Conversion of Ser to Thr residues at the sperm combining-site of mZP3 does not affect sperm receptor activity.
Mammalian eggs are surrounded by a thick extracellular coat, the zona pellucida, that is composed of three glycoproteins, called ZP1-3. Sperm recognize and bind to O-linked oligosaccharides attached to Ser-332 and Ser-334 at the sperm combining-site of mouse ZP3 (mZP3). Mutation of either of these Ser residues to a small aliphatic amino acid results in the loss of sperm binding to mZP3 in vitro. Here, we converted both Ser-332 and Ser-334 to Thr residues by site-directed mutagenesis. Recombinant mutant glycoprotein made by stably transfected EC cells was purified and then assayed for its ability to inhibit binding of sperm to ovulated eggs in vitro. Results of these experiments suggest that Thr residues can replace the two evolutionarily conserved Ser residues as acceptors for essential O-linked oligosaccharides at the sperm combining-site of mZP3 without affecting the glycoprotein's sperm receptor activity.
Williams
Zev
Z
Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574, USA.
Litscher
Eveline S
ES
Wassarman
Paul M
PM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D004527:Egg Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D005260:Female; D066298:In Vitro Techniques; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013084:Sperm-Ovum Interactions / Q000235:genetics / Q000502:physiology; D013912:Threonine / Q000737:chemistry; D014162:Transfection; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000072216:Zona Pellucida Glycoproteins; C000606519:Zp1 protein, mouse; C000606521:Zp3 protein, mouse; C035027:egg surface sperm receptor; D013912:Threonine; D012694:Serine