Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm.
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 (Gal beta 4[Fuc alpha 3]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 (Alexa(568))-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC(50) approximately 180 nM), which at saturation, reduced Alexa(568)-ZP3 binding by approximately 70%. A Lewis A (Le(a))-capped glycan (Gal beta 3[Fuc alpha 4]GlcNAc) was also a potent inhibitor (IC(50) approximately 150-200 nM), but at saturation, it reduced Alexa(568)-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAc beta 4 or Gal beta 4 residues did not compete; neither did sialyl-Le(x) (Neu5Ac alpha 3Gal beta 4[Fuc alpha 3]GlcNAc-Lewis X) nor sulfo-Le(x) (3'-O-SO(3)-Lewis X). However, at saturation, Gal alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc reduced Alexa(568)-ZP3 binding by approximately 70% but with moderate apparent affinity (IC(50) approximately 3000 nM). Fluorescence microscopy revealed that Alexa(568)-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 Alexa(568)-Le(x)-Lac-BSA, and Le(x)-Lac did not inhibit binding of Alexa(568)-Le(a)-Lac-BSA. Finally, Le(x)-Lac and Le(a)-Lac had an additive inhibitory effect on Alexa(568)-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.
Kerr
Candace L
CL
Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205, USA.
Hanna
William F
WF
Shaper
Joel H
JH
Wright
William W
WW
D000177:Acrosome / Q000502:physiology*; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000378:metabolism; D004527:Egg Proteins / Q000378:metabolism*; D005260:Female; D005643:Fucose / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism; D016256:Lewis X Antigen / Q000031:analogs & derivatives; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013075:Sperm Capacitation / Q000502:physiology*; D014312:Trisaccharides / Q000378:metabolism* / Q000494:pharmacology; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D006023:Glycoproteins; D016256:Lewis X Antigen; D008562:Membrane Glycoproteins; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D014312:Trisaccharides; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse; C110530:galactosyl-(1,4)-fucopyranosyl-(1,3)-N-acetylglucosamine; D012710:Serum Albumin, Bovine; D005643:Fucose; D019158:N-Acetylneuraminic Acid; D007785:Lactose; D005690:Galactose