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Lewis X-Containing Glycans are Specific and Potent Competitive Inhibitors of the Binding of ZP3 to Complementary Sites on Capacitated, Acrosome-Intact Mouse Sperm¹

Division of Reproductive Biology,³ Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205 The Johns Hopkins Kimmel Cancer Center and Department of Pharmacology and Molecular Sciences,⁴ Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

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 (Le^x)-containing glycan (Galß4[Fuc3]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 Le^x-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 (Le^a)-capped glycan (Galß3[Fuc4]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 GlcNAcß4 or Galß4 residues did not compete; neither did sialyl-Le^x (Neu5Ac 3Galß4[Fuc3]GlcNAc-Lewis X) nor sulfo-Le^x (3'-O-SO₃-Lewis X). However, at saturation, Gal3Galß4GlcNAcß3Galß4Glc reduced Alexa₅₆₈-ZP3 binding by 70% but with moderate apparent affinity (IC₅₀ 3000 nM). Fluorescence microscopy revealed that Alexa₅₆₈-labeled Le^x-Lac-BSA, Le^a-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le^a-Lac did not inhibit binding of Alexa₅₆₈-Le^x-Lac-BSA, and Le^x-Lac did not inhibit binding of Alexa₅₆₈-Le^a-Lac-BSA. Finally, Le^x-Lac and Le^a-Lac had an additive inhibitory effect on Alexa₅₆₈-ZP3 binding. Thus, Le^x is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le^a binding defines a different but less-abundant class of sites.

¹ This work was supported by the National Institute for Child Health and Human Development (NICHD) 1 R01 HD-35699. W.F.H. and C.L.K. were supported in part by NICHD 5T32 HD-07276.

² Correspondence: William W. Wright, Johns Hopkins University Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, Room 3508, 615 N. Wolfe Street, Baltimore, MD 21205. FAX: 410 614 2356; wwright1{at}jhem.jhmi.edu

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