Characterization of a Mouse ZP3-Derived Glycopeptide, gp55, That Exhibits Sperm Receptor and Acrosome Reaction-Inducing Activityin Vitro

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Abstract
During fertilization, free-swimming mouse sperm bind to mZP3 (∼83 000 Mr), one of three zona pellucida glycoproteins, and once bound undergo the acrosome reaction, a type of cellular exocytosis [Wassarman, P. M., & Litscher, E. S. (1995) Curr. Top. Dev. Biol. 30, 1−19]. Sperm recognize and bind to specific serine/threonine-linked oligosaccharides located at the mZP3 combining site for sperm. Here, we examined certain characteristics of gp55, a ∼55 000 Mr glycopeptide derived from the carboxy-terminal half of mZP3 polypeptide to which sperm bind [Rosiere, T. K., & Wassarman, P. M. (1992) Dev. Biol. 154, 309−317]. gp55 is heterogeneous with respect to Mr (∼47 000−62 000 Mr) and has a relatively low pI (∼4.3−4.5) compared to the polypeptide portion of the glycopeptide (pI ∼6.5). gp55 inhibits binding of sperm to eggs (i.e., exhibits sperm receptor activity) and induces sperm to undergo the acrosome reaction in vitro at about the same concentrations required for intact mZP3 (∼50−200 nM). Each of three different size-fractions of gp55, separated by SDS−PAGE, also exhibits bioactivity in vitro. Removal of asparagine-linked (N-linked) oligosaccharides from gp55, by extensive digestion with N-glycanase, reduces its Mr to ∼21 000 and increases its pI to ∼5.3, but does not significantly affect its ability to inhibit binding of sperm to eggs or to induce sperm to undergo the acrosome reaction. Similarly, digestion of gp55 with either endo-β-galactosidase or neuraminidase alters its Mr and/or pI, but does not significantly affect either of its bioactivities. These observations are consistent with the proposal that neither N-linked oligosaccharides nor sialic acid is an essential element of the mZP3 combining site for sperm. They also indicate that a relatively small mZP3 glycopeptide is able to induce sperm to undergo the acrosome reaction (i.e., cellular exocytosis) in vitro.