Clathrin-associated Protein in Giardia lamblia

FELIZIANI C.; ZAMPONI N.; MIRAS S. L.; ROPOLO A.S.; TOUZ M. C.

Congreso; 47th Annual Meeting. Argentine Society for Biochemistry and Molecular Biology Research; 2011

The presence of clathrin-associated proteins (CLASP) has not yetbeen reported in the protozoan G. lamblia. However, by searching inthe GDB, we found a protein that contains an ENTH domaincharacteristic of the CLASP epsin. The analysis of the participationof the ENTH protein (ENTHp) in the trafficking towardslysosomal?like peripheral vacuoles (PVs) was initiated by cloningand expression of the HA-fusion protein and its mutants in Giardiatrophozoites. Using IFA and confocal microscopy we showed thatENTHp localize mainly in the cytosol and around the nuclei,partially colocalizing with BIP and AP-2. Additionally, wedeveloped monoclonal antibodies directed against clathrin heavychain (CHC) to disclose the interaction and function of theseparticular proteins. Clathrin positive polyclonal Ab (pAb)production was tested in wild-type trophozoites observing that thepAb recognized a protein localized in the PVs and a band of 203 kDacorresponding to the CHC. Using this pAb we observed thecolocalization of ENTHp and CHC in the PVs and also validate theinteraction between both proteins. Antibody-secreting hybridomasfor CLH will allowed a better characterization of clathrin and alsocorroborate whether the ENTHp is a CLASP acting as a dual epsinEpsinR protein. These results will greatly increase ourunderstanding of unique aspects of lysosomal trafficking inGiardia.