Clathrin-associated protein in Giardia lamblia.

FELIZIANI C; ZAMPONI N; MIRAS S; ROPOLO AS; TOUZ MC

San Luis

Congreso; SAIB; 2011

The presence of clathrin-associated proteins (CLASP) has not yet been reported in the protozoan G. lamblia. However, by searching in the GDB, 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 GiardiaG. lamblia. However, by searching in the GDB, 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 GiardiaGiardia trophozoites. Using IFA and confocal microscopy we showed that ENTHp localize mainly in the cytosol and around the nuclei, partially colocalizing with BIP and AP-2. Additionally, we developed monoclonal antibodies directed against clathrin heavy chain (CHC) to disclose the interaction and function of these particular proteins. Clathrin positive polyclonal Ab (pAb) production was tested in wild-type trophozoites observing that the pAb recognized a protein localized in the PVs and a band of 203 kDa corresponding to the CHC. Using this pAb we observed the colocalization of ENTHp and CHC in the PVs and also validate the interaction between both proteins. Antibody-secreting hybridomas for CLH 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..