Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer.
Guanylin is a recently isolated peptide consisting of 15 amino acid residues with four cysteines, which may form two intramolecular disulfide bridges, and stimulates intestinal membrane guanylate cyclase. The position of the disulfide linkages of guanylin was predicted from its structural similarity to a heat stable enterotoxin which is thought to be responsible for secretory diarrhoea. Both guanylin, with disulfide positions 4-12 and 7-15, and its disulfide isomer, with disulfides positions 4-15 and 7-12, were chemically synthesized by the solid-phase method and purified. Two specific disulfides were selectively formed and confirmed by sequencing, mass spectrometry and high-performance liquid chromatography in combination with enzymatic cleavage. The structure of both isomers has been investigated in solution by 1H nuclear magnetic resonance spectroscopy. Guanylin exists as a mixture of two stable conformations which have compact spiral structures, from comparison with literature data. In contrast, the disulfide isomer of guanylin shows only a single conformation with an elongated curved plate-like structure. Binding assays were performed using labelled guanylin with membranes obtained from rat jejunum. Both disulfide isomers were investigated by the cGMP assay. Both binding and cGMP assays indicated that the relevant form of disulfide bridges in the intact guanylin was as predicted.
Nokihara
K
K
Central Research Laboratory, Shimadzu Corp., Kyoto, Japan. noki@ssr.shimadzu.co.jp
Wray
V
V
Ando
E
E
Naruse
S
S
Hayakawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D006152:Cyclic GMP / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry*; D005768:Gastrointestinal Hormones; D006801:Humans; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D045265:Natriuretic Peptides; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D012996:Solutions
D004220:Disulfides; D005768:Gastrointestinal Hormones; D045265:Natriuretic Peptides; D010455:Peptides; D012996:Solutions; C072685:guanylin; D006152:Cyclic GMP