Eg-CLP1: an Echinococcus canadensis cathepsin-L, potentially involved in cyst formation.

NAIDICH, ARIEL; SALAS-SARDUY, EMIR; BOUVIER, LEÓN ALBERTO; NIEMIROWICZ, GABRIELA T.; ALVAREZ, VANINA E.; GUTIERREZ, ARIANA

Congreso; XXVII World Congress of Echinococcosis; 2017

Background and Aim: The lifecycle of the parasite Echinococcus spp. is known since 19th century. Some of their stages have been extensively studied, while others still remain poorly understood. The bibliography about cyst formation is narrowed to few articles based on morphological features, but to date, no molecular characterization has ever been reported. Several molecules, particularly cathepsins, have been found in parasites, involved in roles of extracellular matrix disruption in migration trough tissues or excystment. The aim of the present work was to identify and characterize cysteine peptidases that could eventually be involved in cyst formation.Material and methods: The protoscolices from cysts of pig livers were aseptically collected, washed several times with PBS, and later pepsinized. The RNA was extracted from protoscolices,using Trizol reagent protocol and employed to construct cDNA libraries. Genomic DNA was also extracted from protoscolices, using Trizol protocol. Several PCR reactions were performed with sets of primers designed to amplify a generic cysteine peptidase conserved region and a cathepsin from E. multilocularis, using cDNA libraries previously obtained and genomic parasite DNA as template.The products were sequenced. These sequences were used to perform several bioinformatic studies, including sequence similarity search (BLASTn and tBLASTp), conserved domain search (CDS, Pfam, Prosite), cladogram analysis (Mega 6.0), with similar sequences from related parasites and E. granulosus hosts. Tridimensional modelling of translated sequences (Swiss-Model) and intron-exonprediction for PCR product sequence from genomic DNA (Genscan) was also done.Synthetic DNA for recombinant expression of the obtained putative protein was cloned and expressed in E. coli. The protein was purified under denaturing conditions, and refolded by rapid dilution. Catalytic activity was determined by fluorescent substrate processing, with and without the generic cathepsin inhibitor E-64. The purified recombinant protein was employed for immunizationof rabbits. Studies on protein localization at interphase parasite-host are being performed.Results: Several fragments were amplified in PCRs (longest one named Eg-CLP1). Their sequences yielded similarity with cathepsins-L from several related organisms, as Taenia, Spirometra, Hymenolepis and E. multilocularis. Conserved domain search allowed the identification of a peptidase C1A domain, and inhibitor I29 in the case of the larger fragment. Cladogram analysis alsogrouped the translated sequence of Eg-CLP1 in clades with several E. multilocularis and E. granulosus cathepsin sequences from GenBank. This clade shared branch with Fasciola hepatica cathepsins, and was more dissimilar to other taenias and hosts cathepsins.Structural analysis of 3D models showed the catalytic triad conserved in spatial structure. The catalytic cleft was also present. Recombinant protein showed catalytic activity in good agreement with cathepsin L.Conclusion: We found a molecule with sequence, conserved domains, 3D simulated structure and similarity by cladograms that resulted compatible with a cathepsin L. Same compatibility was showed by recombinant protein in catalytic activity and inhibition. Similar molecules of E. multilocularis showed collagenase and elastase activity. Several studies are currently underway to determine whether different protein components of extracellular matrix can be degraded byrecombinant Eg-CLP1 and to estimate kinetic parameters for the hydrolysis of typical cathepsin L substrates by this enzyme.