The Application Of Truncated/mutated Influenza Haemagglutinin Translocation Domain In Immunoproapoptotic Strategy

The N-terminal23-amino acid peptide of influenza virus hemagglutinin (HA2) which causes endosomal leakage, has been widely used in the development of peptide-based drugs. In our previous study, HA2-included immunoproapoptotic molecules showed rapid accumulation of the effector motif in the cytosol and enhanced antitumor effect in vivo and in vitro. This study was aimed at truncating and mutating HA2in order to obtain the shortest functional derivative and use it in our immunoproapoptotic strategy for enhanced translocation activity. Here we designed6truncatants and3mutatants of HA2, and performed erythrocyte lysis assay, tumor cell killing assay and fluorescence resonance energy transfer (FRET) assay to compare translocation efficiency of all HA2truncatants and mutatants.Firstly, in erythrocyte lysis assay, all HA2truncatants and mutatants were analysed for their capability of releasing hemoglobin(Hb) into supernatant due to disruption of erythrocyte membrane. The higher Hb concentration, the stronger translocation effect. Isolated human erythrocytes were incubated with HA2truncatants and mutatants, with wide type HA2as control. Dose response and time course data indicated that the hemolytic ability of E5C3was stronger than that of HA2, and that hemolysis of E5was decreased in comparison to HA2. Among C-terminal truncatants of HA2, only C3retained hemolytic ability, suggesting that the2helix regions were important for HA2to exert hemolytic function because deficiency in one helix (C8) or two (C15) abrogated HA2function. For N-terminal truncation of HA2, both derivatives failed to induce hemolysis, consistent with the report on the functional importance of the first N-terminus amino acid of HA2. Deletion of the middle pH sensor of HA2gave rise to M7and E5C3M7, which caused much less hemolysis than wide type HA2despite increased dose and prolonged treatment, indicating a contributive role of the middle motif in hemolytic function of HA2. Based on the above data, we finally chose5derivatives of HA2for intracellular functional screening in the next step. Wide type HA2served as positive control. The translocation activity of E5C3was stronger than that of HA2, as compared with the much lower translocation activities of C15, M7and E5C3M7.Secondly, in tumor cell killing assay, the resulting5derivatives of HA2were inserted into our established immunoproapoptotic molecules, and cell killing rates were evaluated to reflect their translocation capability. e23sFv-Fdt-HA2derivatives-tBid were constructed and transfected into HER2-positive BT474and SKBR3cells, followed by apoptosis detection by flow cytometry. The results indicated that the translocation efficiency of E5C3was higher than that of HA2and that E5C3M7, C15and M7had attenuated translocation activities.Lastly, in FRET assay, the derivatives of HA2were inserted between EGFP (FRET donor) and mCherry (FRET acceptor). Theoretically, translocation activity of HA2would result in loss of FRET signals and dispersed cytosol distribution. scFv15-EGFP-Fdt-HA2derivatives-mCherry were constructed and stably transfected into COS7cells. Preliminary data showed that scFv15-EGFP-Fdt-HA2-mCherry secreted by COS7cells, could be internalized into FBsAg-positive hepatocellular carcinoma cells PLC/PRF/5and exhibited a patchy endosomal distribution pattern, which provided clues for further optimizing FRET detection window and observing subcellular location of mCherry fluorescence.The above results indicated that E5C3exhibited enhanced translocation activity as compared with HA2, providing a basis for it’s further application in immunoproapoptotic strategy.