Identification and characterization of two dipeptidyl‐peptidase III isoforms in Drosophila melanogaster

• Published in: The FEBS journal Vol. 273; no. 5; pp. 1056 - 1064
• Main Authors: Mazzocco, Claire, Gillibert‐Duplantier, Jennifer, Neaud, Veronique, Fukasawa, Kayoko M., Claverol, Stéphane, Bonneu, Marc, Puiroux, Jacques
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-03-2006; Wiley
• Subjects: Animals; Base Sequence; Biochemistry; Biochemistry, Molecular Biology; Central Nervous System - enzymology; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - chemistry; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - genetics; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - isolation & purification; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - metabolism; dipeptidyl‐peptidase III; DNA - genetics; Drosophila melanogaster; Drosophila melanogaster - enzymology; enkephalinase; Immunohistochemistry; Insects; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kinetics; Life Sciences; Mass Spectrometry; Molecular Weight; neuropeptide; Peptides; proctolin; Proteins; proteomic; Solubility; proctolin; proteomic; Dipeptidyl-peptidase III; enkephalinase; neuropeptide
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2006.05132.x

Dipeptidyl‐peptidase III (DPP III) hydrolyses small peptides with a broad substrate specificity. It is thought to be involved in a major degradation pathway of the insect neuropeptide proctolin. We report the purification and characterization of a soluble DPP III from 40 g Drosophila melanogaster. Western blot analysis with anti‐(DPP III) serum revealed the purification of two proteins of molecular mass 89 and 82 kDa. MS/MS analysis of these proteins resulted in the sequencing of 45 and 41 peptide fragments, respectively, confirming ≈ 60% of both annotated D. melanogaster DPP III isoforms (CG7415‐PC and CG7415‐PB) predicted at 89 and 82 kDa. Sequencing also revealed the specific catalytic domain HELLGH in both isoforms, indicating that they are both effective in degrading small peptides. In addition, with a probe specific for D. melanogaster DPP III, northern blot analysis of fruit fly total RNA showed two transcripts at ≈ 2.6 and 2.3 kb, consistent with the translation of 89‐kDa and 82‐kDa DPP III proteins. Moreover, the purified enzyme hydrolyzed the insect neuropeptide proctolin (Km≈ 4 µm) at the second N‐terminal peptide bound, and was inhibited by the specific DPP III inhibitor tynorphin. Finally, anti‐(DPP III) immunoreactivity was observed in the central nervous system of D. melanogaster larva, supporting a functional role for DPP III in proctolin degradation. This study shows that DPP III is in actuality synthesized in D. melanogaster as 89‐kDa and 82‐kDa isoforms, representing two native proteins translated from two alternative mRNA transcripts.