Structural characterization of the Mr = 55,000 antigen (ZP3) of porcine oocyte zona pellucida. Purification and characterization of alpha- and beta-glycoproteins following digestion of lactosaminoglycan with endo-beta-galactosidase.

Abstract

The major macromolecular component of the porcine oocyte zona pellucida is a Mr = 55,000 antigen, termed ZP3, comprised of greater than 25 charge isomers. ZP3 was purified to apparent electrophoretic homogeneity from nonreduced, sodium dodecyl sulfate-treated porcine zonae pellucidae by chromatography on Sephacryl S-400 and hydroxylapatite resins. The carbohydrate moiety of purified ZP3 was comprised of a heterogeneous population of acidic lactosaminoglycans as evidenced by the saccharide composition and size distribution of glycopeptides produced by Pronase digestion of ZP3, as well as by the sensitivity of ZP3 to digestion with Escherichia freundii endo-beta-galactosidase. Endo-beta-galactosidase-digested ZP3 was resolved by gel electrophoresis into two components, termed alpha-glycoprotein and beta-glycoprotein, with Mr values (nonreduced) of 46,000 and 42,000, respectively. Each was comprised of fewer and more neutral charge isomers than ZP3. Following purification by reverse phase high performance liquid chromatography, the alpha- and beta-glycoproteins of endo-beta-galactosidase-digested ZP3 were distinguished on the basis of amino acid and carbohydrate compositions, amino-terminal sequence analyses and peptide mapping experiments, thus suggesting differences in the primary structures of their respective polypeptide moieties. Corresponding dissimilarities in the immunoreactivities of the alpha- and beta-glycoproteins toward polyclonal antisera raised against ZP3, alpha-glycoprotein, and beta-glycoprotein were revealed by competitive binding radioimmunoassays as well as by immunoblotting experiments. Collectively, the data were interpreted to indicate that the Mr = 55,000 antigen of porcine oocyte zona pellucida is in fact comprised of overlapping families of charge isomers corresponding to two structurally and immunologically distinct lactosaminoglycan-containing glycoproteins.