title: Proteases from Trypanosoma brucei brucei. Purification, characterisation and interactions with host regulatory molecules.
abstract: African trypanosomes contain proteases that may be released into the bloodstream of their infected hosts. This paper describes a novel, combined isolation of a cysteine proteinase (called trypanopain-Tb) and a serine oligopeptidase (which we call oligopeptidase-Tb) from Trypanosoma brucei brucei, as well as a comparison of the activities of these two enzymes against several host regulatory molecules. The enzymes differed in various respects. Firstly, purified trypanopain-Tb was shown to readily cleave proteins such as gelatin maximally at acidic pH. In contrast, oligopeptidase-Tb, which is optimally active at alkaline pH, did not hydrolyse proteins larger than 4 kDa. However, it readily hydrolysed various polypeptides, including neurotensin and atrial natriuretic factor. The interaction of the two enzymes with mammalian protease inhibitors also differed. Cystatins and alpha2-macroglobulin effectively inhibited trypanopain-Tb, with the Ki values for cystatin C and low-molecular-mass kininogen (approximately 10(-11) M) predicting, that trypanopain-Tb is likely to be effectively controlled by these inhibitors if released into the host bloodstream. In contrast, oligopeptidase-Tb was not inhibited by serpins or (a2-macroglobulin, suggesting that it may remain active if released into the host bloodstream. In support of these in vitro results, the blood of trypanosome-infected rats displayed no trypanopain-Tb-like activity, but exhibited high oligopeptidase-Tb-like activity. Thus, while trypanopain-Tb seems likely to be confined to an intracellular role within the parasite, oligopeptidase-Tb has the potential to remain active in the host bloodstream and so contribute directly to pathogenesis.
authors.affiliation: Department of Biochemistry, University of Natal, Scottsville, South Africa.
authors.forename: L
authors.initials: L
authors.lastname: Troeberg
authors.forename: R N
authors.initials: RN
authors.lastname: Pike
authors.forename: R E
authors.initials: RE
authors.lastname: Morty
authors.forename: R K
authors.initials: RK
authors.lastname: Berry
authors.forename: T H
authors.initials: TH
authors.lastname: Coetzer
authors.forename: J D
authors.initials: JD
authors.lastname: Lonsdale-Eccles
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055312:Cystatin A; D055313:Cystatin B; D055316:Cystatin C; D015891:Cystatins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007704:Kininogens / Q000494:pharmacology; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D012697:Serine Endopeptidases / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000201:enzymology*; D014353:Trypanosomiasis, African / Q000201:enzymology*
chemical_list: C525032:Cst3 protein, rat; D055312:Cystatin A; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D007704:Kininogens; D010455:Peptides; D011506:Proteins; D055313:Cystatin B; D010447:Peptide Hydrolases; C028759:oligopeptidase; D012697:Serine Endopeptidases; C100685:oligopeptidase Tb; D003546:Cysteine Endopeptidases; C100684:trypanopain Tb