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.
Highlights
From the Department of Obstetrics and @necobgy, Wayne StateUniversity School of Medicine, The C
The carbohydrate moiety of purified Z P 3 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 Z P 3, as well asby the sensitivity of Z P 3 to digestion with Escherichia freundii endo-&galactosidase
Immunological evidence indicates that thezona pellucida glycoproteins represent a unique class of extracellular matrix macromolecules since both polyclonal and monoclonal antibodies directed against zona pellucida antigens arehighly tissue-specific (1113)
Summary
A method for the purification to apparent electrophoretic homogeneity of the M, = 55,000 family of charge isomers (ZP3) of porcine oocyte zona pellucida is described in detail for the first time. A primary concern is whether the differences in the biochemical and immunological properties of the a- and @-glycoproteins of ZP3(EBGD) represent differences in the primary structures of the polypeptide and carbohydrate moieties of separate gene products or differences resulting from either proteolytic modification or variationsinpost-translational glycosylation of a common polypeptide precursor. The presence of @-glycoproteincould conceivably beattribis largely a consequence of the occurrence of overlapping families of charge isomers corresponding to two structurally and immunologically distinct lactosaminoglycan-containing glycoproteins. Zona Pellucida Glycoproteins of 38,000 and 35,000 for the core polypeptides of a-and @- 10. B., Faltynek, C.R., Hof, L., Reichert, L.E., and cal assistance of Sue-Ya Zhang, James DeMates, and JihadZahran
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