Subtilisin-like enzymes in Trypanosoma cruzi.

NIEMIROWICZ, GABRIELA TERESA; CAZZULO, JUAN JOSÉ

Woods Hole

Congreso; 2011 (22nd) Annual Molecular Parasitology Meeting; 2011

Family S8, also known as the subtilase family, is the second largest serine protease family both in terms of number of sequences and characterized peptidases to date. Subtilases are widespread, being found in eubacteria, archaebacteria, eukaryotes and viruses. The vast majority of them are nonspecific endopeptidases, with a preference to cleave after hydrophobic residues. In particular, the genome of Trypanosoma cruzi, the causative agent of Chagas disease, encodes two such enzymes named TcSUB and TcCUB. Both proteins display 18% identity along the catalytic domain and are characterized by being mosaic proteins. Thus, TcSUB contains a C-terminal extension that shows no sequence similarity to any other known protein, whereas TcCUB present three EGF-like domains and a structural motif found almost exclusively in extracellular and plasma membrane-associated proteins, the CUB domain. In this work we employed the baculovirus-insect cell expression system to produce a recombinant C-terminal His-tagged version of TcCUB. In this system we demonstrated that maturation of TcCUB involves a processing event in which the precursor protein is converted to a 116 kDa form. Mutation of the predicted active site serine (S613A) abolished this processing, suggesting that it is autocatalytic. Overexpression experiments using the epimastigote form of the parasite, and a HA-tagged version of the peptidase cloned in the pTEX vector allowed us to localize this enzyme in small vesicular-like structures that did not co-localize with classical vesicular markers such as those for reservosomes, glycosomes or acidocalcisomes. Thus, this might be a new subcellular compartment that will need to be characterized.