Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases.

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.