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Molecular Human Reproduction vol.2 no.10 pp. 767-774, 1996 The role of carbohydrate in sperm-ZP3 adhesion Neil R.Chapman1-2 and Christopher L.R.Barratt2-3 Departments of 1Molecular Biology and Biotechnology, Firth Court, University of Sheffield, Sheffield S10 2UH and Obstetrics and Gynaecology, Jessop Hospital for Women, Leavygreave Road, Sheffield S3 7RE, UK •'To whom correspondence should be addressed 2 Key words: carbohydrate/cell adhesion spermatozoa/human zona pellucida/ZP3 Introduction Fertilization is the most important cell adhesion event in the life time of any organism. In a number of organisms ranging from echinoderms to mammals, the recognition of carbohydrate epitopes by complimentary protein receptors has been proposed to be a critical factor in gamete interaction (Glabe et al, 1982; Florman and Wassarman, 1985; DeAngelis and Glabe, 1987). The following article briefly describes both how glycoproteins arise in vivo and lists some potential biological roles of the carbohydrate moieties of the given examples. The majority of the article will be concerned with the involvement of proteincarbohydrate interactions in mammalian sperm—zona binding. What is protein glycosylation? Glycoproteins arise in vivo following the covalent linkage of carbohydrate moieties to certain amino acid residues within the polypeptide chain of a newly synthesized protein. There are two main classes of glycosylation, each defined by the nature of the covalent linkage of the carbohydrate residues to the polypeptide backbone. The first type is AMinked glycosylation. The carbohydrate moieties in these structures are attached to the protein backbone through covalent linkages to asparagine (Asn) residues. These Asn residues are located in an amino acid consensus sequence Asn-X-Ser/Thr (where X is any amino acid except Pro) within the protein to be glycosylated. Recently, it has been demonstrated that the amino acid at the X position of this sequon is an important determinant of core glycosylation efficiency (Shakin-Eshleman' et al, 1996). Residues such as Tip, Asp, Glu and Leu were associated with poor N-glycosylation, while Lys, Arg and His had a positive effect on Nglycosylation (Shakin-Eshleman et al, 1996). However, it must be emphasized that the actual presence of this tripeptide within the primary amino acid sequence does not necessarily imply that the protein will be yV-glycosylated at that site (Komfeld and Komfeld, 1985). It is believed that other factors including the nature of neighbouring amino acids and local conformation of the protein are also important determinants of whether a consensus sequence becomes modified. All Nlinked carbohydrates are derived from a common precursor © European Society for Human Reproduction and Embryology which is the sequence of sugar residues as follows (terminal residue first): Manal-3(Manal-6)Manpl^NAGpi^NAGAsn. (Man = mannose, NAG = A/-acetylglucosamine, Asn = Asparagine.) There are three major types of A'-glycosylation: (i) high mannose, where all subsequent sugars attached to the core sequence are mannose; (ii) complex, where both the a l - 3 and a l - 6 terminal mannose residues of the core are linked to Nacetylglucosamine; and (iii) hybrid where the glycosylated structure contains elements of both (i) and (ii). (For a more detailed review of A/-linked glycosylation, see Komfeld and Komfeld, 1985.) The second type of glycosylation is O-linked. In this form of modification, the carbohydrate chain is covalently attached through a glycosidic linkage to a (J-hydroxy amino acid such as serine or threonine. As with AMinked glycosylation, it is believed that local protein structure may be an important determinant as to which Ser or Thr residues are modified (Elliott et al, 1994). In contrast to A'-glycosylation, there is no consensus amino acid sequence flanking the modified amino acid (Wilson et al, 1991). However, comparisons of glycosylated and non-glycosylated amino acids within an Olinked glycoprotein demonstrate that proline residues are often found at the - 1 , - 3 , -6 and +3 positions when this form of glycosylation occurs (Wilson et al, 1991). Glycosylation can therefore give rise to three classes of proteins, those that contain only O-linked glycosylation, those that contain only A'-linked glycosylation and those that possess both classes of carbohydrate modifications. A more detailed review of protein glycosylation can be found in Dwek (1994). Why glycosylate proteins? Proteins appear to become glycosylated for a number of reasons. For example, O-glycosylation appears to be important for the maintenance of structure in the polypeptide chain of ovine submaxillary mucin (Gerken et al, 1989). A'-glycosylation appears to have a number of roles in protein function ranging from the correct folding of polypeptide subunits such as the a-subunit of the nicotinic acetylcholine receptor from Torpedo californica (Rickert and Imperiali, 1995) to the 767 N.R.Chapman and C.L.R.Barratt secretion and dimerization of human interferon-v (Sareneva etal., 1994). At the molecular level, the survival of an organism can depend upon the success of interactions between proteins and carbohydrates; for example cell adhesions between leukocytes and endothelial cells of blood vessels and capillaries are vital for leukocyte extravasation into damaged tissue giving rise to inflammation. Conversely, these protein-carbohydrate interactions may also bring an untimely end to the organism's existence, examples being the binding of some viral particles (influenza) and bacterial toxins (cholera toxin from Vibrio cholerae and pertussis toxin from Bordetella pertussis) to the plasma membrane of eukaryotic cells which is mediated by protein-carbohydrate interactions (see Karlsson, 1995 for a detailed review of microbial recognition of target-cell glycoconjugates). Glycosylation and fertilization In the context of mammalian fertilization, it is generally accepted that a glycoprotein within the zona pellucida termed ZP3 is responsible for specifically binding to complementary receptor molecules located on the head of the spermatozoa. This has been demonstrated to be the case in mice (Bleil and Wassarman, 1980), hamsters (Moller et al., 1990) and pigs (Sacco et al, 1989). Florman and Wassarman (1985) initially demonstrated that the sperm-binding capacity of mouse ZP3 resided within the O-glycosylation present on this glycoprotein since removal of O-linked carbohydrates alone was sufficient to prevent spermzona interaction. Removal of AMinked carbohydrates was found to have negligible effect on sperm-binding activity. More recently, Kinloch et al. (1995) have demonstrated that when five Ser residues located in the C-terminus of mouse ZP3 were mutated to either Gly, Val or Ala then inactive recombinant mouse ZP3 was secreted from transfected embryonal carcinoma cells implying that O-linked carbohydrate located within the C-terminal portion of the ZP3 polypeptide chain was vital for sperm-binding activity. Further work demonstrated that enzymatic removal of terminal galactose or fructose residues from carbohydrate attached to mouse ZP3 with either a-galactosidase or cc-fucosidase respectively caused a reduction in sperm-zona pellucida interaction. The observation that sperm-zona pellucida interactions could be perturbed by the oxidation of the C6 position alcohol of terminal galactose residues present in the 0-linked carbohydrate of mouse ZP3 was also noted in this study (Bleil and Wassarman, 1988). Concomitant with the subsequent reduction of this sugar aldehyde, sperm-binding activity was restored (Bleil and Wassarman, 1988). Taken together, these observations imply that terminal galactose residues of 0-linked oligosaccharides are essential for initiating mouse gamete interaction. In contrast to the observations of Bleil and Wassarman (1988), however, other groups have demonstrated that the amino sugar A'-acetylglucosamine (NAG) is the key terminal monosaccharide involved in sperm—zona interaction in the mouse (Lopez etal., 1985). This view is supported by evidence that implies mouse spermatozoa express a membrane-bound 768 p-galactosyltransferase overlying the acrosomal region (Shur and Neely, 1988). This enzyme utilizes UDP-galactose in binding to NAG moieties terminally located on ZP3 0-linked oligosaccharides (Miller et al., 1992). The involvement of terminal galactose residues in an aglycosidic linkage to penultimate sugar moieties in gamete interaction of the mouse has been questioned recently. Litscher et al. (1995) utilized oligosaccharides with defined structures and sequences to inhibit binding of mouse spermatozoa to unfertilized eggs in vitro. A total of 15 individual oligosaccharides were synthesized; each oligosaccharide differed in chain length and configuration of the glycosidic linkage at the anomeric carbon of the terminal sugar residue. This group demonstrated that a pentasaccharide based on a blood group type-B oligosaccharide was capable of causing 95% inhibition of sperm-zona interaction when used at a concentration of 5 raM. Furthermore, at this concentration, this oligosaccharide structure was seen to promote head-to-head aggregation of mouse spermatozoa. It is possible these observations may be artefacts of the experimental conditions employed, i.e. the spermatozoa were exposed to a very high local concentration of oligosaccharide resulting in a non-specific inhibition of mouse gamete interaction. The observed head-to-head aggregates of mouse spermatozoa may arise through the ability of mouse spermatozoa to recognize and subsequently bind the reducing (anomeric) carbon of the sugar residue. In vivo, this anomeric carbon would be conjugated to the polypeptide backbone of ZP3 via an a-glycosidic linkage: it would therefore be unavailable for interaction with free swimming spermatozoa as in the present study. The study by Litscher et al. (1995) also demonstrated an apparent lack of specificity concerning the binding of these oligosaccharides to spermatozoa. Bi- and tetra-antennary oligosaccharides containing at least six monosaccharide units with the terminal galactose residue in either the a- or pVanomeric configuration inhibited spermatozoa-zona interaction. Furthermore none of the oligosaccharide molecules were able to elicit the spermatozoal acrosome reaction; regarding the latter, it has been suggested by a number of authors that an intact ZP3 polypeptide chain is required for the acrosome reaction to proceed (Florman et al., 1984; Florman and Wassarman, 1985; Leyton and Saling, 1989). The absence of the ZP3 polypeptide backbone in the study by Litscher et al. (1995) is a possible explanation for the lack of spermatozoal acrosome reaction observed with these oligosaccharides. Recently it has been demonstrated that a galactose-al,3galactose (galal,3gal) epitope implicated in sperm-binding to mouse ZP3 is not required for fertilization. The enzyme UDPgalactose:P-D-galactose-al,3-galactose-galactosyltransferase (al,3GT) is responsible for gal-al,3-gal synthesis. Female mice homozygous for the al,3GT transgene developed normally and were able to produce oocytes capable of being fertilized naturally (Thall et al., 1995). Two implications arise from this observation: (i) the absence of terminal a-galactose moieties from O-Iinked carbohydrates in mouse ZP3 are not detrimental to successful sperm-zona pellucida interaction (Nlinked glycosylation has been shown not to actively participate in mouse sperm-ZP3 binding (Florman and Wassarman, 1985), The role of carbohydrate in sperm-ZP3 adhesion therefore the absence of the galal,3gal epitope from this type of glycosylation should not effect mouse gamete interaction to any extent); (ii) if initial gamete interaction in the mouse is carbohydrate mediated, then other carbohydrate epitopes are being recognized by ZP3 receptors located on the spermatozoa, e.g. either NAG (Miller et ai, 1992) and/or fucose (Bleil and Wassarman, 1988). The latter is based on the observation that treatment of mouse ZP3 with a-fucosidase reduced spermzona binding (although fucose was not detected by high performance liquid chromatography analysis). The data concerning al,3GT cannot be extrapolated to human gamete interactions for the simple reason that, although a human homologue of the gene encoding this enzyme exists, it is in fact a pseudogene (an inactive gene) which is not expressed in humans and other Old World primates (Joziasse et ai, 1991). The data concerning the involvement of either N- or Olinked glycosylation in other mammals are equivocal: with regard to gamete interaction in the pig, it is clear that the glycan chains associated with pig ZP3a (renamed pZPB by Gupta et ai, 1995) are responsible for mediating sperm-zona recognition (Sacco et ai, 1989). Whether these glycans are of the N- or 0-linked type is still open to debate since evidence for the involvement of both classes has been put forward: Yurewicz et al. (1991) demonstrated a role for 0-linked glycosylation in sperm-zona pellucida interaction, while more recently N-linked glycosylation has been proposed to be important for successful pig gamete recognition (Yonezawa etal., 1995). Data relating to a role for carbohydrate in human spermzona binding are rare due to the lack of native zonae pellucidae. Initially it was believed that cloning of the human ZP3 gene (Chamberlin and Dean, 1990) would circumvent this lack of material since a 'constant' supply of recombinant material would be available. However, although biologically-active human recombinant ZP3 produced by Chinese hamster ovary (CHO) cells was first reported in 1993 (Barratt et al., 1993), subsequent purification of biologically active material that provides consistent results has presented significant problems in both our own and in other laboratories (Van Duin et ai, 1994; Barratt and Hornby, 1995). We believe characterization of active glycosylated recombinant human ZP3 is made difficult due to the fact that a number of different glycoforms have the potential of being synthesized depending on which cell line is chosen to express the ZP3 cDNA. There are a number of examples of this occurring with other glycoproteins that have been documented in the literature, e.g. tissue plasminogen activator (t-PA). When t-PA was isolated from a Bowes melanoma cell line and a human colon fibroblast cell line, it was shown to contain TV-linked carbohydrate: the nature of which depended upon the cell line used (Parekh et ai, 1989a; Wittwer et ai, 1989a). Furthermore, the in-vitro activities of these t-PA glycoforms were also shown to differ. The reason for the differences in activity correlates with the nature of the specific N-glycosylation site on the polypeptide which was found to differ between the cell lines used (Parekh et ai, 1989b; Wittwer et ai, 1989b). It is the authors' opinion, that sperm-ZP3 interaction in the mouse is an event where a protein receptor(s) binds to a number of different epitopes within the ZP3, i.e. gamete interaction is a process where recognition of gametes relies on multivalent ligand interactions. With regard to the molecular basis of cell adhesion, individual protein-carbohydrate interactions are thought to be very weak and often of broad specificity (Kiessling and Pohl, 1996). However, it should be mentioned that bacterial sugar-binding proteins have high affinities for their substrates, e.g. the arabinose binding protein has a K<j == 1 uM for L-arabinose (Quiocho and Vyas, 1984). Structural studies illustrate that the high affinity of the arabinose binding protein for its substrate arises from the deep binding cleft within its structure. Conversely, some cell adhesion proteins (such as E-selectin) contain only very shallow grooves on their surfaces. These grooves act as the carbohydrate binding sites resulting in a much lower substrate affinity (Graves etal., 1994). Kiessling and Pohl (1996) have suggested that recognition of carbohydrate epitopes by protein receptors is a process where the receptor protein binds to a great many sugar residues. Individual monosaccharide-protein interactions would be very weak but, as the number of interactions increases, then the strength of the entire protein—oligosaccharide interaction also increases. This results in a strong and highly specific interaction. The concept of multivalent ligand binding in high affinity interactions has been suggested for the binding event between the L- and P-selectin proteins and a tetrasaccharide structure termed the sialyl Lewis x (sLex) (Rosen and Bertozzi, 1996). Based on the observations stated above regarding the number of different sugar residues reported to be essential for mouse sperm-ZP3 binding, we suggest that if the sperm-ZP3 binding interaction relies on recognition of ZP3-based carbohydrate epitopes by cognate protein receptors on the spermatozoa, then it is possible the protein receptors involved are recognizing a range of epitopes, rather than a single sequence, e.g. the protein receptor has the ability to bind galactose, NAG and fucose moieties when displayed in a specific geometric pattern on ZP3 (i.e. a multivalent ligand as opposed to a monovalent ligand). In this hypothesis, the ligands (the glycans displayed on ZP3) have a number of different sites with which the protein receptor on the spermatozoa will interact. In relation to human sperm-zona recognition, a study conducted by BarShira Maymon et ai (1994) employed lectins to determine the distribution and identity of carbohydrate moieties on human zonae pellucidae. A number of sugar residues including mannose, p-galactose and N-acetylglucosamine were shown to be present within the human zona pellucida. At present one can only speculate as to whether human gamete interaction also utilizes a spermatozoa-based receptor protein to recognize and specifically bind to a multivalent carbohydrate epitope within ZP3. A number of studies have demonstrated that a polysaccharide termed fucoidan from Fucus vesiculosus has the ability to inhibit a number of cell adhesion events including selectinmediated cell-cell interactions (Foxall et ai, 1992) and spermzona pellucida binding (Oehninger et ai, 1990). Fucoidan is a complex polymer of ocl-3 linked fucose residues, with sulphate covalently attached to the 4-position of certain fucose moieties (Patankar et ai, 1993). To our knowledge, the 769 N.R.Chapman and C.L.R.Barratt mechanism by which fucoidan inhibits human gamete interaction has yet to be fully addressed. According to Oehninger et al. (1991), fucoidan is acting in a competitive manner and as such is masking the zona pellucida receptor(s) located on spermatozoa. It is possible that the nature by which fucoidan inhibits human sperm-zona pellucida interaction depends upon its ability to present a number of sulphated fucose residues in relatively fixed, spatial orientations to the receptor(s) located on the spermatozoa, thereby filling binding sites normally used by ZP3 associated glycans. This would imply that the zona receptor located on the spermatozoa has the ability to recognize a number of sulphated fucose ligands simultaneously, i.e. fucoidan is acting as a multivalent ligand. It is possible to immobilize sugar residues on inert matrices and subsequently examine their effect upon particular binding events. For example, in an attempt to probe the mechanisms underlying intercellular recognition and adhesion, Weigel et al. (1979) demonstrated that rat hepatocytes were able to specifically bind to polyacrylamide gels modified to contain galactose moieties at fixed intervals along the polymer backbone. This system provided a synthetic polyvalent ligand with which the hepatocytes could interact in a specific manner (other monosaccharides such as NAG were utilized but failed to promote hepatocyte adhesion to the gel support). More recently, the use of polyacrylamides to present multiple epitopes to a protein receptor has been successfully employed in the inhibition of influenza virus haemagglutinin attachment to erythrocytes (Spaltenstein and Whitesides, 1991). This group demonstrated that polyacrylamides with multiple a-sialic acid residues attached were more effective inhibitors of haemagglutination than structurally similar monosaccharides. Neuraminidase-treatment of these sialic acid-derived gels completely abolished the inhibition of haemagglutination. It would be of great interest to utilize these types of modified gel to examine the nature of the carbohydrate involved in mammalian sperm-ZP3 interactions. Monosaccharides of interest could be conjugated to acrylamide as described by Kallin (1994). Once poured and set, adhesion of spermatozoa to the glycosylated gel is examined in a similar fashion to that employed by Weigel et al. (1979) for adhesion of rat hepatocytes to immobilized sugars. As stated above, this system would provide a polyvalent carbohydrate ligand to which spermatozoa could bind, with the immobilized sugar acting as a 'terminal' monosaccharide (see Figure 1A-C). However, there are a number of drawbacks associated with a study of this nature. The first problem is concerned with how one defines whether a spermatozoon has bound to the glycosylated gel or not. The study by Weigel et al. (1979) employed rat hepatocytes, which were unable to undergo exocytosis upon binding to their ligand. This is in contrast to spermatozoa from some mammals (e.g. mice) which, in vivo, undergo the acrosome reaction (a specialized form of cell exocytosis) shortly after binding to their ligands (Florman and Storey, 1982). It is possible acrosome-reacted spermatozoa would be removed from the gel during the experminental procedure and give rise to a false negative result, i.e. a proportion of spermatozoa bind the ligand, undergo the acro770 some reaction and are subsequently lost with the removal of non-specifically bound debris. Secondly, it is debatable whether the polyacrylamide used in studies of this nature would be an ideal substitute for the polypeptide chain, i.e. would a polyacrylamide backbone be of sufficient flexibility to facilitate the clustering of receptors on each spermatozoon. Thirdly, the spatial separation of monosaccharide ligands covalently linked to polyacrylamide may not reflect the geometric orientation utilized in the natural ligand. This could potentially lead to a sub-optimal number of receptors on the spermatozoon being occupied by immobilized ligand, preventing tight binding of spermatozoa to the glycosylated gel and causing failure of acrosomal exocytosis (Figure 1D-E). The final major drawback of this system is that only monosaccharides are used. Thus, the effects that penultimate and other deeper saccharides have on both the terminal sugar and spermatozoa binding are not observed. These other carbohydrate residues (including the terminal saccharide) may form part of a sequence with which the spermatozoa interacts; if they are not present the spermatozoa will either bind with a much lower affinity or not bind at all to the immobilized sugar giving the impression that this sugar is not vital for gamete interaction. An appropriate analogy for this hypothetical scenario is obtained from studies into the mechanism of lysozyme. This enzyme is found in a number of body secretions where its function is to hydrolyse the polysaccharide component of bacterial cell walls. The enzyme hydrolyses the glycosidic bond between Af-acetylmuramic acid and A'-acetylD-glucosamine within an active site cleft that is able to accommodate six sugar residues. Binding of carbohydrate to all six subsites is important for successful catalysis since the enzyme will hydrolyse a hexasaccharide 107 times more rapidly than it does a disaccharide (Creighton, 1993). However, despite these caveats, glycosylated gels have been successfully employed in a number of studies investigating the functions of carbohydrates in cell adhesion (see above and references therein). Furthermore, we believe experminents utilizing monosaccharides immobilized on polyacrylamide backbones could prove useful tools to investigate the identity of critical carbohydrate residues involved in mammalian gamete interactions. Is sulphate important actions? in sperm-ZP3 inter- Sulphated proteins arise in vivo through the covalent attachment of sulphate groups to tyrosine residues within the polypeptide chain forming tyrosine O-sulphate and also by modifying carbohydrate residues within certain glycoproteins (Huttner, 1984). Sulphated tyrosine residues and sulphated carbohydrate moieties can exist within the same protein (Huttner, 1984). Shimizu et al. (1983) demonstrated that it was possible to maintain mouse oocytes when grown in follicle culture. This study investigated the biosynthesis of the three mouse zona pellucida glycoproteins. Zonae pellucidae grown in this system were shown to incorporate [35S]-sulphate. Subsequent analysis of individual zona glycoproteins illustrated that [35S] had been incorporated only into ZP1 and ZP2, not ZP3. From a study The role of carbohydrate in sperm-ZP3 adhesion Spermatozoon plasms 11KJIIUJ altt Acrosome Fig. 1A. Poh/acrylamide backbone with covaJently attached monosaccharide units binds spermatozoa •"" = Carbohydrate nx)iety —( = Spermatozoa receptor forZP3hgand Fig. IB Fig. ID Figure 1. Potential use of polyaerylamide gels to investigate the involvement of selected immobilized monosaccharide moieties in spermzona pellucida interaction. This figure illustrates two hypothetical experimental situations. The polyaerylamide backbone presents a number of monosaccharides in arelativelyfixedposition to the spermatozoa. (A) The immobilized sugars bind to receptors located on the spermatozoa, the polyaerylamide backbone facilitates the subsequent clustering of thesereceptors.(B) Acrosomal exocytosis ensues. (C) In contrast, the synthetic backbone could preclude tight binding to the immobilized monosaccharides. The distance between each sugar bound to the polyaerylamide backbone may not reflect the spatial separation occurring between individual monosaccharides in vivo (D-E). of this nature, it is tempting to extrapolate in-vitro observations to an in-vivo situation and suggest that sulphation of ZP3 in vivo is not required for successful mouse gamete interaction. This speculation was not addressed in this study. It would be of interest to investigate the role played by sulphate in the sperm-zona interaction. For example, a possible structure of the mouse zona pellucida was proposed by Greve and Wassarman (1985). This structure has ZP2 and ZP3 closely juxtaposed within the intact zona. It is possible that sulphate within the ZP2 glycoprotein is affecting sperm-ZP3 interaction in an indirect manner. Pronase digested [35S]-sulphate and [3H]-glucosamine-labelled material co-migrated during gel filtration experiments. From this observation it was suggested that the sulphate was attached to the carbohydrate moiety of this glycoprotein. However, whether tyrosine sulphation was present or not within these zonae glycoproteins was not addressed. It has been suggested that [35S]-sulphate incorporation into carbohydrate moieties of glycoproteins can obscure the presence of tyrosine O-sulphate in these macromolecules (Huttner, 1982). Recently, it has been observed that tyrosine sulphation is required for high affinity binding to P-selectin (Wilkins et al, 1995), while sulphation of carbohydrate moieties of the glycoprotein GlyCAM-1 (the ligand for L-selectin) is also required for a productive interaction between this receptor-ligand complex (Rosen and Bertozzi, 1996). The nature of interaction (if any) that sulphate has with cognate receptors on the spermatozoa is unclear, e.g. is this interaction mediated through the anionic nature of the sulphate groups along the ZP2 glycoprotein and any cationic groups (Arg, Lys and possibly His residues) located on the spermatozoal receptor for ZP3? The effect of a high NaCl concentration on the stability of the sperm-zona pellucida interaction would indicate whether ionic interactions are required in this binding process. Sulphation of cell adhesion molecules involved in fertilization in echinoderms has been shown to be critical for the binding of fucose-containing polysaccharides to spermatozoa from the sea urchin Strongylocentrotus purpuratus (DeAngelis and Glabe 1987). Removal of the sulphate moieties from these polysaccharides destroyed the binding activity of the fucans. Sperm-fucan interaction could subsequently be restored by chemical re-sulphation of the fucose-containing polysaccharides. To study the potential role of sulphation in gamete adhesion one would try and prevent the incorporation of sulphate into the molecule of interest (in this case ZP2). Selenate has been shown to be an inhibitor of sulphation of heparan sulphate in rabbit endothelial cells (Dietrich et al, 1988) and also an inhibitor of sulphation of a ligand for P-selectin (also CD62) in myeloid cell lines THP-1 and HL60 (Aruffo et al, 1991). It would be of interest to utilize an inhibitor of this kind in the follicle culture system described by Shimizu et al. (1983). One could then isolate individual zonae pellucidae and examine the effect that sulphate depletion has on sperm-zona interaction. A major drawback of using such an inhibitor in this culture system is the possibility that non-specific inhibition of the sulphation of critical cellular macromolecules may lead to 771 N.R-Chapman and C.L.R.Barratt poor or abnormal growth of the cells in question. Such inhibition has been demonstrated with the amoeba Dictyostelium discoideum. Inclusion of sodium selenate in the growth medium of the D.discoideum arrested vegetative growth of this organism (Davies and Wheldrake, 1986). What is the role of the ZP3 polypeptide backbone in mammalian gamete interaction? The precise function of the polypeptide backbone of ZP3 in fertilization is unclear at present. In the mouse it has been proposed to mediate the aggregation of protein receptors located within the plasma membrane overlying the acrosome in spermatozoa subsequent to the initial attachment of spermatozoa via the O-glycosylation of ZP3 (Florman et ai, 1984; Leyton and Saling, 1989). The protein backbone in humans (and pigs) is likely to play a more significant role. For example, several studies have demonstrated that antibodies against the polypeptide backbone of zona proteins significantly reduce sperm binding to the zona pellucida in humans (Koyama et ai, 1994). Interestingly, Oehninger et al. (1996), using a specific peptide antibody to human ZP3, showed that abnormalities in the polypeptide backbone of the human zona pellucida are a significant cause of fertilization failure during in-vitro fertilization. Furthermore, in pigs, monoclonal antibodies to peptide sequences in porcine ZPC (formerly known as ZP3 beta) significantly block the attachment of boar spermatozoa to the zona (Gupta et ai, 1995). In contrast, antibodies against mouse ZP3 do not block spermatozoa binding to the mouse zona (Vasquez et ai, 1989). We therefore think that the protein backbone of ZP3 may have a more significant role to play in spermatozoa binding in the human (and pig) than the mouse. Presently our group is attempting to produce recombinant human ZP3 in Escherichia coli. The use of bacteria to express mammalian zona proteins has been demonstrated by Prasad et ai (1995). This group demonstrated that it was possible to express the rabbit 55 kDa protein in E.coli, to purify this protein and subsequently use it to elicit an immune response (and hence give a source of polyclonal antiserum in rabbits). The biological activity of the £.co//-derived 55 kDa protein was not examined in this study. The advantages of a bacterial system are that a great deal of information is available concerning both the genetics and physiology of E.coli. It is financially attractive relative to other cell culture systems and, owing to the problems relating to post-translational modifications and the heterogeneous nature of glycosylation described above, we believe that this bacterial system should give a more defined population of ZP3 molecules that are uniformly non-glycosylated. The latter would thereby circumvent the need to use relatively harsh measures to remove the carbohydrate from the polypeptide chain, allowing experimental analysis of the role of the polypeptide chain in sperm binding. To ensure that specific binding of ZP3 is observed spermatozoa must also be exposed to control proteins. Ideally, these proteins should be of a similar molecular weight and pi. If £.co/j-derived ZP3 alone binds to human spermatozoa, it would suggest that glycosylation of ZP3 is not vital for successful human gamete interaction. 772 However, like any system employed to express recombinant proteins, E.coli has its drawbacks (Marston and Hartley, 1990). The major disadvantage is that many recombinant proteins are often expressed as insoluble aggregates, which can interfere with affinity purification protocols. The second major drawback of a bacterial expression system is that it is unclear whether the recombinant protein is folded in a native manner (this is particularly important if the biological activity of a recombinant protein is difficult to assay). For example, the cytoplasm of E.coli does not favour the formation of disulphide bonds (Pollit and Zalkin, 1983). Human ZP3 polypeptide contains 15 Cys residues (two of which reside within the putative /V-terminal signal sequence) (Chamberlin and Dean, 1990). It is possible that at least some of these are required for intra-molecular disulphide bond formation to maintain the tertiary structure of the molecule. To circumvent this difficulty, recombinant proteins can be directed to the periplasmic space of E.coli where disulphide bond formation usually takes place (Takahara et ai, 1988). Secretion vectors have been developed that take advantage of the natural secretory pathways of bacteria. The cDNA encoding the desired protein is placed at the 3' end of a sequence encoding an E.coli secretion signal peptide (such as pelB or OmpA). It may also be possible to use a strain of E.coli that permits cytoplasmic disulphide bond formation. Such a strain has been described by Derman et al. (1993) and is now commercially available. This strain of E.coli should facilitate cytoplasmic disulphide bond formation and hence stimulate the folding of the heterologous protein. However, the ultimate test of whether or not a recombinant protein is correctly folded is its biological activity. In the case of human ZP3, this would involve the determination of specific binding to spermatozoa and the subsequent initiation of the acrosome reaction. Our preliminary results show that rhuZP3 produced in E.coli stimulates a significant increase in phosphorylation of a 95 kDa membrane protein (possibly ZRK, see Burks et ai, 1995; Moore, 1995) as assessed by the in-vitro kinase assay and Western blotting using anti-phosphotyrosine antibodies (Brewis et ai, 1995). However, no induction of the acrosome reaction has yet been observed (Barratt and Homby, 1995). To further examine the role of protein-protein interaction in human sperm-zona binding we have produced recombinant human ZP3 using a commercially-available in-vitro transcription and translation system. This is widely used for rapidly producing and purifying small quantities of protein, and, in mutagenesis experiments, examining interaction between proteins (Boyd et al., 1994; Paroush et ai, 1994; Snyder et ai, 1994; King et ai, 1995). In-vitro translated ZP3 is not glycosylated. Limited proteolytic digestion experiments suggest that the recombinant human ZP3 protein (rhuZP3) has a similar folding pattern to that of mouse native ZP3 (Whitmarsh et ai, 1994) thus overcoming the problem of protein folding which may be apparent in our E.coli. system. In addition, we have immobilized this rhuZP3 on agarose beads. This recombinant product is biologically active, as significantly higher levels of sperm binding to rhuZP3-coated beads were observed compared with controls, and significant induction of the acrosome reaction was recorded (Barratt and Hornby, The role of carbohydrate in sperm-ZP3 adhesion 1995). Thus, we are encouraged to believe that the polypeptide backbone of human ZP3 does possess some biological activity. Conclusions Clearly, a more detailed characterization of the molecules involved in initial gamete interaction is warranted. 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