O-linked trisaccharide and N-linked poly-N-acetyllactosaminyl glycans are present on mouse ZP2 and ZP3

Mammalian oocytes are surrounded by an extracellular glycocalyx, the zona pellucida (ZP). In the mouse, the ZP is composed of three glycoproteins, designated mZP1, mZP2, and mZP3. Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) receptors for spermatozoa. In this paper we present evidence for the occurrence of poly-N-acetyllactosaminyl glycans and an O-linked trisaccharide on mZP2 and mZP3. When exhaustively digested with endo-beta-galactosidase, an enzyme known to cleave repeating units of acetyllactosamine (3Gal beta 1, 4GlcNAc beta 1), mZP2 and mZP3 showed an apparent reduction in size by 23 kDa and 16 kDa, respectively. Experimental evidence included in this report indicates that polylactosaminyl glycans are present on N-linked sugar chains. In addition, O-linked sugar chains of mZP3 have been characterized. First, treatment of de-N-glycosylated mZP3 with O-glycanase in the presence of exo-glycosidases (sialidase, alpha-L-fucosidase, and N-acetylglucosaminidase) caused an apparent reduction in its size by 2-3 kDa as determined by SDS-PAGE. Second, treatment of the de-N-glycosylated mZP3 with mild alkali in the presence of 1 M NaB3H4 released radiolabeled oligosaccharide (OS) that eluted from a high-resolution Bio-Gel P-4 column at the position of a trisaccharide. The radiolabeled OS had the following structure: GlcNAc-->Gal beta 1,3GalNAcol. The structure was established by sizing on the Bio-Gel P-4 column, followed by examination of the susceptibility of the OS to exo-glycosidases and by its absorbability to immobilized lectin (PNA). Potential roles of N-linked and O-linked sugar chains in sperm-egg interaction are herein discussed.