Catabolism of the octadecaneuropeptide ODN by prolyl endopeptidase: identification of an unusual cleavage site.
The octadecaneuropeptide ODN (QATVGDVNTDRPGLLDLK), a biologically active fragment of diazepam-binding inhibitor, exerts a number of behavioral and neurophysiological activities. The presence of a proline residue in the sequence of ODN led us to investigate the role of proline endopeptidase (PEP) in the catabolism of this neuropeptide. The effect of PEP on the breakdown of ODN and related analogs was studied by combining RP-HPLC analysis and MALDI-TOF MS characterization. Incubation of ODN with PEP generated two products, i.e. ODN3-18 and ODN5-18 which resulted from cleavage of the Ala-Thr and Val-Gly peptide bonds. S 17092, a specific PEP inhibitor, significantly reduced the PEP-induced cleavages of ODN. Similarly, [Ala2]OP showed S 17092-sensitive post-alanine cleavage, while [pGlu1]ODN and OP (ODN11-18) were not catabolized by the enzyme. For all these peptides, cleavage of the Pro-Gly peptide bond by PEP was never observed, even after prolonged incubation times. In contrast, PEP hydrolyzed human urotensin II at the canonical post-proline site. Collectively, these data suggest that the Ala2 residue is the preferential cleavage site of ODN and that the Pro-Gly bond of ODN is not hydrolyzed by PEP. In addition, this study reveals for the first time that the endoproteolytic activity of PEP can specifically take place after a valine moiety.
Leprince
Jérôme
J
INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, Mont-Saint-Aignan, France.
Cosquer
David
D
Bellemère
Gaëlle
G
Chatenet
David
D
Tollemer
Hélène
H
Jégou
Sylvie
S
Tonon
Marie-Christine
MC
Vaudry
Hubert
H
D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D026261:Diazepam Binding Inhibitor; D005417:Flavobacterium / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments; D010455:Peptides / Q000737:chemistry; D000085822:Prolyl Oligopeptidases; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014579:Urotensins / Q000737:chemistry
D026261:Diazepam Binding Inhibitor; D009479:Neuropeptides; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D014579:Urotensins; C067529:diazepam binding inhibitor (33-50); C029677:urotensin II; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases