CLATHRIN-ASSOCIATED PROTEIN IN GIARDIA LAMBLIA

FELIZIANI C; ZAMPONI N,; MIRAS SL,; ROPOLO A S; TOUZ MC.

potrero de los funes

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2011

The presence of clathrin-associated sorting proteins (CLASP) has not yet been reported in the protozoan Giardia lamblia. However, by searching in the Giardia Genome Database, we found a protein that contains an ENTH domain characteristic of the CLASP epsin. The analysis of the participation of the ENTH protein (ENTHp) in the trafficking towards lysosomal?like peripheral vacuoles (PVs) was initiated by cloning and expression of the HA-fusion protein and its mutants in Giardia trophozoites. Using IFA and confocal microscopy we showed that ENTHp localize mainly in the cytosol and in the nuclei, partially colocalizing with the ER protein BIP and a PV marker protein. Additionally, we developed monoclonal antibodies directed against the N-terminal region of clathrin heavy chain (CHC) to provide a unique set of tools for understanding the interaction and function of these particular proteins. Clathrin positive polyclonal Ab (pAb) production was tested by IFA and immunoblotting in wild-type trophozoites observing that the pAb recognized a protein localized in the PVs and a band of 206 kDa, respectively, corresponding to the CHC. Using this pAb in IFA and IPP assays we were able to observe the colocalization of ENTHp and CHC in the PVs and also validate the interaction between both proteins. Antibody-secreting hybridomas for CHC will allowed a better characterization of clathrin and also corroborate whether the ENTHp is a CLASP acting as a dual epsin-EpsinR protein. These results will greatly increase our understanding of unique aspects of lysosomal trafficking in Giardia.