Synthesis, biological activity and isomerism of guanylate cyclase C-activating peptides guanylin and uroguanylin.
Recently, the peptides guanylin and uroguanylin were identified as endogenous ligands of the membrane-bound guanylate cyclase C (GC-C) that is mainly expressed in the intestinal epithelium. In the present study, bioactive guanylin and uroguanylin have been prepared by solid-phase methodology using Fmoc/HBTU chemistry. The two disulfide bonds with relative 1/3 and 2/4 connectivity have been introduced selectively by air oxidation of thiol groups and iodine treatment of Cys(Acm) residues. Using this strategy, several sequential derivatives were prepared. Temperature-dependent HPLC characterization of the bioactive products revealed that guanylin-related peptides exist as a mixture of two compounds. The isoforms are interconverted within approximately 90 min, which prevents their separate characterization. This effect was not detected for uroguanylin-like peptides. Synthetic peptides were tested for their potential to activate GC-C in cultured human colon carcinoma cells (T84), known to express high levels of GC-C. The results obtained show that both disulfide bonds are necessary for GC-C activation. The presence of the amino-terminally neighboring residues of Cys104 for guanylin and Cys100 for uroguanylin has been found to be essential for GC-C stimulation. Unexpectedly, a hybrid peptide obtained from substitution of the central tripeptide AYA of guanylin by the tripeptide VNV of uroguanylin was not bioactive.
Klodt
J
J
Niedersächsisches Institut für Peptid-Forschung (IPF), Hannover, Germany.
Kuhn
M
M
Marx
U C
UC
Martin
S
S
Rösch
P
P
Forssmann
W G
WG
Adermann
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002712:Chlorides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D005768:Gastrointestinal Hormones; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D008969:Molecular Sequence Data; D045265:Natriuretic Peptides; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D014407:Tumor Cells, Cultured
D002712:Chlorides; D005768:Gastrointestinal Hormones; D045265:Natriuretic Peptides; D010455:Peptides; C072685:guanylin; C084362:uroguanylin; D006162:Guanylate Cyclase; D006152:Cyclic GMP