Comparative studies of the purified cysteine proteases of trypanosoma brucei and trypanosoma congolense
Date
2011-04-04
Authors
Konde, Victor
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Abstract
The cysteine protease enzymes from T. congolense and T. brucei
were purified from pure blood stream forms of the trypanosomes grown in
adult albino rats. The trypanosomes were harvested when the number of
trypanosomes had reached 108/ml of infected blood or higher. The rats
were supplied with 10% glucose in drinking water when the parasiteamia
o was 10 /ml which enabled the rats to survive for at least two extra days. In
case of T. brucei infections, the level of stumpy forms in blood was
monitored and when the number of stumpy forms reached 50% or more
the trypanosomes were harvested. This resulted in an increase in cysteine
protease recovery by at least two fold.
The enzymes were successfully purified using egg white cystatin
immobilised on sepharose. There were no significant differences in the
yields of the two cysteine proteases and both enzymes had similar activity.
The activity of the two enzymes was assayed with carbobenzoxyphenylalaninyl-
argininyl 7-amido-4-methylcoumarin by monitoring the
fluorescence at 365 nm. The reactions were performed at pH 6 at room
temperature (28 - 30°C). T. congolense cysteine protease was purified
using a monoclonal antibody, prepared against the T. congolense cysteine
protease, immobilised on sepharose. The enzyme was of similar molecular
weight as that purified by the cystatin-sepharose column.
The enzyme was incubated with a variety of host macromolecules,
mainly connective tissue proteins and the digestion products were
followed using sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE). hi other cases the proteins were copolymerized
in SDS-PAGE and then digestion monitored in zymograms.
The enzymes expressed proteolytic activity against collagen type I,
collagen type IV, glycoproteins, fibronectin, laminin and to a small extent
against elastin. Fibronectin was the best substrate of all the connective
tissue proteins while elastin was least reactive. All the above connective
tissue proteins were not denatured prior to digestion as reported in similar
experiments.
The two trypanosome cysteine proteases failed to round off and
detach cells in culture at our working enzyme concentration (160 ug/ml).
The enzymes had greater effect on the cell membrane than on the
intercellular matrix, an effect that is similar to papain. The enzymes have
high proteolytic activity against cell surface proteins and this may account
for the failure to round off cells, as the enzymes are likely to preferentially
attack cell surface proteins rather than proteoglycans in the extracellular
matrix.
The enzymes were inhibited by trans-epoxysuccinyl-1-leucylamido
(4-guanidino) butane (E-64), egg white cystatin, and Noc-p-tosyl-1-l-lysine
chloromethyl ketone (TLCK) while phenylmethylsulphonyl-fluoride
(PMSF) and aprotinin failed to inhibit the enzymes to any noticeable
extent.
The enzymes digested bovine serum albumin (BSA), fibrinogen
and casein and their proteolytic products were resolved on sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The protein
bands were resolved at similar regions and the patterns of bands produced
by the two trypanosome cysteine proteases were identical. This strongly
suggests that the trypanosome cysteine proteases under study have
preference for the same peptide bonds. The two trypanosome enzymes
have similar proteolytic properties and are cysteine proteases in nature as
shown by their inhibition by cysteine protease inhibitors
Description
Keywords
: Cysteine proteinases , : Trypanosoma brucei