Identification of an unusual cleavage site for prolyl endopeptidase:investigation of the breakdown of the octadecaneuropeptide ODN

摘要

Prolyl endopeptidase(PEP)is a serine protease that preferentially cleaves proline-containing peptides such as oxytocin,thyrotropin-releasing hormone,alpha-melanocyte-stimulating hormone and substance-P at the carboxyl-side of proline residues.PEP can also specifically hydrolyze post-alanine bonds,a type of cleavage that is thought to contribute to the formation of the P-amyloid peptide,The octadecaneuropeptide(ODN,QATVGDVNTDRPGLLDLK),a proteolytic fragment of diazepam-binding inhibitor,exhibits multiple behavioral and neurobiological activities.In particular,ODN induces anxiety,attenuates pentylenetetrazol-evoked convulsions,suppresses apomorphine-induced yawning and inhibits food intake.However,the mechanism involved in its inactivation is currently unknown.The presence of both proline and alanine residues in the ODN sequence led us to investigate in vitro the effect of PEP on the breakdown of ODN and related peptides,by combining reversed phase HPLC analysis and MALDI-TOF mass spectrometry characterization.