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1 September 2004 Lewis X-Containing Glycans are Specific and Potent Competitive Inhibitors of the Binding of ZP3 to Complementary Sites on Capacitated, Acrosome-Intact Mouse Sperm
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Abstract

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Lex)-containing glycan (Galβ4[Fucα3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888–1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Lex-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa568)-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC50 ∼180 nM), which at saturation, reduced Alexa568-ZP3 binding by ∼70%. A Lewis A (Lea)-capped glycan (Galβ3[Fucα4]GlcNAc) was also a potent inhibitor (IC50 ∼150–200 nM), but at saturation, it reduced Alexa568-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAcβ4 or Galβ4 residues did not compete; neither did sialyl-Lex (Neu5Acα 3Galβ4[Fucα3]GlcNAc-Lewis X) nor sulfo-Lex (3′-O-SO3-Lewis X). However, at saturation, Galα3Galβ4GlcNAcβ3Galβ4Glc reduced Alexa568-ZP3 binding by ∼70% but with moderate apparent affinity (IC50 ∼3000 nM). Fluorescence microscopy revealed that Alexa568-labeled Lex-Lac-BSA, Lea-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Lea-Lac did not inhibit binding of Alexa568-Lex-Lac-BSA, and Lex-Lac did not inhibit binding of Alexa568-Lea-Lac-BSA. Finally, Lex-Lac and Lea-Lac had an additive inhibitory effect on Alexa568-ZP3 binding. Thus, Lex is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Lea binding defines a different but less-abundant class of sites.

Candace L. Kerr, William F. Hanna, Joel H. Shaper, and William W. Wright "Lewis X-Containing Glycans are Specific and Potent Competitive Inhibitors of the Binding of ZP3 to Complementary Sites on Capacitated, Acrosome-Intact Mouse Sperm," Biology of Reproduction 71(3), 770-777, (1 September 2004). https://doi.org/10.1095/biolreprod.103.023812
Received: 30 September 2003; Accepted: 1 April 2004; Published: 1 September 2004
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