The role of carbohydrate moieties at the N-linked glycosylation sites of guanylate cyclase C (GC-C), a receptor protein for guanylin, uroguanylin and heat-stable enterotoxin, in ligand binding and structural stability was examined using site-directed mutagenesis of the putative N-linked glycosylation sites in the extracellular domain (ECD) of porcine GC-C. For this purpose, eight mutant proteins of ECD (N9A, N20A, N56A, N172A, N261A, N284A, N334A and N379A) and six mutant proteins of the complete GC-C (N9A, S11A, N172A, T174A, N379A and T381A) were prepared, in which Ala replaced Asn, Ser and Thr at the N-linked glycosylation consensus sites. All the mutant proteins showed a ligand-binding affinity (K(d)) similar to those of the wild-type proteins, although the deletion of a carbohydrate moiety at each of the N-linked glycosylation sites affected the ligand-binding ability of ECD or GC-C to some degree. However, the mutant proteins of ECD (N379A) and GC-C (N379A and T381A) showed considerably decreased binding ability in the context of maximum capacity (B(max)) to a ligand, despite the fact that the expression levels of these mutant proteins were nearly the same as the wild-type proteins. Moreover, the mutant protein of ECD (N379A) was considerably less stable to a denaturant. These results clearly indicate a crucial role for the carbohydrate moiety at N379, which is located near the transmembrane region, in structural stability, the ability to bind to a ligand and the cyclase catalytic activity of GC-C, and provide a route for the elucidation of the mechanism of the interaction between GC-C and a ligand.