Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases.
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2011
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Abstract
African animal trypanosomosis or nagana is a disease in livestock caused by various
species of protozoan parasites belonging to the genus Trypanosoma particularly T.
congolense, T. vivax and T. b. brucei. Nagana is the most important constraint to livestock
and mixed crop-livestock farming in tropical Africa. Trypanosomes undergo part of their
developmental life in their insect vector, the tsetse fly and part in their mammalian host.
Measures for eradicating the continent of the tsetse fly vector include insecticidal spraying,
targeting and trapping. Vaccine development has been hampered by the generation of an
inexhaustible collection of variant surface glycoproteins that trypanosomes possess and
allow for evasion of the host immune system. Anti-disease vaccines aimed at reducing the
symptoms of the disease rather than killing the parasite itself have been demonstrated as an
alternative approach. Trypanotolerant cattle are able to protect themselves from the
disease-associated symptoms. They are able to mount a better antibody response to the
CATL-like cysteine peptidase, TcoCATL, compared to trypanosusceptible breeds. Bovine
trypanosomosis, however, continues to be controlled primarily by trypanocidal compounds
such as isometamidium chloride, homidium and diaminazene that have been developed
more than 50 years ago and consequently drug resistance is widespread. Trypanosomal
cysteine peptidases have also been proven to be effective targets for chemotherapeutics.
TcrCATL, inhibited by the vinyl sulfone pseudopeptide inhibitor K11777, was effective in
curing or alleviating T. cruzi infection in preclinical proof-of-concept studies and has now
entered formal preclinical drug development investigation.
Understanding enzymatic as well as structural characteristics of pathogenic peptidases is
the first step towards successful control of the disease. To date no such characterisation of
the major cysteine peptidases from T. vivax has been conducted. Although the major
cysteine peptidase from T. vivax, TviCATL, has not been proven as a pathogenic factor yet,
its high sequence identity with the pathogenic counterparts such as TcrCATL and
TcoCATL hold much speculation for TviCATLs role in pathogenocity.
In the present study, native TviCATL was isolated from T. vivax Y486, purified and
characterised. TviCATL showed to have a general sensitivity to E-64 and cystatin and has a
substrate specificity defined by the S2 pocket. TviCATL exhibited no activity towards the
CATB-like substrate, Z-Arg-Arg-AMC but was able to hydrolyse Z-Phe-Arg-AMC, the
CATL-like substrate. Leu was preferred in the P2 position and basic and non-bulky
hydrophobic residues were accepted in the P1 and P3 positions respectively. Similar
findings were reported for TcoCATL. The substrate specificity of TviCATL and TcoCATL
does argue for a more restricted specificity compared to TcrCATL. This was based on the
Glu333 in TcrCATL substituted with Leu333 in TviCATL and TcoCATL. In the case of
TcrCATL, the Glu333 allows for the accommodation of Arg in the P2 position. Like other
trypanosomal cysteine peptidases, TviCATL was inhibited by both chloromethyl ketones,
Z-Gly-Leu-Phe-CMK and H-D-Val-Phe-Lys-CMK. Determining further structural and
functional characteristics as well as whether TviCATL, like the T. congolense homolog,
TcoCATL, acts as a pathogenic factor, would be important information to the designing of
specific chemotherapeutic agents.
To date, TcrCATL and TbrCATL (from T. b. rhodesiense) are the only trypanosomal
CATL-like cysteine peptidases been crystallised and their tructures solved. This advantage
has allowed for the directed design of synthetic peptidase inhibitors. The crystal structure
of TcoCATL will be of major significance to the design of specific chemotherapeutic
agents. Furtherrmore, understanding the dimeric conformation of TcoCATL is important
for vaccine design as immune responses are likely to recognise the dimer specific epitopes.
In the current study, the catalytic domain of TcoCATL and TviCATL, were recombinantly
expressed in Pichia pastoris and purified to homogeneity. The T. congolense cysteine
peptidase pyroglutamyl peptidase (PGP), also proven to be pathogenic in T. b. brucei, was
recombinantly expressed in E. coli BL21 (DE3) cells and also purified to homogeneity.
Purified cysteine peptidases along with previously purified TcoCATL dimerisation
mutants, TcoCATL (H43W) and TcoCATL (K39F; E44P), possessing mutated residues
involved in TcoCATL dimerisation, as well as the mutant proenzyme TcoCATL (C25A),
were screened for crystallisation conditions using the Rigaku robotic crystallisation suite.
One-dimensional needle-like crystals were found for TcoCATL (K39F; E44P).
Optimisation of the TcoCATL (K39F; E44P) crystals were analysed for X-ray diffraction.
The poor diffraction pattern prompted further optimisations for better crystal quality,
which is presently underway. The crystal structure of TcoCATL, with some of the residues
involved in dimerisation mutated, will be pivotal in understanding the dimerisation model.
Furthermore, the information about the structure will be valuable for vaccine design and
chemotherapeutics development.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.
Keywords
African trypanosomiasis., Trypanosomiasis in animals--Immunotherapy., Cysteine proteinases--Structure., Theses--Biochemistry.