The protozoan parasite Trypanosoma brucei brucei causes nagana in cattle and is a widely used
model for human sleeping sickness. The major lysosomal cysteine proteinases (trypanopains) of
African trypanosomes may contribute to pathogenesis by degrading proteins in the mammalian
bloodstream and also appear to be essential for the viability of T. cruzi and T. congolense. This
study describes the first purification to electrophoretic homogeneity of trypanopain-Tb from
T. b. brucei and the first reported characterisation of its enzymatic properties. Trypanopain-Tb
was purified from bloodstream forms of T. b. brucei by a combination of three phase
partitioning (between ammonium sulfate and tertiary butanol), and chromatography on
quaternary amine or pepstatin A-Sepharose resins.
Trypanopain-Tb was found to be a typical cysteine proteinase, in that it is inhibited by typical
cysteine proteinase inhibitors and requires reducing agents for full activity. Trypanopain has
cathepsin L-like specificity for synthetic substrates and readily degrades various proteins.
In vitro analysis of the kinetics of trypanopain interaction with cystatins suggested that these are
likely to inhibit any trypanopain released into the mammalian bloodstream. Furthermore, no
trypanopain-like activity was detectable in the blood of infected hosts, so it appears that
trypanopain is unlikely to contribute directly to pathogenesis by degrading bloodstream host
proteins.
Antibodies against a peptide corresponding to a region of the trypanopain active site were
produced in rabbits and chickens. Both enzyme activity-enhancing and enzyme activity inhibiting
antibodies were produced and these effects varied with the substrate tested. Thus, the
in vivo effects of anti-trypanopain antibodies will only become clearly understood once the
physiological substrates of trypanopain have been identified.
Various cysteine proteinase inhibitors, including peptidyl diazomethylketones, killed cultured
bloodstream forms of T. b. brucei. Use of biotinylated derivatives of peptidyl
diazomethylketone and fluoromethylketone inhibitors suggested that trypanopain is the likely
intracellular target of these inhibitors, indicating that the enzyme is essential for parasite
viability. Furthermore, chalcones (a class of reversible cysteine proteinase inhibitors) killed in
vitro cultured parasites and also prolonged the life of T. b. brucei-infected mice. Thus,
trypanopain-Tb seems to be a possible target for new anti-trypanosomal drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9278 |
| Date | January 1997 |
| Creators | Troeberg, Linda. |
| Contributors | Coetzer, Theresa Helen Taillefer., Lonsdale-Eccles, John D. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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