Abstract
The fertilization layer of Xenopus laevis eggs is formed by the cortical granule lectin (CGL) binding to its ligand. This lectin is released from the egg cortical granules at fertilization and biologically functions in providing a block to polyspermy, an essential step in the fertilization process. Xenopus laevis CGL has a calcium requiring binding specificity for galactose, and exists as a large oligomeric complex composed of 10-12 CGL subunits. The carbohydrate moiety consists of three types of N-linked glycans: high mannose, hybrid, and complex types. In addition, sialic acid residues are known to be present on the hybrid and complex glycans. Because sialic acids often play an important role in binding interactions, this study was undertaken to evaluate the hypothesis that the binding of the Xenopus laevis CGL glycoprotein to its ligand counterpart is dependent on its N-linked hybrid/complex oligosaccharides. Elucidation of the role of the xlCGL glycans on binding will be not only informative for understanding the block to polyspermy, but for a variety of other functions performed by homologues of xlCGL such as pathogen surveillance and allergic responses in humans. In the current study, two different strategies were employed to modify the N-linked oligosaccharides of the xlCGL and evaluate their role in binding. The first strategy used enzymatic methods to remove different types of N-linked oligosaccharides from xlCGL, whereas the second modified the N-linked oligosaccharides found on xlCGL by expressing a recombinant form of CGL in a yeast (Pichia pastoris) expression system which could only make simple mannose structures and not hybrid/complex oligosaccharides. Modified CGL was then evaluated for binding to its jelly ligand using a plate binding assay. Yeast CGL (yCGL) clones generated previously were evaluated for secretion and purification to enable the binding studies. Although the yCGL clone was found to have an extra 21 amino acids added to its C-terminus from the vector, the clone was found to secrete yCGL into the culture medium at a level of ~1mg/ml, and was partially purified by ammonium sulfate precipitation. Immunoblotting analysis revealed that deglycosylated yCGL (PNGaseF treated) was the appropriate size for the polypeptide (~35kD) whereas glycosylated yCGL had an apparent size of ~65kD, significantly larger than xlCGL (~45kD). Ligand binding activity of yCGL was found to be significantly reduced, but since the preparation was only partially purified due to difficulties, a quantitative analysis was not feasible. Complete removal of xlCGL N-linked glycans by PNGaseF resulted in a 91% decrease of binding indicating that the N-glycans were indeed important. Treatment with two sialidases that remove sialic acid residues reduced binding by 86% with the α2-3 linkage of sialic acid being the most important. Competitive inhibition of xlCGL binding to its ligand using a sialic acid analog, N-acetylneuraminic acid, decreased binding to ~80% at a 2mM concentration, indicating that sialic acid plays a direct role in binding. It is likely that sialic acid is involved in coordinating calcium and facilitating oligomer formation, since calcium promotes oligomer formation and yCGL was unable to form oligomers. Treatment with two mannosidases to remove mannose residues in a variety of linkages (α1-2,3,6) also reduced binding by 86%, which indicated that mannose residues were also important for binding. It is unclear as to the role mannose residues play in binding, but it is hypothesized that they play a structurally supportive role for proper presentation of the binding pocket. Taken together, enzymatic, genetic, and inhibition experiments demonstrate that the N-glycans of the Xenopus laevis cortical granule lectin are required for binding to its ligand. Specifically, terminal sialic acid residues on CGL play an important role in the binding mechanism to its ligand. These experiments have enhanced our knowledge of the CGL-ligand binding mechanism, and are likely to be applicable to the binding interactions of other CGL homologues.