Structural Characterization of the N‐Linked Carbohydrate Chains of the Zona Pellucida Glycoproteins from Bovine Ovarian and Fertilized Eggs

Abstract

The N-linked oligosaccharides that were released by hydrazinolysis from glycoproteins of zonae pellucidae of bovine ovarian eggs, were composed of neutral (23%) and acidic (77%) carbohydrate chains; almost all the acidic chains were neutralized by sialidase digestion. Sugar mapping analysis of pyridylaminated N-linked chains by reverse-phase and normal-phase HPLC and 500-MHz 'H-NMR spectroscopy revealed that the major neutral chain is a high-mannose-type oligosaccharide and the acidic chains are di-, tri-, and tetra-antennary, fucosylated complex-type chains that have N-acetyllactosamine repeats in the non-reducing regions. The structures of the neutral chain and the core regions of the acidic chains of N-linked oligosaccharides from the zona proteins of fertilized eggs, which were obtained by the in vitro fertilization method, were essentially the same as those of the ovarian egg zonae. The amount, however, of the acidic chains decreased to 32 mol/l00 mol in the fertilized egg zonae, which suggests that a sialidase released from the oocyte during fertilization operates on the zona glycoproteins.Keywords: zona pellucida; carbohydrate structure ; glycoprotein ; mammalian fertilization ; egg.The mammalian oocyte is surrounded by an extracellular matrix called the zona pellucida. The zona plays important roles during fertilization including species-specific sperm binding, blocking of polyspermy, and protection of the embryo. The zona of most mammals consists of three glycoprotein components, which have N-linked and 0-linked carbohydrate chains. It has been proposed that oligosaccharides of the zona protein are involved in sperm binding [I, 21. In the mouse, the a-galactosyl residue [3] or N-acetylglucosaminyl residue [4] at the non-reducing end of the 0-linked chain of the ZP3 glycoprotein possesses sperm receptor activity. In the pig, neutral N-linked chains of pZP3a glycoprotein have been shown to have receptor activity (5, 61.We have elucidated the structure1 features of the N-linked chains of the porcine PZP3 family and mouse ZP2 and ZP3 components [S, 7, 81. N-linked chains of PZP3 consist of di-, tri-, and tetra-antennary, complex-type neutral (28 %) and acidic (72%) chains. The acidic chains have sialic acids at the nonreducing ends of N-acetyllactosamine repeats and sulfates at the C6 position of GlcNAc residues of the repeats. core regions. The sulfate contents in the mouse ZP2 and ZP3 are much smaller than those in the porcine PZP3 family, and , 8-GalNAc and a-Gal residues, which are absent from the porcine chains, are found at the non-reducing ends of mouse N-linked chains.We characterized the protein moieties of zona glycoproteins from the unfertilized and fertilized bovine eggs [9]. Furthermore, we have observed that N-linked chains of bovine zona proteins possess the sperm receptor activity; the number of sperm binding to the bovine zona is reduced after fertilization (Mitsui, s., Katsumata, T., Yonezawa, N. and Nakano, M., unpublished results). In this study, we analyzed the structures of N-linked ...

Localization of N‐linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida

“…The pyridylaminated neutral fraction of N-linked chains was chromatographed on a Shim-pack CLC-ODS column (6 · 150 mm; Shimadzu) by a linear gradient from 0.1 to 0.25% 1-butanol in 10 mM sodium phosphate (pH 3.8) for 60 min at a flow rate of 1 mLAEmin )1 at 55°C [14]. The fluorescence intensity was monitored with excitation at 320 nm and emission at 400 nm.…”

“…The fluorescence intensity was monitored with excitation at 310 nm and emission at 375 nm. The pyridylaminated acidic fractions were also analyzed using both columns after digestion with Arthrobacter ureafaciens sialidase (Nacalai tesque, Kyoto, Japan) in 0.1 M ammonium acetate (pH 5.0) at 37°C for 24 h. The glucose units of the pyridylaminated N-linked chains were calculated from their retention times using the pyridylaminated glucose oligomers as the standard [14,21].…”

“…In ZPC, the triantennary/tetraantennary chains are mainly localized at Asn271 in the C-terminal region [13]. The sperm-binding activity of porcine ZPA, a minor component, has not been assessed because its purification is difficult.The N-linked chains of the bovine zona protein mixture include neutral (23%) and acidic (77%) chains [14]. We have determined the structures of the neutral chain and the core regions of the acidic chains [14].…”

“…The N-linked chains of the bovine zona protein mixture include neutral (23%) and acidic (77%) chains [14]. We have determined the structures of the neutral chain and the core regions of the acidic chains [14].…”

“…We have determined the structures of the neutral chain and the core regions of the acidic chains [14]. The acidic chains are diantennary, triantennary and tetraantennary, fucosylated complex-type chains with a tandem N-acetyllactosamine repeat in the nonreducing regions to which the main sialic acids are attached.…”