Porcine oocyte zona pellucida M(r) 55,000 glycoproteins: identification of O-glycosylated domains

• Published in: Molecular reproduction and development Vol. 33; no. 2; p. 182
• Main Authors: Yurewicz, E C, Pack, B A, Sacco, A G
• Format: Journal Article
• Language: English
• Published: United States 01.10.1992
• Subjects: Amino Acid Sequence; Animals; Chromatography, Affinity; Egg Proteins; Glycopeptides - analysis; Glycosylation; Mammals; Membrane Glycoproteins - isolation & purification; Molecular Sequence Data; Molecular Weight; Peptide Mapping; Protein Processing, Post-Translational; Receptors, Cell Surface; Sequence Alignment; Swine - metabolism; Zona Pellucida - chemistry; Zona Pellucida Glycoproteins
• ISSN: 1040-452X

The distribution of O-linked oligosaccharides on the M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of the porcine oocyte zona pellucida was examined. Purified preparations of endo-beta-galactosidase digested ZP3 alpha and ZP3 beta were reduced and carboxamidomethylated and digested with trypsin. When the trypsin digests were mapped by HPLC, each glycoprotein yielded only one N-acetylgalactosamine containing glycopeptide. Purification of the O-glycopeptides was achieved by a two-step protocol. Tryptic digests were applied to jacalin-agarose and specifically-bound O-glycopeptides (alpha OGP and beta OGP) were eluted with buffer containing 50 mM alpha-methylgalactoside as the haptenic sugar. Further purification of each O-glycopeptide was accomplished by reverse phase HPLC. Purified O-glycopeptides were characterized with respect to amino acid and carbohydrate compositions and sequenced by automated Edman degradation; alpha OGP was a 41-residue glycopeptide with three O-linked sugar chains. Sequence comparisons revealed a 75% identity between alpha OGP and a corresponding segment of rabbit rec55 zona protein; beta OGP was a 25-residue glycopeptide characterized by the presence of one N-linked and five O-linked sugar chains and a trypsin-resistant internal arginine residue. Sequence alignments revealed an 80% or greater identity between beta OGP and internal peptides of mouse, hamster and human ZP3 zona proteins. These studies demonstrate that in the case of ZP3 alpha and ZP3 beta, the pig homologues of rabbit rec55 and mouse ZP3, respectively, O-linked oligosaccharides are confined within delimited domains rather than widely dispersed on the polypeptide backbone. Such clustering of O-linked oligosaccharides may represent an essential determinant of the structure and biological activity of zona proteins.