RESUMO
Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N-terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II-GrBa fusion protein genes were constructed by replacing N-terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280-364/358) by recombinant PCR, and then cloned into pIND inducible expression vector. The resulting pIND-PE II-GrBa expression vectors were co-transfected with assistant plasmid pVgRXR into HeLa cells through lipofectamine, followed by selection on G418 and zeocin. The resistant cells were collected and induced with ponasterone A. Western blot analysis demonstrated that ponasterone A induction caused the expression of PE II-GrBa fusion proteins, and indirect immunofluorescence detected giant sized multinucleated cells, suggesting cytoskeletal and mitotic abnormalities as reported in our previous studies. Western blot, enzymatic activity assay and cell counting analysis indicated that two types of PE II-GrBa fusion proteins were capable of cleaving both endogenous and exogenous substrates of granzyme B, and inhibiting the growth of cells. The PE II (aa 280-358)-GrBa was shown to have higher serine protease activity and stronger growth inhibitory effect. Such inhibition was presumably associated with G2 arrest as determined by cell cycle analysis. These data prove that PE II-GrBa fusion proteins have cell inhibitory effect similar to GrBa, and that the shorter PE-derived peptide exerts less influence on GrBa activity. This study helps to optimize the construction of recombinant protein comprising translocating peptides and cytotoxic molecules for tumor cell killing.