The relevance of N-linked glycosylation to the binding of a ligand to guanylate cyclase C.
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.
Division of Protein Organic Chemistry, Institute for Protein Research, Osaka University, Osaka, Japan.
M
M
Hasegawa
Y
Y
Hidaka
A
A
Wada
T
T
Hirayama
Y
Y
Shimonishi
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry; D016367:Baculoviridae / Q000737:chemistry; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D016208:Databases, Factual; D004305:Dose-Response Relationship, Drug; D004768:Enterotoxins / Q000737:chemistry; D029968:Escherichia coli Proteins; D006031:Glycosylation; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014508:Urea / Q000494:pharmacology
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008024:Ligands; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C034151:heat stable toxin (E coli); D014508:Urea; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D006152:Cyclic GMP