Purification and characterization of a cyclooxygenase-like enzyme from Trypanosoma brucei

Abstract

The protozoan hemoflagellate Trypanosoma brucei is the causative agent of African trypanosomosis in humans and nagana in domestic animals. The human disease is a health concern in many African countries. The infection of mammalian-host by African trypanosomes is characterised by an up-regulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid (CSF), which modulates the host responses and causes symptoms of the infection. It has been shown that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from arachidonic acid (AA) and its metabolite, prostaglandin 2 alpha (PGH2α). In view of the AA cascade metabolism, the cyclooxygenase (COX) or PGH synthase (PGHS) is the enzyme that catalyses the rate-limiting step in the biosynthesis of the PGs. This work involved the purification and characterization of a COX-like enzyme from trypanosomes using chromatographic techniques such as Gel filtration and Ion exchange chromatography under the control of AKTA-FPLC system, running an SDS polyacrylamide gel and Western blot analysis for immunodetection of a COX-like enzyme from trypanosomes and cloning the COX- like gene in bacteria and/or insect cell system. A probable COX-like enzyme from the trypanosomes lysate was identified specifically in the cytosolic fraction by western blot analysis using a polyclonal anti-ovine-COX antibody. Proteome data analysis from the 10% SDS-PAGE and silver stain of the complete trypanosomes lysate, gel-digested at range of 50-95KDa generated 23 proteins probably putative COX-like protein candidates. The cytosolic fraction from trypanosomes lysate was used in an attempt to design purification strategies of the COX-like enzyme from T. brucei using gel chromatography. Ion exchange chromatography was done as an intermediate step and the fraction from the strong cation exchange gave absorbance in the COX-fluorometric assay, indicating the presence of a COX-like enzyme from trypanosomes. This suggested that trypanosome COX is positively charged. These observations together with the data from chromatographic steps, western blotting and COX-fluorometric assay, suggest that a COX-like enzyme exists in T. brucei as a cytosolic protein. Further experiments need to be done to optimize the purification, SDS-PAGE and Western blot analysis, and biochemical characterization of the recombinant COX-like enzyme. Functional and structural biology studies and phylogenetic analysis should be done to determine whether TbCOX is completely distinct from mammalian COXs. The finding that PG-producing enzymes are distinct from their mammalian counterparts makes them suitable drug targets.