Optimization of Liquid Chromatography-Electrospray Tandem Mass Spectrometry for the characterization of Bifunctional Sphingolipid D4-Desaturases/C4-Hydroxylases in Trypanosomatids

VACCHINA, PAOLA; TRIPODI, KARINA; ESCALANTE, ANDREA; UTTARO, ANTONIO D.

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protzoología

Sphingolipids (Spl) are important class of lipids and essential components of eukaryotic membranes. In addition to their structural role, they are involved in cellular processes such as growth regulation, apoptosis and stress response. In Tritryp database, we found six genes encoding putative sphingolipid desaturases: one in Trypanosoma brucei (TbSLdes), one in T. cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4, 99-100% identical). The six amino acid sequences have the characteristic long spaced histidine boxes, which group them as pure desaturases or bifunctional desaturases/hydroxylases, along with the fungi enzymes to which they are phylogenetically related. We functionally characterized these trypanosomatid enzymes by their heterologous expression in the Saccharomyces cerevisiae sur2D mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (as dinitrophenyl derivatives) of each yeast mutant transformed with one of the different parasites´ genes was analysed by HPLC. Whereas TbSLdes was capable of efficiently desaturate endogenous sphingolipids, T. cruzi and L. major enzymes showed only negligible activity. Alternatively, the use of the same HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry allowed us to detect significant percentages of desaturated and hydroxylated sphingoid bases in membrane extracts of all transformed yeast mutants. These results let us to conclude that T. brucei, T. cruzi and L. major enzymes are all bifunctional. In order to confirm these results, we applied the same approach to parasite membrane extracts of L. major, T. brucei and T. cruzi and we were able to detect both, desaturated and hydroxylated sphingoid bases. In addition, we analyzed the effect of a strong inhibitor of D4-SplDes activity –GT11- on different cultures of trypanosomes. Interestingly, we found that epimastigotes of T. cruzi and promastigote of L. major were more resistant to the drug, showing an EC50 of 11,25 mM and 11,75 mM respectively. In contrast, T. brucei bloodstream form cultures were significantly more susceptible to the action of the inhibitor (EC50 5 mM). Moreover, the desaturating as well as the hydroxylating activity in T. cruzi epmastigotes incubated with 1 mM of GT11 dropped to the 20% of untreated cultures. These results highly support our previous finding confirming the dual activity of these enzymes.