title: Proteolytic activity in Encephalitozoon cuniculi sporogonial stages: predominance of metallopeptidases including an aminopeptidase-P-like enzyme.
abstract: A fraction enriched in spore precursor cells (sporoblasts) of the microsporidian Encephalitozoon cuniculi, an intracellular parasite of mammals, was obtained by Percoll gradient centrifugation. Soluble extracts of these cells exhibited proteolytic activity towards azocasein, with an alkaline optimum pH range (9-10). Prevalence of some metallopeptidases was supported by the stimulating effect of Ca2+, Mg2+, Mn2+ and Zn2+ ions, and inhibition by two chelating agents (EDTA and 1,10-phenanthroline), a thiol reductant (dithiothreitol) and two aminopeptidase inhibitors (bestatin and apstatin). Zymographic analysis revealed four caseinolytic bands at about 76, 70, 55 and 50 kDa. Mass spectrometry of tryptic peptides from one-dimensional gel slices identified a cytosol (leucine) aminopeptidase homologue (M17 family) in 50-kDa band and an enzyme similar to aminopeptidase P (AP-P) of cytosolic type (M24B subfamily) in 70-kDa band. Multiple sequence alignments showed conservation of critical residues for catalysis and metal binding. A long insertion in a common position was found in AP-P sequences from E. cuniculi and Nosema locustae, an insect-infecting microsporidian. The expression of cytosolic AP-P in sporogonial stages of microsporidia may suggest a key role in the attack of proline-containing peptides as a prerequisite to long-duration biosynthesis of structural proteins destined to the sporal polar tube.
authors.affiliation: Parasitologie Moléculaire et Cellulaire, LBP, UMR-CNRS 6023, Université Blaise Pascal-Clermont 2, 63177 Aubière Cedex, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Chavant
authors.forename: Vanessa
authors.initials: V
authors.lastname: Taupin
authors.forename: Hicham
authors.initials: H
authors.lastname: El Alaoui
authors.forename: Ivan
authors.initials: I
authors.lastname: Wawrzyniak
authors.forename: Christophe
authors.initials: C
authors.lastname: Chambon
authors.forename: Gérard
authors.initials: G
authors.lastname: Prensier
authors.forename: Guy
authors.initials: G
authors.lastname: Méténier
authors.forename: Christian P
authors.initials: CP
authors.lastname: Vivarès
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D009670:Encephalitozoon cuniculi / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D005656:Fungal Proteins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000378:metabolism; D045726:Metalloproteases / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D016415:Sequence Alignment
chemical_list: D002364:Caseins; D005656:Fungal Proteins; D008670:Metals; D011480:Protease Inhibitors; C026018:azocasein; D045726:Metalloproteases; D000626:Aminopeptidases; D007931:Leucyl Aminopeptidase; C022750:X-Pro aminopeptidase