BIOCHEMICAL-CHARACTERIZATION OF HAMSTER OVIDUCTIN AS A SULFATED ZONA-PELLUCIDA-BINDING GLYCOPROTEIN

Oviductins are a family of glycoproteins. synthesized and released by oviductal secretory cells, which bind to the zona pellucida of the ooc yte after ovulation. Hamster oviductin migrates as diffuse species of 160-350 kDa during SDS/PAGE under reducing as well as non-reducing con ditions. In this report. we describe the one-step purification of hams ter oviductin using either immuno- or lectin-affinity chromatography. Probing with specific lectins showed that the glycoprotein contains te rminal alpha-D-GalNAc, and either terminal alpha-D-NeuAc or non-termin al beta-D-(GlcNAC)2 residues, but fails to react with concanavalin A a nd Ulex Europeus A-1 lectins which are specific for branched alpha-D-m annose and alpha-L-fucose moieties respectively. Intraovarian oocytes do not contain this glycoprotein and we demonstrate here that the immu noaffinity-purified oviductin readily binds to their zonae pellucidae in vitro, thus mimicking the in vivo phenomenon. Two major immunologic ally related forms of hamster oviductin (named alpha and beta) were ch aracterized using one- and two-dimensional gel electrophoresis. The al pha-form (160-210 kDa) has an acidic pl of 3.5-4.5 and the beta-form ( approx. 210-350 kDa) is localized at the cathodic site in the isoelect ric focusing dimension; in between these two major forms lies a smear of minor-charge isomers. Peptide mapping of both major forms with papa in and Staphylococcus aureus V8 protease yielded fragments of identica l size. Moreover, the two forms share the same N-terminal sequence whi ch display no significant homology with other reported proteins. Treat ment with trifluoromethanesulphonic acid showed that a protein with th e size and pI of the alpha-form can be generated from the beta-form. B oth the alpha- and beta-forms are sulphated on 0-linked oligosaccharid e side chains but are not phosphorylated. Collectively, these results suggest that the hamster oviductin polymorphism observed in two-dimens ional PAGE is a consequence of different glycosylation patterns and no t the polypeptide chain itself, Hamster oviductin is mostly O-glycosyl ated and contains a few N-linked oligosaccharide side chains (approx. 10 kDa). We propose that hamster oviductin is a mucin-type glycoprotei n which might act as a protective secretion influencing the first step s of the reproductive process necessary for the normal triggering of f ertilization and early embryonic development.