töpfer-Petersen et al., 2002). However, these suggestions are not in agreement with the findings that a small portion of only AWN spermadhesin adsorbed to the sperm surface reaches the oviduct (Calvete et al., 1997). The mannan-binding fraction of boar seminal plasma contains components that were also present in the non-interacting fraction. The observed phenomenon may be explained by the presence of different aggregated forms that differ in their binding properties (Jonáková et al., 2000; Maˇ ásková et al., 2000). n Acknowledgements This work was supported by the Grant Agency of the Czech Republic, Grants Nos. 303/02/0433 and 303/02/P069; by the Ministry of Education of the Czech Republic, Grant No. MSM 1131-00001 Grant No. NJ/7463-3 from the Grant Agency of the Ministry of Health of the Czech Republic. References Amari, S., Yonezawa, N., Mitsui, K., Katsumata, T., Hamano, S., Kuwayama, M., Hashimoto, Y., Suzuki, A., Takeda, Y., Nakano, M., 2001. Essential role of the non-reducing terminal -d-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm–egg binding. Mol. Reprod. Dev. 59, 221–226. Bleil, J.L., Wassarman, P.M., 1988. Galactose at the non-reducing terminus of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein ZP3 is essential for the glycoprotein sperm receptor activity. Proc. Natl. Acad. Sci. U.S.A. 95, 6778–6782.
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