doi:10.1016/j.peptides.2005.11.024
Copyright © 2005 Elsevier Inc. All rights reserved.
Catabolism of the octadecaneuropeptide ODN by prolyl endopeptidase: Identification of an unusual cleavage site
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Jérôme Leprince1, David Cosquer1, Gaëlle Bellemère, David Chatenet, Hélène Tollemer, Sylvie Jégou, Marie-Christine Tonon and Hubert Vaudry
, 
INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, Mont-Saint-Aignan, France
Received 13 October 2005;
revised 28 November 2005;
accepted 28 November 2005.
Available online 6 January 2006.
Abstract
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.
Keywords: Prolyl oligopeptidase; EC 3.4.21.26; PEP; Neuropeptide catabolism; Endozepines; Urotensin II
Abbreviations: DBI, diazepam-binding inhibitor; hUII, human urotensin II; ODN, octadecaneuropeptide; PEP, prolyl endopeptidase
Fig. 1. Schematic representation of the primary structure of rat diazepam-binding inhibitor (DBI) and two of its processing fragments, the triakontatetraneuropeptide TTN (DBI17–50) and the octadecaneuropeptide ODN (DBI33–50). Vertical bars indicate the basic amino acids which represent potential cleavage sites for prohormone convertases. The sequence of rat ODN is shown.
Fig. 2. RP-HPLC analysis showing the time-course of ODN breakdown induced by recombinant PEP. Synthetic ODN (7 μg) was incubated at 37 °C in the absence (A) or presence of 3 μg PEP for 6 h (B) and 15 h (C). The incubation mixtures were then analyzed by RP-HPLC and the resolved peptides were monitored by UV detection at 215 nm. The arrows indicate the retention times of synthetic intact ODN, [pGlu1]ODN and ODN13–18. The dotted lines show the percentage of acetonitrile in the eluting solvent.
Fig. 3. MALDI-TOF MS spectra of the RP-HPLC peaks isolated in Fig. 2C. The m/z ions revealed that peak 1 (A) contained both intact ODN (MH+ exptl = 1911.7 vs. MH+ calcd = 1912.0) and ODN3–18 (MH+ exptl = 1712.4 vs. MH+ calcd = 1712.9), peak 3 (B) contained ODN5–18 (MH+ exptl = 1512.9 vs. MH+ calcd = 1512.8), and peak 2 (C) contained [pGlu1]ODN (MH+ exptl = 1895.3 vs. MH+ calcd = 1895.0).
Fig. 4. Effect of S 17092 on ODN breakdown induced by recombinant PEP. Synthetic ODN (7 μg) was incubated with 3 μg of PEP, at 37 °C for 15 h, in the presence (A and C) or absence (B) of the selective PEP inhibitor S 17092 (4 μM). The incubation mixtures were analyzed by RP-HPLC and the resolved peptides were monitored by UV detection at 215 nm. The arrows indicate the retention times of synthetic intact ODN13–18, ODN, [pGlu1]ODN and ODN3–18. The dotted lines show the percentage of acetonitrile in the eluting solvent.
Fig. 5. Effect of recombinant PEP on [pGlu1]ODN breakdown. Synthetic [pGlu1]ODN (7 μg) was incubated with 3 μg of PEP at 37 °C for 15 h. The incubation mixture was then analyzed by RP-HPLC and the resolved peptides were monitored by UV detection at 215 nm. The arrows indicate the retention times of synthetic ODN13–18, ODN5–18 (peak 3), ODN3–18 (peak 1) and [pGlu1]ODN (peak 2). The dotted line shows the percentage of acetonitrile in the eluting solvent.
Fig. 6. Effect of recombinant PEP on OP breakdown. Synthetic OP (7 μg) was incubated with 3 μg of PEP at 37 °C for 15 h. (A) The incubation mixture was then analyzed by RP-HPLC and the resolved peptides were monitored by UV detection at 215 nm. The arrows indicate the retention times of synthetic OP and ODN13–18. The dotted line shows the percentage of acetonitrile in the eluting solvent. (B) MALDI-TOF MS spectrum of the OP/PEP mixture after a 15-h incubation time. The m/z ion revealed the presence of intact OP (MH+ exptl = 911.3 vs. MH+ calcd = 911.6), and the absence of ODN13–18 (MH+ calcd = 658.4).
Fig. 7. Effect of recombinant PEP on [Ala2]OP breakdown. Synthetic [Ala2]OP (7 μg) was incubated with 3 μg of PEP at 37 °C for 15 h, in the absence (A) or presence (B) of the selective PEP inhibitor S 17092 (1 μM). The incubation mixtures were analyzed by RP-HPLC and the resolved peptides were monitored by UV detection at 215 nm. The arrows indicate the retention times of synthetic [Ala2]OP and ODN13–18. The dotted lines show the percentage of acetonitrile in the eluting solvent.
Fig. 8. Effect of recombinant PEP on hUII breakdown. Synthetic hUII (7 μg) was incubated with 3 μg of PEP at 37 °C for 6 h, in the absence (A) or presence (B) of the selective PEP inhibitor S 17092 (1 μM). The incubation mixtures were analyzed by RP-HPLC and the resolved peptides were monitored by UV detection at 215 nm. The arrows indicate the retention times of synthetic hUII and hUII4–11. The dotted lines show the percentage of acetonitrile in the eluting solvent.
Fig. 9. Schematic representation of PEP-induced breakdown of rat ODN. The arrows point to the observed positions of cleavage and the arrow with an × through it indicates that PEP does not cleave ODN at this canonical post-proline site.
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1 They contributed equally to this work.
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