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

Abstract

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.

Protein-Carbohydrate Interactions during Fertilization

“…The O-glycosylated domain of the pZPB protein only retained sperm receptor activity, indicating that both ZP glycoproteins may have a different O-glycosylation pattern. The poly-N-acetyllactosamine units, however, do not appear to play a role in sperm binding [Yurewicz et al, 1992]. The amino acid sequences of the O-glycosylated regions are highly conserved within the ZPB and ZPC protein families.…”

“…According to the deduced cDNA-encoded amino acid sequence, each of the proteins may possess up to five potential N-glycosylation sites [Harris et al, 1994]. Carbohydrate composition analysis, however, suggested that the pZPB and pZPC proteins contain three or four N-linked oligosaccharide chains and additionally three (pZPB) and six (pZPC) O-linked glycan chains [Yurewicz et al, 1992].…”

Molecular Mechanisms Underlying Pig Oocyte Maturation and Fertilization

“…In the pig, preincubation of boar sperm with ZP3 or O-linked glycans in hibits s perm att achm en t to zo na pellucida, and ligand competition bioassays suggest that O-glycans mediates, at least in part, the sperm adhesive properties of ZP3 [63]. In pZP3α and pZP3β, the homologues of rabbit rec55 a n d m o u s e Z P 3 , r e s p e c t i v e l y , O -l i n k e d oligosaccharides are confined within delaminated domains rather than widely dispersed on the polypeptide backbone [64]. However, the role of pZP3 in primary recognition is still not defined [65].…”

Molecular Basis of Mammalian Gamete Binding

“…Conclusions about the biological activity of purified porcine zona proteins have this caveat. Another difference between the porcine and mouse zona is evidence that the sperm-binding oligosaccharides may be linked to the porcine zona protein through both asparagine and serine/threonine linkages (Yurewicz et al, 1992;Yonezawa et al, 1997;Nakano and Yonezawa, 2001). Further study is necessary to determine whether the glycosylation differences may contribute to species specificity during spermoocyte binding.…”