Molecular analysis of a carbohydrate antigen involved in the structure andfunction of zona pellucida glycoproteins

A lactosaminoglycan-associated antigen is associated with a carbohydrate mo iety of all three zona pellucida (ZP) glycoproteins of pig and rabbit but i s absent in the mouse and rat. A monoclonal antibody (PS1) recognizing this determinant was obtained by immunizing mice with a porcine ZP glycoprotein isoform purified by two-dimensional polyacrylamide gel electrophoresis. Co nditions known to remove O-linked or sialic acid carbohydrate moieties (alk aline reduction; O-glycanase or neuraminidase enzymatic cleavage) did not r emove the carbohydrate epitope. However, treatment with endo-beta -glycosid ase, endoglycosidase F, or combinations of neuraminidase plus beta -galacto sidase, totally removed the determinant, indicating that it is associated w ith a poly-N-acetyl lactosaminoglycan structure present on an N-linked olig osaccharide. Molecular morphology studies using immunofluorescence and conf ocal microscopy techniques demonstrate that the PS1 antigen is localized at the surface of the ZR Confirmation of this localization was obtained throu gh studies that show that this antibody will inhibit homologous sperm bindi ng to the pig ZP. Additional analyses using modular contrast microscopy and 3immunocytochemistry demonstrate that this carbohydrate-associated antigen is localized in discrete layers throughout the ZP matrix. These studies ar e the first to demonstrate the presence of a lactosaminoglycan type carbohy drate moiety in all three ZP proteins using a monoclonal antibody that appe ars to be involved in sperm recognition and structural organization.