Purification of transglutaminase enzyme from Leishmania infantum promastigotes

Transglutaminases (TGase) catalyze post-translational proteins modifications including lysineglutamine isopeptide bonds formation. This activity seems ubiquitous and has been reported in Leishmania species, protozoan parasites living in sandflies gut and in macrophages of the mammalian host, causing serious infections in a large part of the world. It was reported important for parasite proliferation. One identified substrate is the virulence factor GP63, a zinc-dependent metalloprotease known also as leishmanolysin or Leishmania Major Surface Protease, the most represented protein in the parasite released exosomes, providing immunosuppressive and pro-parasitic action. Several Leishmania strains genomes were sequenced but no apparent TGase-like sequences can be found. Thus we aimed to characterize and purify TGase from L. infantum promastigotes. We confirmed TGase activity in L. infantum through in vivo and in vitro incorporation of fluorescein isothiocyanate-cadaverin into parasite proteins and a commercial microplate assay. In addition, we observed a calcium activation and a GTP millimolar inhibition of the enzymatic activity. The presence of TGase in L. infantum promastigotes was further confirmed by the positive immunostaining of cells found using polyclonal antibodies against a conserved peptide of human TGase 2 within the catalytic core. Based on reported purification procedures we used DEAE-Sepharose and Heparin-Sepharose chromatography obtaining a band with an apparent molecular weight of 54 kDa., which was confirmed to be a TGase by Western Blot analysis. In addition, this analysis enabled us to identify another band with an apparent molecular weight of 66 kDa. 2D electrophoresis and mass spectrometry will help us to identify a sequence useful to clone the enzyme. Testing its antigenicity might unveil if it can be useful in diagnosis or vaccine studies, as well as differences from mammal TGase could open new avenues as a possible drug target.