tailieunhanh - Báo cáo khoa học: Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster

A Drosophila melanogaster cDNA clone (GH01916) encoding a putative 723-residue long (82 kDa) protein (CG 7415) and displaying 50% identity with mammalian cytosolic dipeptidyl aminopeptidase (DPP) III was func-tionally expressed in Schneider S2 cells. Immunocytochemi-cal studies using anti-(rat liver DPP III) Ig indicated the expression of this putative DPP III at the outer cell mem-brane and into the cytosol of transfected cells. | Eur. J. Biochem. 270 3074-3082 2003 FEBS 2003 doi Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster Claire Mazzocco1 I Kayoko M. Fukasawa2 Patrick Auguste3 and Jacques Puiroux1 t 1Laboratoire des Regulations Neuroendocriniennes Université Bordeaux I Talence France department of Oral Biochemistry Matsumoto Dental College Shioriji Nagano Japan 3Laboratoire des Facteurs de Croissance et de la Differenciation Cellulaire INSERM EPI-0113 Universite Bordeaux I Talence France A Drosophila melanogaster cDNA clone GH01916 encoding a putative 723-residue long 82 kDa protein CG 7415 and displaying 50 identity with mammalian cytosolic dipeptidyl aminopeptidase DPP III was functionally expressed in Schneider S2 cells. Immunocytochemical studies using anti- rat liver DPP III Ig indicated the expression of this putative DPP III at the outer cell membrane and into the cytosol of transfected cells. Two protein bands 82 and 86 kDa were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells. Western blot analysis of partially purified D. melanogaster DPP III confirmed the overexpression of these two protein bands into the cytosol and on the membranes of transfected cells. Despite the identification of six potential glycosylation sites PAGE showed that these protein bands were not shifted after deglycosylation experiments. The partially purified enzyme hydrolysed the insect myotropic neuropeptide proctolin Arg-Tyr-Leu-Pro-Thr at the Tyr-Leu bond Km w 4 M . In addition low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation IC50 im . These results constitute the first characterization of an evo-lutionarily conserved insect DPP III that is expressed as a cytosolic and a membrane peptidase involved in proctolin degradation. Keywords enkephalinase genome sequencing insects neuropeptides proctolin. .