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  • br Materials and methods br Results br Discussion Recently


    Materials and methods
    Discussion Recently it has been found that prostaglandins play an important role in modulating the host immune system [1,3,22,23], and some parasites produce these eicosanoid mediators to survive into their hosts [6,[10], [11], [12],24,25]. The discovery that parasites produce prostaglandins as do their mammalian hosts, besides having the enzymatic machinery for the biosynthesis of prostaglandins [2,[10], [11], [12], [13], [14],21,23] has led to a shift in the understanding of the roles of prostaglandins during parasitic infections. Studies have been conducted to search for the vesicular monoamine transporter involved in the generation of prostaglandins in parasites [[11], [12], [13],26]. Studies have revealed that the three-dimensional structures of prostaglandin synthase of T. brucei and prostaglandin synthases of mammalian organisms are similar [21]. Synthase prostaglandin F2α gene of T. brucei has been searched in the Leishmania database and it was possible to identify a single copy of p100/11e gene that is upregulated in promastigotes vesicular monoamine transporter and of unknown function [27]. The DNA from L. major, L. donovani and L. tropica for p100/11e gene was cloned, sequenced and overexpressed in bacteria [13], which produced a cytosolic recombinant protein of 34 kDa that catalyzes the reduction of PGH2 to PGF2α. PGF synthase from L. major was 61%, 99.3% and 99.3% identical to PGF synthase of T. brucei, L. donovani and L. tropica respectively, however the search for the gene and protein of this prostaglandin synthase by RT-PCR and western blotting did not give positive results in Leishmania species of the New World [13]. In this work we focus on identifying the protein responsible for the COX-2 like activity in Leishmania mexicana, as previous studies have reported the presence of different prostaglandins in several Leishmania species [13,14,27]. We demonstrate the presence of a protein with an approximate molecular weight of 66 kDa, which presents cross-reaction with an anti-mouse COX-2 polyclonal antibody. This type of cross-reaction was reported also by Dey et al. [15], using an anti-sheep COX-1 antibody that recognized a protein in E. histolytica. The identity of the protein with COX activity in L. mexicana was analyzed by different strategies. In this context, studies with the fractions obtained by various methods of column chromatography, and the analysis of peptide mapping and immunoprecipitation assays established the identity of the protein as gp63. Comparative analysis at the primary sequence level of several COX-1, COX-2 and COX-like enzymes showed a very low identity among them; however, comparisons at the structural level showed small structural portions conserved between human COX-2 and L. mexicana gp63. In spite of the low identity among these enzymes, the parasite has COX-2 activity. In this regard, the existence of proteins with cyclooxygenase activity in other protozoa, whose sequence is lacking the important domains and residues for the activity, has been reported. Among these residues, the proton acceptor histidine at position 193, the metal-binding histidine at position 374 and the tyrosine at position 371 within the active site can be mentioned. The mechanism by which these “non-classical” enzymes perform a cyclooxygenase activity has not been elucidated. Finally, analysis by UniProtKB allowed us to identify the presence of a glutamic acid and three metal binding (zinc) histidine residues at positions 332, 263 and 267 of gp63 that could interact with tyrosine residues in positions 353 or 354 that are near the active site (TopMatchWeb comparison), which could imply the presence of a COX type activity in this parasite. Several findings are now further reinforcing the critical role played by the zinc-metalloprotease gp63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. Thus the status of gp63 has reached the level of a multifunctional protein; consequently, it is possible to suggest that gp63 is a protein that has the ability to perform multiple additional or secondary functions to its main function. Furthermore, it has been reported that the search for sequence alignment in the parasite genome sequence shows no significant homology to classical COX [2]. However as has been reported for some synthases of prostaglandins, similarities between enzymes of higher and lower organisms is due to three dimensional structure of the protein and not to the linear sequence [21]. Gp63 was described as the main surface antigen expressed on promastigotes of several species of Leishmania [[28], [29], [30], [31], [32]], and it is mentioned that due to its protein glycosylation state its molecular weight is in the 60–66 kDa range, data that coincides with the protein recognized by the polyclonal anti-mouse COX-2 polyclonal antibody. Moreover, a recombinant gp63 protein had COX activity. In view of the above, gp63 protein from Leishmania is a virulence factor with multiple functions to which, based on results reported in this paper, the COX activity is now added.