Characterization of possible palmitoyl transferases in the protozoan parasite Giardia lamblia

MERINO MC; VRANYCH CV; MIRAS S; TOUZ MC; ROPOLO AS

Ascochinga

Congreso; XXIV Reunión Científica Anual Sociedad Argentina de Protozoología.; 2010

Posttranslational modifications such as phosphorilation, glycosilation, sumoylation, palmitoylation, are important to regulate molecular mechanisms involved in cell homeostasis and intracellular signalling. Despite palmitoylation has been involved in protein trafficking, cell signaling and localization to lipid rafts, its biological function has not been completely elucidated. Very little is known about palmitoylation in the intestinal protozoan parasite Giardia lamblia. In a previous work from our laboratory it has been reported that variable specific surface proteins from Giardia lamblia are palmitoylated, determining the site of palmitoylation. In the same report, Touz y col., identified two palmitoyl transferases of Giardia lamblia and determined some of the possible biological consequences of palmytoilation in this parasite. The aim of the present work is to characterize other possible enzymes with palmitoyl transferase (PAT) activity in G. lamblia. Proteins that promote palmitoylation have been identified in Saccharomyces cerevisiae including effector of Ras function (Erf2) and the SNARE protein Ykt6 which have a common Asp-His-His-Cys-cysteine-rich domain (DHHC-CRD) motif that likely confers PAT activity. Looking at Giardia lamblia genome, we found other possible enzymes that may have PAT activity. These proteins display the typical DHHC-CRD motif found in others palmitoyl transferases. DNA from WB 1267 G. lamblia trophozoites was isolated and PCR was performed using specific primers, determining that four of the possible enzymes are present in G. lamblia genome. PCR products were then cloned in pTub-ApaH7HApac or pINDG-V5 vectors. Plasmidic DNA was sequenced and WB 1267 trophozoites were then transfected. Inmunofluorescense analysis of the epitope-tagged proteins showed that they localize mainly in the cytoplasm or around the nuclei. The characterization of proteins which may act as palmitoyl transferases in G. lamblia would contribute to clarify the molecular mechanisms used by this protozan parasite in antigenic variation and/or encystation.