Porcine oocyte zona pellucida Mr 55,000 glycoproteins: Identification of O‐glycosylated domains

<jats:title>Abstract</jats:title><jats:p>The distribution of O‐linked oligosaccharides on the M<jats:sub>r</jats:sub> 55,000 glycoproteins, ZP3α and ZP3, of the porcine oocyte zona pellucida was examined. Purified preparations of endo‐β‐galactosidase digested ZP3α and ZP3β 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 (αOGP and βOGP) were eluted with buffer containing 50 mM α‐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; αOGP was a 41‐residue glycopeptide with three O‐linked sugar chains. Sequence comparisons revealed a 75% identity between αOGP and a corresponding segment of rabbit rec55 zona protein; 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 βOGP and internal peptides of mouse, hamster and human ZP3 zona proteins. These studies demonstrate that in the case of ZP3α and ZP3β, 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. © 1992 Wiley‐Liss, Inc.</jats:p>