title: Recombinant expression and biochemical characterisation of two alanyl aminopeptidases of Trypanosoma congolense.
abstract: Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed in procyclic metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro.
authors.affiliation: Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg Campus, Private Bag X01, Scottsville 3209, South Africa; CNRS, Université Bordeaux Segalen, Microbiologie fondamentale et Pathogénicité, UMR 5234, F-33000 Bordeaux, France.
authors.forename: Davita
authors.initials: D
authors.lastname: Pillay
authors.forename: Alain F V
authors.initials: AF
authors.lastname: Boulangé
authors.forename: Virginie
authors.initials: V
authors.lastname: Coustou
authors.forename: Théo
authors.initials: T
authors.lastname: Baltz
authors.forename: Theresa H T
authors.initials: TH
authors.lastname: Coetzer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D018826:CD13 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D008670:Metals / Q000494:pharmacology; D051379:Mice; D058849:Protein Refolding; D016053:RNA, Protozoan / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014348:Trypanosoma congolense / Q000201:enzymology* / Q000235:genetics / Q000276:immunology
chemical_list: D000953:Antigens, Protozoan; D002413:Cations, Divalent; D008670:Metals; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D018826:CD13 Antigens
keywords: 4-(2-aminoethyl)benzenesulfonyl fluoride; 4′,6-diamidino-2-phenylindole; AAT; AEBSF; AMC; AMT; AP; African animal trypanosomosis; BSF; DAPI; DMSO; DTT; E-64; L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane; LC–MS/MS; M1 type aminopeptidase; MBS; PBS; PSG; Protozoan parasite; RNAi; RT; Trypanosoma congolense; VSG; Z; acetate-MES-Tris; amino peptidase; aminomethylcoumarin; benzyloxycarbonyl; bloodstream form; dimethyl sulfoxide; dithiothreitol; liquid chromatography coupled to tandem mass spectrometry; m-maleimidobenzoyl-N-hydroxysuccinimide ester; phosphate buffered saline, pH 7.2; phosphate-buffered saline containing glucose; room temperature; variable surface glycoprotein