Mol. Hum. Reprod. Advance Access originally published online on March 27, 2006
Molecular Human Reproduction 2006 12(4):275-282; doi:10.1093/molehr/gal028
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Identification and characterization of oviductal glycoprotein-binding protein partner on gametes: epitopic similarity to non-muscle myosin IIA, MYH 9
1National Institute for Research in Reproductive Health, Indian Council for Medical Research, Mumbai, Maharashtra, India
2 To whom correspondence should be addressed at: Department of Biochemistry, National Institute for Research in Reproductive Health, Indian Council for Medical Research, Jehangir Merwanji Street, Parel, Mumbai 400012, Maharashtra, India. E-mail: ushan3{at}gmail.com
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Abstract |
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The mammalian estrogen induced oviductal glycoprotein (OGP) has been known to associate with capacitated sperm, oocytes and developing embryos. This study aimed to identify the putative binding partner of OGP on gametes using N-terminal peptide of bonnet monkey (Macaca radiata) OGP, Nmon, as bait. A protein(s) of molecular size
54 kDa was detected by far-western blot analysis of detergent solubilized human sperm proteins. MALDI-TOF mass spectra analysis of 54 kDa tryptic peptides gave a significant hit to non-muscle myosin heavy chain. Biochemical characterization of 54 kDa was done with antibodies specific to non-muscle myosin IIA, MYH9. The 54 kDa protein, possible breakdown product of MYH9, immunoreacted with MYH9 antibody in western blot analysis. OGP binding to 54 kDa could also be demonstrated in far-western blot analysis of detergent solubilized human sperm proteins and nuclear matrix intermediate filament (NM-IF) preparations from human sperm and mouse oocytes. Far-western blot analysis of MYH9 enriched by immunoprecipitation identified the native 220 kDa protein as OGP-binding partner. The identical and characteristic immunogold localization pattern of Nmon and MYH9 on sperm NM-IF preparation substantiated these findings. The results suggest that OGP binds to both gametes through its interaction with MYH9 through the non-glycosylated N-terminal conserved region of OGP, spanning the residues 11–137. Key words: cytoskeleton/NM-IF/MYH9/oviductal glycoprotein
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Introduction |
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Mammalian oviduct, including human fallopian tube, is the physiological site for sperm capacitation, fertilization and early embryonic development (Yanagimachi, 1994
). Oviduct epithelial cells secrete a major estrogen-induced oviductal glycoprotein, OGP, also called MUC 9. Advances made in understanding the biology of OGP, especially hormonal regulation, cDNA cloning, the gene organization and functional significance, have been the subject of several reviews (Verhage et al., 1998; Buhi, 2002; Bhatt et al., 2004).
Several lines of evidence implicate involvement of OGP in fertilization and protection of oocytes and early embryos. OGP has been reported to have embryotrophic effects (McCauley et al., 2003), enhance sperm capacitation and viability (King and Killian, 1994; Boatman and Magnoni, 1995 McCauley et al., 2003), increase zona binding and penetration (Boatman and Magnoni, 1995; O’Day-Bowman et al., 1996) thereby improving fertilization rate (King and Killian, 1994; McCauley et al., 2003) via specific binding sites on sperm (King and Killian, 1994; Boatman and Magnoni, 1995; O’Day-Bowman et al., 1996; Natraj et al., 2002). OGP-binding sites can be detected only on capacitated sperm (King and Killian, 1994). Further, immunodetection is feasible following cell permeabilization with detergent, deoxycholine. Additionally, the association/binding on permeabilized sperm seems to be of a higher affinity since OGP persists despite extensive washing (McNutt et al., 1992). It is likely that it may be true in other animal systems including human sperm. However, the exact role of OGP in modulating gamete functions is yet to be established.
In vitro studies in our laboratory using purified recombinant bonnet monkey (Macaca radiata) OGP have shown the presence of OGP-binding sites on head and midpiece regions of monkey and human sperm (Natraj et al., 2002) consistent with that of bovine sperm (King and Killian, 1994). Even though the presence of OGP-binding sites on gametes has been known as early as 1994, the biochemical identity of the binding partner remained unexplored. The identification of binding partner/receptor of OGP on gametes would be of value in not only understanding the physiological significance of a highly conserved protein like OGP but also expanding our knowledge on the mechanisms of gamete and embryo viability in the oviduct.
The objective of this study is to identify and characterize the putative OGP-binding partner(s) on sperm.
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Materials and methods |
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Antibodies
Following well-characterized antibodies were used: Rabbit polyclonal antibodies to N-terminal bonnet monkey (Nmon) OGP (Natraj et al., 2002
), human OGP (Rapisarda et al., 1993) and non-muscle myosin IIA (MYH9) (Phillips et al., 1995). Goat anti-rabbit polyclonal antibody tagged with 10 nm gold particles (AuroprobeTM, Amersham Pharmacia, Buckinghamshire, UK).
Proteins
The methodology for preparation of recombinant N-terminal conserved domain of OGP from bonnet monkey (M. radiata) (residues 11–137), described here as Nmon (Natraj, 1999: Natraj et al., 2002), and isolation of enriched human OGP from hydrosalphinx fluid is the same as detailed elsewhere (Rapisarda et al., 1993; Natraj et al., 2002).
Sperm sample preparation
The human semen samples obtained by masturbation after 2–3 day abstinence from 3 normal fertile donors were analysed for sperm count and motility as per World Health Organization (1999) criteria. The leucocyte free sperm samples obtained by swim up technique (Lopata et al., 1976) were analysed for count and washed twice with 0.1 M phosphate buffered saline (PBS) pH 7.4 at 400x g and used immediately.
Sperm sample preparation for SDS-PAGE
Two x 106 sperm per well were treated with detergents (0.5% deoxycholic acid, 0.1% digitonin, 1%Triton X-100) individually or as a mixture in 10 mM Tris, 1 mM EDTA pH 8.0, CocktailTM protease inhibitor (Sigma-Aldrich, USA) for 1 h at 4°C. The cell debris was separated by centrifugation at 16000x g at 4°C. The detergent extracted sperm proteins from the crude membrane extract were dialysed overnight at 4°C against 10 mM Tris-HCl pH 7.0. The ensuing protein pellets were resolved by SDS-PAGE.
Nuclear matrix intermediate filament preparation
Nuclear matrix intermediate filament (NM-IF) preparation of sperm samples was carried out as reported (Markova, 2004). Briefly, swim up sperm samples were adjusted to 10 x 106/ml and incubated with all the buffers in a minimum volume. All the incubations were at 4°C unless specified. Initially, the washed pellet was incubated for 10 min in (100 µl) cytoskeletal buffer (10 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] [HEPES] pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5% Triton X-100, 1X CocktailTM protease inhibitor [Sigma-Aldrich, USA]). Subsequently, spermatozoa were incubated for 10 min in (100 µl) extraction buffer [10 mM HEPES pH 6.8, 250 mM (NH4)2SO4, 300 mM sucrose, 3 mM MgCl2, 0.5% Triton X-100, 1X CocktailTM protease inhibitor]. The sample was then digested by incubation at room temperature for 30 min in (100 µl) digestion buffer (10 mM HEPES pH 6.8, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5% Triton X-100, 1X CocktailTM protease inhibitor, 100–200 µg/ml DNase I [Sigma-Aldrich, USA]). Finally, (NH4)2SO4 was added to a final concentration of 250 mM and incubated for 5 min at room temperature. The digested spermatozoa were washed at 400x g with 0.01 M PBS pH 7.4
NM-IF preparation of zona free mouse oocytes
The procedure used for the preparation of zona-free mouse ovarian oocytes was as described previously (Kyurchiev et al., 1988). Briefly, ovaries harvested from superovulated mice were teased and cumulus-free oocytes were treated with 1% SDS at 72°C, 1 h. The ensuing zona proteins were separated by centrifugation at 400x g and the oocytes were used for NM-IF preparation as described above.
Identification of OGP-binding sites by far-western blotting
The NM-IF was resolved on SDS-PAGE and immunoblotted with phosphate buffer (Natraj et al., 2002). The immunological identification of OGP bound to sperm and NM-IF protein was done by far-western blot analysis (Sambrook and Russell, 2001) using 10 µg/ml Nmon OGP as bait, 1 h at room temperature. Following extensive washing, the blots were developed with primary antibody to Nmon (1 : 1000) followed by peroxidase labelled second antibody (1 : 1000). Detection of the immune reaction was carried out by assaying enzyme using chromogenic substrate 3, 3-diaminobenzidine or ECL (Rodrigues, 1999). Positive control was inclusion of enriched human OGP as bait and antibodies to human OGP as probe. The blots incubated without protein and probed with either normal rabbit serum (NRS) or antibodies to OGP served as other controls.
Mass spectrometric analysis
Far-western blot analysis of detergent solubilized sperm proteins with Nmon identified a 54 kDa band as OGP-binding protein. The band corresponding to 54 kDa was excised from a Coommassie stained preparative (6%) SDS-PAGE gel and subjected to Mass Spectrometric (MALDI-TOF/MS) analysis at Indian Institute of Science, Bangalore, India. Briefly, band corresponding to 54 kDa was excised from a stained polyacrylamide gel (6%) and subjected to in-gel digestion. The protein(s) were reduced and carbamidomethylated using a standard protocol. Trypsin digestion was carried out with one volume of 0.1 mg/ml trypsin (Pierce, Rockford, IL, USA) solution, 5 min followed by 12–16 h at 37°C post rehydration. The purified concentrated peptides were loaded on an Ultraflex TOF/MALDI TOF mass spectrometer (Bruker-Daltonix flex Analysis, Bremen, Germany) and resolved on sinapinic acid matrix.
In silico analysis
Database mining of peptide mass fragment (PMF) spectra obtained by MALDI-TOF was done with following URLs: ProFound version 4.10.5, Peptide Search, MS-Fit Protein Prospector 4.0.5. Masses analysed: 2465.428, 2065.155, 1724.019, 1708.968, 1515.813, 1439.855, 1406.767, 1405.770, 1370.736, 1172.555, 931.653, 924.656, 908.672, 893.659, 893.151, 877.173, 871.653, 785.544, 719.721, 698.602, 682.968, 673.216, 667.278, 655.974, 651.594 and 644.053. Parameters: enzyme trypsin, cysteine modification – Carbamidomethyl-Cys and possible missed cleavage was stated to be 1. Other possible modifications were: N-terminal Gln to pyro Glu, oxidation of methionine and N-terminal acetylation. Mass tolerance was set to 0.1 Da. MS-Fit checked 161 074 entries from SwissProt. 2005.01.06 database with a MOWSE P Factor: 0.4 while ProFound checked NCBInr (2005/06/01) Mammalia (mammals) database.
Cross-linking assay
Protein was electro-eluted by a procedure suggested by the manufacturers (LKB Bromma 2014 Extraphor electrophoretic concentrator, Uppasala, Sweden) from a band corresponding to 54 kDa, excised from a 6% SDS-PAGE gel. Cross-linking was done by a protocol provided by the manufacturer (Pierce Biotechnology, Rockford, IL, USA). Briefly, desalted proteins, Nmon OGP (2 µg/µl) and 54 kDa sperm protein (2 µg/µl), in PBS pH 7.4, were allowed to interact in solution in a minimal volume (10 µl), 60 min at 37°C. The reacted proteins were cross-linked with DSS (Disuccinimidyl suberate, Pierce Biotechnology, USA) to a final concentration of 1 mM, 15 min at 37°C. Reaction was terminated with 1 M Tris pH 7.4 (1 µl) and resolved on SDS-PAGE (10%). Controls included sperm protein and Nmon as uncross-linked proteins as well as DSS cross-linked Nmon. This was followed by immunoblot analysis with specific antibodies to Nmon OGP and MYH9. Detection was done by ECL.
Immunoprecipitation
The interaction of myosin and Nmon was further analysed by immunoprecipitation as detailed by Bonifacino and Dell’Angelica (2003). Sperm proteins were obtained using the Alternate Protocol 2. Myosin was enriched from total sperm extract (20 x 106 sperm/ml) using MYH9 antibody bound to Protein A. Briefly, the protein A was allowed to interact with 2 µl of MYH9 antibody for 1 h at 4°C. It was then equilibrated following washing with non-denaturing lysis buffer (1% Triton X-100, 50 mM Tris.Cl, pH 7.4, 300 mM NaCl, 5 mM EDTA, 10 mM iodoacetamide, 10X Cocktail protease inhibitor). The sperm sample was lysed with denaturing lysis buffer (1% SDS, 50 mM Tris.Cl, pH 7.4, 5 mM EDTA, 10 mM DTT, 10X Cocktail protease inhibitor, 15 U/ml DNase I) and the lysate was precleared with protein A. Simultaneous negative control experiment was set up with NRS. Immunoprecipitation was carried out for 2 h at 4°C. The beads were washed as recommended and heated with Laemmli (1970) buffer. The supernatant following centrifugation at 16 000x g at 4°C was resolved on 7.5% SDS-PAGE and immunoblotted (Towbin et al., 1979). Far-western blot analysis was carried out with Nmon.
EF-EM on sperm NM-IF
Sperm NM-IF preparations were incubated in 10 µg/ml Nmon in PBS pH 7.4, 30 min at 4°C followed by blocking (5% normal goat serum, 0.8% BSA, 0.1% IGSS [gelatin, Amersham Pharmacia, Buckinghamshire, UK] in 50 mM glycine-PBS pH 7.4). This was followed by incubation with primary antibody (1 : 200) against Nmon. Simultaneously, sperm samples following blocking were also incubated with antisera to MYH9 in Incubation buffer (1% normal goat serum, 0.8% BSA, 0.1% IGSS in 10 mM glycine-PBS pH 7.4), 30 min at 4°C, excess antibody was washed and the sample was fixed with 2% paraformaldehyde with 0.05% glutaraldehyde in PBS, 20 min at 4°C. The washed sample was layered on formvar-coated 200 mesh nickel grids overnight at 4°C. Similarly, sperm incubated with BSA in control experiments were incubated either with Nmon antibody or normal rabbit serum and coated on grids. Subsequently, the spermatozoa were incubated with (1 : 5) secondary antibody conjugated to 10 nm gold particles, 75 min at 4°C. The extensively washed grids were contrasted with 0.1% buffered OsO4, 5 min. The grids were viewed in a TEM (Tecnai G2-12, FEI, Eindhoven, Netherlands) at an accelerating voltage of 80 kV. Photographs were captured using Mega View III Sis Ccd camera. Quantitative digital image analysis was performed using analySIS software, version 3.1, supplied by manufacturer. The size of the particles was evaluated on 100 micrographs from 5 grids per sample for each experiment. Labelling density expressed as number of particles/µm2 was calculated.
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Results |
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Detection of OGP-binding partner on sperm
Far-western blot analysis of detergent extracted sperm proteins for the presence of OGP-binding protein (Figure 1) showed that the
54 kDa protein immunoreacted with antibodies to monkey and human OGP only in the presence of OGP as bait. No immune reaction was detected when OGP was omitted or non-specific protein (BSA) was included (data not shown). The OGP-binding sperm protein could be extracted in the presence of both ionic and non-ionic detergents.
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Identification of OGP-binding protein
The PMF spectra of the 54 kDa protein eluted from the 1D SDS-PAGE gel generated 30 peptides, of which 11 peptides gave high intensity peaks. Among the peptides, 2045.006, 1881.006, 1567.818 and 1479.853 are contaminants and were omitted from database mining. Of the remaining 26 peptides, 5 peptides gave a significant hit to cytoskeletal protein myosin heavy chain Va, IIX or myoxin (myosin 12) with 10% total sequence coverage of MYH9 from three different algorithms (Table I). All three algorithms identified myosin heavy chain.
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OGP interaction with cytoskeletal protein MYH9
The ability of Nmon to interact with cytoskeletal protein was demonstrated by far-western blot analysis of sperm and oocyte NM-IF preparation which revealed the presence of 54 kDa protein, (Figure 2A). The immunoreactivity of the 54 kDa protein with MYH9 antibody was demonstrated by western blot analysis of electro-eluted 54 kDa protein and sperm NM-IF preparations (Figure 2B). Far-western blot analysis with Nmon and western blot analysis of sperm whole cell lysate with MYH9 antibody gave a faint band at 220 kDa and a prominent band at 54 kDa (Figure 2C).
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Cross-linking assay
The western blot analysis of Nmon cross-linked to 54 kDa protein and probed with MYH9 antibody revealed a mobility shift in the sperm protein from 54 to 70 kDa after interaction with 14 kDa Nmon. Additionally, the shift was detected only by antibody to MYH9 (Figure 3). Self cross-linked Nmon seemed to lose antigenicity (data not shown).
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Immunoprecipitation
Far-western blot analysis of MYH9 enriched by immunoprecipitation revealed the presence of native MYH9 with a molecular size of 220 kDa (Figure 4) as OGP-binding partner. The negative controls are patently devoid of any band at the corresponding position.
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OGP and MYH9 react at the same site on human spermatozoa
Sperm pre-incubated with Nmon showed intense labelling on head (Figure 5a–c) and midpiece. Similar localization pattern was seen with antibodies to MYH9 (Figure 5d–f). Negative controls included, sperm incubated with BSA and probed with Nmon antibody or with normal rabbit serum showed negligible stray particles (Figure 5g–i). Sperm samples incubated without Nmon and developed with Nmon antibody or second antibody alone also did not show any localization as expected. The characteristic labelling pattern of myosin, 350 nm periodic label, is appreciable at a higher magnification in midpiece (Figure 5c and f).
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The size of the particles (mean ± SEM) on the sperm was 10.13 ± 0.46 nm. An average of 200 particles per sperm cell were counted in test grids. Additionally, the distance between two particles could be better analysed only in the midpiece and was found to be 350 ± 6.06 nm (n = 12). Labelling densities are as indicated in Graph I.
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Discussion |
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The presence of OGP-binding sites on sperm, oocytes and recently on oviductal cilia (McBride et al., 2004
) has been demonstrated by indirect immunogold labelling studies. In the present study we have provided evidence that an interacting protein for OGP in sperm could be MYH9 or a protein with immunological similarity to MYH9.
Far–western blot analysis of detergent extracted sperm using either Nmon (non-glycosylated partial protein) or enriched human OGP (glycosylated native protein) as bait identified 54 kDa sperm protein as an OGP interacting protein. Further, we have also provided data that other OGP specific antibodies (human OGP antibody) could also immunostain the same protein ruling out possible uniqueness or peculiarity of Nmon OGP antibody. Our data with mouse testis and epididymis also revealed that an integral membrane protein of 54 kDa interacts with Nmon OGP (Bhatt, 2003).
Despite the excellent PMF spectra obtained, no similarity to known membrane proteins was observed. This could be attributed to two possibilities: (i) it could be a novel protein or (ii) that OGP-binding protein could be anchored to the membrane through phospho or glycolipid linkages. The latter possibility could be likely since a partial sequence homology to myosin heavy chain with a relevant MOWSE score of 71.2 was obtained (Table I). We were encouraged to speculate that MYH9 could very well be a putative partner to OGP based on several direct and indirect evidences. In vitro, OGP-binding sites were visualized following partial permeabilization and on capacitated sperm (King and Killian, 1994; Natraj et al., 2002) indicating that the membrane is modified so as to allow interaction with OGP, possibly with intracellular proteins. It has been well established that membrane reorganization with lipid rearrangement is known to occur during capacitation. Further, in vitro capacitated sperm have been reported to have actin exposed on the surface of head (Liu et al., 2002, 2005) supporting the possibility that cytotoskeletal proteins are exposed following capacitation. Even though there are no known myosin receptors on the membrane, myosin isoforms do interact with the membrane through negatively charged phospholipids indicating a mechanism for myosin anchoring to membranes (Barylko et al., 2005). Lastly, OGP co-localizes with f-actin at the cleavage furrow of developing blastocysts (Murray and Messinger, 1994) where myosin IIA is known to be present (Simerly et al., 1998). Therefore, identification of cytoskeletal protein as OGP interacting partner which could be membrane associated is highly conceivable. The identity of the electro-eluted 54 kDa protein as MYH9 was verified with the use of MYH9 specific antibodies in a western blot analysis wherein it reacted positively.
Myosin II is known to be enriched in detergent-resistant membranes being tightly bound to these membranes and/or to integral membrane proteins rendering such rafts resistant to extraction with non-ionic detergents such as Triton X-100. The extraction conditions used here are known to extract peripheral membrane proteins (Lasserre et al., 2003) as also exemplified by the database mining results which included GTPase-activating protein and GTP binding protein. Lipid rafts and detergent-resistant membranes contain resident integral membrane and a subset of cytosketetal proteins including actin, myosin II, fodrin, etc. (Chen et al., 2003). Since myosins are reported to localize at the plasma membrane in addition to the cortex (Nebl et al., 2002), it is likely that OGP-binding partner is MYH9. Accordingly, an analysis for OGP-binding sites on nuclear matrix preparation was conducted which enabled analysis with the cytoskeletal proteins alone since the preparation is obtained by extraction of other cellular proteins. The presence of OGP-binding protein in the nuclear matrix preparation from human sperm and zona-free mouse ovarian oocytes established the presence of OGP-binding 54 kDa protein by far-western blot analysis. A western blot analysis of NM-IF also detected 54 kDa protein with antibodies to MYH9.
Interaction with Nmon (14 kDa) in solution detected as a mobility shift from 54 kDa of electro-eluted sperm protein to 70 kDa provides additional supporting data.
The specific interaction of Nmon with MYH9 was further strengthened with far-western blot analysis of MYH9 enriched by immunoprecipitation. A single immunoreactive band was seen at 220 kDa.
Furthermore, the localization pattern of OGP on sperm head and midpiece mirrors the characteristic localization pattern of myosin in fibroblast cells (Verkhovsky et al., 1995) wherein myosin label is localized at regular 350 nm intervals and reinforces the conclusions drawn here.
Thus, these studies indicate that OGP interacts with myosin on gametes. That OGP interacts with a cytoskeletal protein is further corroborated from our studies on bonnet monkey oviduct wherein OGP is localized on oviductal cilia (unpublished results) in a pattern similar to that of myosin as reported previously (Sandoz et al., 1982).
One caveat in this study is the disparity of molecular weight of MYH9. The expected molecular weight of myosin IIA is around 200–250 kDa. However, under the extraction conditions used in this study, we consistently observed the binding of OGP and MYH9 antibody with a protein of molecular size 54 kDa. Indeed, myosin appears to denature from 220 kDa band of myosin to 54 kDa in sperm NM-IF preparations as reported previously (Ocampo et al., 2005). It could be inferred that either the extraction procedure may lead to a degradation of MYH9 to 54 kDa and or the amount of native myosin present in the preparation may not be sufficient to be detected in these blots. Alternately, MYH9 in gametes may be a truncated form of low molecular weight. This premise was verified by conducting experiments that enriched MYH9 preparation. This was achieved by including ten-fold excess concentration of protease inhibitor and immunoprecipitation of detergent solubilized sperm extract with antibodies to MYH9 bound to Protein A Sepharose beads. Other modifications effected were change in the transfer buffer, higher concentration of MYH9 antibody and biotin-avidin signal amplification system. Even though inclusion of higher concentration of protease inhibitor revealed the presence of native MYH9 along with a more prominent band at 54 kDa (Figure 2C), convincing data was obtained with immunoprecipitation which detected native MYH9 with molecular size 220 kDa as OGP-binding protein (Figure 4).
Presence of several myosin isoforms, myosin binding proteins and actin binding proteins to regulate key parameters of cell function is well known. Of the several functions attributed to myosin/actin, those relevant to gametes and their physiological environment, namely oviduct, are: cell shape, motility, acrosome reaction, oocyte activation and cytokinesis. These membrane-related events are mediated by the cytoskeleton and calcium ion concentration (Moreno-Fierros et al., 1992; Murray and Messinger, 1994; Kim et al., 1996; Maciver, 1996; Vlad et al., 1996; Barnett et al., 1997; Breitbart and Spungin, 1997; DiMaggio et al., 1997; Matsumoto et al., 1998; Simerly et al., 1998; Wang et al., 1999; Hernandez-Gonzalez et al., 2000; Terada et al., 2000), and hence it can be speculated that a fallout of OGP–myosin interaction could modulate downstream biological effects possibly through changes in internal calcium ion concentrations. Our results suggest that the conserved N-terminal region of OGP, specifically the region 11–137 residues, interacts with MYH9 and is independent of glycosylation since both glycosylated and non-glycosylated proteins interacted with 54 kDa NM-IF preparation indicating OGP–MYH9 interaction could be part of an evolutionarily conserved mechanism of OGP functions.
In summary, we propose that capacitation-induced changes on the sperm membrane leads to exposure of membrane-associated cytoskeletal protein, myosin, which acts as a protein partner to OGP and the interacting domain could be the non-glycosylated N-terminal conserved region of OGP, spanning the residues 11–137.
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Acknowledgements |
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This study was supported by funds from the Indian Council for Medical Research. Thanks are due to Dr C.P. Puri, Director, for his encouragement during the course of this work. We acknowledge the keen interest and encouragement of Dr Vijaya Raghavan, Head of the Department. We highly appreciate the generous and timely gift of MYH9 antibodies by Dr Robert Adelstein, NIH, USA. We gratefully acknowledge Prof H. G. Verhage, University of Illinois, Chicago, for the gift of antibodies to human OGP and Prof D. Chatterjee, IISc, Bangalore, for the MALDI-TOF analysis. We thank Drs Deepak Modi and Purvi Bhatt for their valuable suggestions. Thanks are also due to Mrs Sushma Khavale for technical help. The Council for Scientific and Industrial Research is also gratefully acknowledged for Senior Research Fellowship awarded to Ms K. M. Kadam (Manuscript number NIRRH/MS/18/2005).
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Submitted on January 9, 2006; resubmitted on February 13, 2006; accepted on February 18, 2006.
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