Biochemical characterization of hamster oviductin as a sulphated zona pellucida-binding glycoprotein

Abstract

Oviductins are a family of glycoproteins, synthesized and released by oviductal secretory cells, which bind to the zona pellucida of the oocyte after ovulation. Hamster oviductin migrates as diffuse species of 160-350 kDa during SDS/PAGE under reducing as well as non-reducing conditions. In this report, we describe the one-step purification of hamster oviductin using either immuno- or lectin-affinity chromatography. Probing with specific lectins showed that the glycoprotein contains terminal alpha-D-GalNAc, and either terminal alpha-D-NeuAc or non-terminal beta-D-(GlcNAc)2 residues, but fails to react with concanavalin A and Ulex Europeus A-1 lectins which are specific for branched alpha-D-mannose and alpha-L-fucose moieties respectively. Intraovarian oocytes do not contain this glycoprotein and we demonstrate here that the immunoaffinity-purified oviductin readily binds to their zonae pellucidae in vitro, thus mimicking the in vivo phenomenon. Two major immunologically related forms of hamster oviductin (named alpha and beta) were characterized using one- and two-dimensional gel electrophoresis. The alpha-form (160-210 kDa) has an acidic pI of 3.5-4.5 and the beta-form (approx. 210-350 kDa) is localized at the cathodic site in the isoelectric focusing dimension; in between these two major forms lies a smear of minor-charge isomers. Peptide mapping of both major forms with papain and Staphylococcus aureus V8 protease yielded fragments of identical size. Moreover, the two forms share the same N-terminal sequence which display no significant homology with other reported proteins. Treatment with trifluoromethanesulphonic acid showed that a protein with the size and pI of the alpha-form can be generated from the beta-form. Both the alpha- and beta-forms are sulphated on O-linked oligosaccharide side chains but are not phosphorylated. Collectively, these results suggest that the hamster oviductin polymorphism observed in two-dimensional PAGE is a consequence of different glycosylation patterns and not the polypeptide chain itself. Hamster oviductin is mostly O-glycosylated and contains a few N-linked oligosaccharide side chains (approx. 10 kDa). We propose that hamster oviductin is a mucin-type glycoprotein which might act as a protective secretion influencing the first steps of the reproductive process necessary for the normal triggering of fertilization and early embryonic development.

Oviductins possess chitinase‐ and mucin‐like domains: A lead in the search for the biological function of these oviduct‐specific ZP‐associating glycoproteins

“…Oviductins are heterogeneous glycoproteins. Indeed, hamster oviductin was shown to consist of at least two principal charge isoforms, the a and p forms, that possess different levels of glycosylation (Malette and Bleau, 1993). We have recently proposed (Malette and Bleau, 1993) that the heterogeneity of hamster oviductin, also reported in other species (Gandolfi et al, 1989;Buhi et al, 1990;Boice et al, 1990a;Donnelly et al, 1991;Rapisarda et al, 1993), might target different populations of the glycosylated molecules to distinct cellular localizations.…”

“…Extensive charge heterogeneity is a common property of all oviductins hitherto characterized (see Table 2): the presence of predominantly acidic and basic isoelectric variants was also reported for the baboon , human (Rapisarda et al, 1993), and porcine (Buhi et al, 1990) that a protein with the PI of the a form can be generated from the p form (Malette and Bleau, 1993).…”

“…Analyses of post-translational modifications using lectin binding experiments revealed that the mouse (Kapur and Johnson, 1985), bovine (Sendai et al, 1993), and hamster (Malette and Bleau, 1993) oviductins react with the WGA lectin thus indicating the presence of terminal a-D-NeuAc and/or nonterminal p-D-(GlcNAc)2 residues in their oligosaccharide side chains. Bovine oviductin binds peanut lectin (Wegner and Killian, 1992) which is specific for galactosyl (p1,3)Nacetylgalactosamine residues, confirming that it bears Sequence comparison of the repeated motifs found in oviductins.…”

“…N-linked carbohydrate side chains. Hamster oviductin possesses terminal N-acetyl-a-D-galactosamine residues (Malette and Bleau, 1993), which are specific for O-linked glycans.…”

“…In this review, we present the complete amino acid sequence of the processed hamster oviductin polypeptide, critically examine the relationships between the biochemical and immunological characteristics of oviductin molecules from various species, and report on the striking finding of chitinase-like and Sermhr rich, repeated mucin-type units in these glycoprotein structures. Finally, we propose that oviductins might poten- Buhi et al, 1990, 1994Buhi et al, 1990 Malette and Bleau, 1993;Merlen et al, 1994 and 8.0 70.890d 7 Suzuki et al, 1994 aApparent molecular mass on reducing SDS-PAGE. bRefers to the number of potential sites of N-glycosylation.…”