Genetic grafting of membrane-acting peptides to the cytotoxin dianthin augments its ability to de-stabilize lipid bilayers and enhances its cytotoxicpotential as the component of transferrin-toxin conjugates

Three chimeric proteins were obtained by fusing together the dianthin gene and DNA fragments encoding for the following membrane-acting peptides: the N-terminus of protein G of the vesicular stomatitis virus (KFT25), the hi t erminus of the HA2 hemagglutinin of influenza virus (pHA2), and a membrane- acting peptide (pJVE). Chimeric dianthins (KFT25DIA, pHA2DIA and pJVEDIA) r etained full enzymatic activity in cell-free assays and showed increased ab ility to induce pH-dependent calcein release from large unilamellar vesicle s (LUVs). pHA2DIA and pJVEDIA also showed faster kinetics of interaction wi th LUVs, while KFT25DIA and pHA2DIA displayed a reduced cytotoxicity as com pared to wild-type dianthin. Conjugates made by chemically crosslinking KFT 25DIA or pJVEDIA and human transferrin (Tfn) showed greater cell-killing ef ficiency than conjugates of Tfn and wild-type dianthin. As a consequence, b y fusion of membrane-acting peptides to the dianthin sequence the specifici ty factor (i.e., the ratio between non-specific and specific toxicity) of T fn-KFT25DIA, Tfn-pHA2DIA and Tfn-pJVEDIA was increased with respect to that of Tfn-based conjugates made with wild-type dianthin. Taken together, our results suggest that genetic fusion of membrane-acting peptides to enzymati c cytotoxins results in the acquisition of new physicochemical properties e xploitable for designing new recombinant cytotoxins and to tackle cell-into xication mechanisms. Int. J. Cancer 86:582-589, 2000. (C) 2000 Wiley-Liss, Inc.