Serine and metallocarboxypeptidases from Trypanosoma cruzi.

GABRIELA NIEMIROWICZ; FABIOLA PARUSSINI; JUAN JOSÉ CAZZULO

Portoroz

Simposio; Xth International Symposium on Proteinase Inhibitors and Biological Control; 2007

Trypanosoma cruzi, the protozoan parasite which is the causative agent of the American Trypanosomiasis, Chagas Disease, contains at least three carboxypeptidases: a serine carboxypeptidase (TcSCP) belonging to the S10 family (1), and two metallocarboxypeptidases (TcMCPs) belonging to the M32 family, found up to now only in prokaryotes (2).  TcSCP is a lysosomal monomeric glycoprotein with a molecular mass of about 54 kDa, acting on hydrophobic C-terminal residues at acidic pH values, and strongly inhibited by 3,4-dichloroisocoumarin.  The enzyme, which is encoded by a number of genes arrayed in head-to-tail tandems, has been expressed as an active recombinant protein in the baculovirus-insect cell system.  The activity of the recombinant enzyme was, however, considerably lower than that of the native one.    Assuming that part of the small predicted pro-domain might not have been processed, and considering that TcSCP co-localizes with cruzipain in the reservosomes, we treated the recombinant TcSCP with the endoproteinase.  Activity increased by 3-fold, thus suggesting that cruzipain might be responsible for pro-TcSCP processing in vivo.The genome of the CL Brener clone of T. cruzi encodes two TcMCPs with 64 % identity between them, TcMCP1 and 2.  Both genes are present as single copies per haploid genome, and are differentially expressed in the parasite?s life cycle.  Whereas TcMCP1 is expressed by the four main stages, TcMCP2 seems to be present only in the stages present in the insect vector.  The purified recombinant proteins differed also in specificity;  whereas TcMCP1 preferred a C-terminal basic residue, TcMCP2 preferred aromatic and aliphatic non polar residues.   Immunofluorescence experiments showed that both TcMCPs are localized in the cytosol.   If the activity of these enzymes proves to be essential for the parasite, they would be good candidates as possible targets for the development of new drugs against Chagas Disease, since the M32 family is absent from all eukaryotic genomes sequenced so far, with the only exception of trypanosomatids