title: Ghrelin degradation by serum and tissue homogenates: identification of the cleavage sites.
abstract: The endogenous ligand for the GH secretagogue receptor is ghrelin, a peptide recently purified from the stomach. Ghrelin is n-octanoylated on the Ser(3) residue, and this modification is essential for its interaction with the receptor. The degradation of ghrelin by rat and human serum, purified commercial enzymes, and tissues homogenates was analyzed by combining HPLC and mass spectrometry. In serum, ghrelin was desoctanoylated, without proteolysis. The desoctanoylation was significantly reduced by phenylmethylsulfonyl fluoride, a serine proteases and esterases inhibitor. In rat serum, the carboxylesterase inhibitor bis-p-nitrophenyl-phosphate totally inhibited ghrelin desoctanoylation, and a correlation was found between ghrelin desoctanoylation and carboxylesterase activity. Moreover, purified carboxylesterase degraded ghrelin. Thus, carboxylesterase could be responsible for ghrelin desoctanoylation in that species. In human serum, ghrelin desoctanoylation was partially inhibited by eserine salicylate and sodium fluoride, two butyrylcholinesterase inhibitors, but not by bis-p-nitrophenyl-phosphate and EDTA. Purified butyrylcholinesterase was able to degrade ghrelin, and there was a correlation between the butyrylcholinesterase and ghrelin desoctanoylation activities in human sera. This suggested that several esterases, including butyrylcholinesterase, contributed to ghrelin desoctanoylation in human serum. In contact with tissues homogenates, ghrelin was degraded by both desoctanoylation and N-terminal proteolysis. We identified five cleavage sites in ghrelin between residues -Ser(2)-(acyl)Ser(3)- (stomach and liver), -(acyl?)Ser(3)-Phe(4)- (stomach, liver, and kidney), -Phe(4)-Leu(5)- (stomach and kidney), -Leu(5)-Ser(6)- and -Pro(7)-Glu(8)- (kidney). In all cases, the resulting fragments were biologically inactive.
authors.affiliation: Department of Biochemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Bat G/E, CP 611, 808 route de Lennik, B-1070 Brussels, Belgium.
authors.forename: Carine
authors.initials: C
authors.lastname: De Vriese
authors.forename: Francoise
authors.initials: F
authors.lastname: Gregoire
authors.forename: Roger
authors.initials: R
authors.lastname: Lema-Kisoka
authors.forename: Magali
authors.initials: M
authors.lastname: Waelbroeck
authors.forename: Patrick
authors.initials: P
authors.lastname: Robberecht
authors.forename: Christine
authors.initials: C
authors.lastname: Delporte
mesh_terms: D000818:Animals; D043303:Aryldialkylphosphatase / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism*; D002091:Butyrylcholinesterase / Q000378:metabolism; D043182:Carboxylesterase / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D005753:Gastric Mucosa / Q000378:metabolism; D054439:Ghrelin; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D036361:Peptide Hormones / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D044967:Serum / Q000201:enzymology
chemical_list: D001798:Blood Proteins; D054439:Ghrelin; D036361:Peptide Hormones; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; C019734:arylesterase; D002091:Butyrylcholinesterase; D043303:Aryldialkylphosphatase