Palmitoylation in Giardia lamblia: possible palmitoyl transferases and target proteins

MERINO, MC; VRANYCH, CV; MIRAS, SL; TOUZ, MC; ROPOLO, AS

Mar del Plata

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

Sociedad Argentina de Protozoología

Palmitoylation refers to the addition of palmitate to a cysteine residue of proteins. Despite palmitoylation has been involved in protein trafficking, cell signaling and localization to lipid rafts, its biological function has not been completely elucidated. In a previous work from our laboratory it has been reported that variant-specific surface proteins from G. lamblia are palmitoylated, determining the site of palmitoylation. In the same report, Touz et al., identified two palmitoyl transferases of G. lamblia and determined some of the possible biological consequences of palmitoylation in this parasite. The goals of the present work are to characterize other possible enzymes with palmitoyl transferase (PAT) activity and the target proteins. Proteins that promote palmitoylation have been identified in S. 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 genome, we found other possible enzymes that may have PAT activity. These proteins display the typical DHHC-CRD motif found in other PATs. 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. Immunofluorescense analysis of the epitope-tagged proteins showed that they localize mainly in the cytoplasm or around the nuclei. In order to know the target proteins of palmitoylation in this parasite we are carrying out experiments with palmitic acid labeled with tritium. We observed that when palmitoylation was inhibited by adding 2-Fluoro palmitic acid to the encystation media, the percentage of cells expressing Cyst wall protein 1+ decreased (CWP 1+). Moreover, we observed an increase of CWP1+ parasites in encysting G. lamblia trophozoites that overexpressed a PAT compared to wild type G. lamblia. Due to the many intracellular signals involved in encystation it is possible that palmitoylation participates in that process. Further studies are needed to find out the molecular mechanisms in which palmitoylation may be involved. The characterization of proteins which may act as PATs and target proteins of palmitoylation in G. lamblia would contribute to clarify the molecular mechanisms used by the parasite in Key biological events such as antigenic variation and/or encystation.