ABSTRACT
Cyclooxygenase-2 (COX-2) is an enzyme responsible of prostaglandins production, such as prostaglandin E2 (PGE2), an immune response modulator that regulates the immune system to inhibit Th1 and to promote Th2 cytokines production. Many parasites modulate their host immune response through PGE2 effects; however, in parasites, only one protein with COX activity has been described, the α-actinin of Entamoeba histolytica. Prostanoids production has been reported in some species of Leishmania but not the enzymes responsible of their production. To identify the protein responsible for COX activity in Leishmania mexicana, we examined total extracts of promastigotes and samples with COX activity were subjected to ion exchange column purification and precipitation with ammonium sulphate; fractions with activity were analyzed by SDS-PAGE and Western blot using an anti-mouse COX-2 polyclonal antibody. Results showed that in those samples with enzymatic activity, the anti-mouse COX-2 polyclonal antibody recognized a protein with an approximate molecular weight of 66â¯KDa. Bands recognized by the antibody were subjected to mass spectrometry analysis and the results showed that several peptides from the bands purified by two different methods, and that were recognized by the anti-mouse COX-2 polyclonal antibody corresponded to the Leishmania mexicana gp63 surface protease. L. mexicana gp63 was purified by a Concanavalin A (Con-A) affinity column and subjected to immunoprecipitation with a commercial anti-Leishmania gp63 polyclonal antibody; the immunoprecipitated sample was analyzed for COX activity showing that the anti-gp63 antibody did immunoprecipitate the COX activity. The presence of COX activity was further confirmed in amastigotes extracts of L. mexicana. Moreover, a recombinant gp63 protein was produced and its COX activity tested, confirming that gp63 is the molecule responsible for COX activity.
