Selective Blockade of Trypanosomatid Protein Synthesis by a Recombinant Antibody Anti-Trypanosoma cruzi P2β Protein

Abstract

The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.

Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion

“…A cell invasion assay in the presence of distinct molecules (mAb-10D8, scFv-10D8, and negative controls) was performed to confirm scFv-10D8 function. Accordingly, scFv-10D8 showed a clear capacity to specifically reduce parasite invasion, which is compatible with the results described by Ayub et al [43]. This result was similar to that of the experiment without purified protein.…”

“…The scFv expression analysis was performed in a pET22b+ vector, which allowed for the insertion of the pelB leader sequence for assessing expression in the periplasm, which is an environment conducive to the formation of the disulfide bonds that allow the correct conformation of proteins including scFvs [45]. The scFv-10D8 expression levels observed in the periplasm in the present study are consistent with those reported for other scFvs, which are usually low [43,46,47]. Instead of the low level of expression at the periplasm, we were able to show the binding capacity of scFv-10D8 through Western blotting, confirming the same specificity described for mAb-10D8 [48].…”

“…cruzi cell surface are previously characterised mAbs because they can be humanised for use in human therapy. However, to exploit their potential, they must be engineered as recombinant molecules, such as scFvs and diabodies [42,43]. Since hybridoma cell lines are quite unstable and frequently loose the expression of functional antibody, the sequencing mAb-10D8 variable segments (VL and VH) in the present study allowed preserving the antibody expression in other systems.…”