title: Identification of activated protein C as a ghrelin endopeptidase in bovine plasma.
abstract: Ghrelin is a natural GH secretagogue first identified in the stomach. The ghrelin peptide is 28 amino acids long with an octanoic acid attached to Ser(3) near the N-terminus. This lipid modification is essential for the interaction between ghrelin and the ghrelin-specific receptor GH secretagogue receptor type 1a (GHSR1a), whereas the five or more residues of the N-terminus seem to be sufficient to activate GHSR1a to the same level as those of full-length ghrelin. In this study, we found that ghrelin was converted into smaller fragments during incubation with animal plasma in vitro and in a mouse model. Mass spectrometric analysis revealed that both acyl and desacyl ghrelin were hydrolyzed at the peptide bond between Arg(15) and Lys(16), generating an N-terminal peptide consisting of the first 15 residues. Next, we partially purified a ghrelin endopeptidase from bovine plasma and identified the enzyme as an anticoagulant serine protease-activated protein C. Octanoyl-truncated ghrelin(1-15) activated GHSR1a-dependent signaling similar to the full-length peptide, as assayed using the cell-based early-growth factor 1 reporter system. Moreover, administration of the protein C-activating agent, ProTac, to mice enhanced the production of octanoyl ghrelin(1-15) in circulation. These results indicate that ghrelin is processed into shorter peptides in circulation under thrombotic and inflammatory conditions, although high doses of the short-form or full-length ghrelin did not have any obvious effects on thromboplastin time or platelet aggregation in human plasma. Truncation of ghrelin might be responsible for altering structural characteristics such as stability, hydrophobicity, and affinity with circulating macromolecules.
authors.affiliation: Department of BiochemistryDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of PhysiologyKurume University School of Medicine, Kurume, Fukuoka 830-0011 JapanDepartment of Infection Control and Clinical Laboratory MedicineDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of BiochemistryNational Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors.forename: Motoyasu
authors.initials: M
authors.lastname: Satou
authors.affiliation: Department of BiochemistryDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of PhysiologyKurume University School of Medicine, Kurume, Fukuoka 830-0011 JapanDepartment of Infection Control and Clinical Laboratory MedicineDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of BiochemistryNational Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nishi
authors.affiliation: Department of BiochemistryDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of PhysiologyKurume University School of Medicine, Kurume, Fukuoka 830-0011 JapanDepartment of Infection Control and Clinical Laboratory MedicineDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of BiochemistryNational Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Hishinuma
authors.affiliation: Department of BiochemistryDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of PhysiologyKurume University School of Medicine, Kurume, Fukuoka 830-0011 JapanDepartment of Infection Control and Clinical Laboratory MedicineDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of BiochemistryNational Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hosoda
authors.affiliation: Department of BiochemistryDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of PhysiologyKurume University School of Medicine, Kurume, Fukuoka 830-0011 JapanDepartment of Infection Control and Clinical Laboratory MedicineDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of BiochemistryNational Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kangawa
authors.affiliation: Department of BiochemistryDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of PhysiologyKurume University School of Medicine, Kurume, Fukuoka 830-0011 JapanDepartment of Infection Control and Clinical Laboratory MedicineDokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, JapanDepartment of BiochemistryNational Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan h-sugi@dokkyomed.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sugimoto
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D010450:Endopeptidases / Q000097:blood / Q000378:metabolism*; D004789:Enzyme Activation; D054439:Ghrelin / Q000097:blood / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016716:PC12 Cells; D010949:Plasma / Q000201:enzymology*; D011486:Protein C / Q000378:metabolism*; D051381:Rats
chemical_list: D001798:Blood Proteins; D054439:Ghrelin; D011486:Protein C; D010450:Endopeptidases
keywords: ghrelin; plasma; protein C; protein purification