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Genetic grafting of membrane‐acting peptides to the cytotoxin dianthin augments its ability to de‐stabilize lipid bilayers and enhances its cytotoxic potential as the component of transferrin‐toxin conjugates
Author(s) -
Lorenzetti Irene,
Meneguzzi Alessandra,
Fracasso Giulio,
Potrich Cristina,
Costantini Laura,
Chiesa Elena,
Legname Giuseppe,
Menestrina Gianfranco,
Tridente Giuseppe,
Colombatti Marco
Publication year - 2000
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/(sici)1097-0215(20000515)86:4<582::aid-ijc22>3.0.co;2-i
Subject(s) - cytotoxicity , biochemistry , biology , anthrax toxin , vesicular stomatitis virus , hemagglutinin (influenza) , immunotoxin , peptide , microbiology and biotechnology , recombinant dna , fusion protein , vesicle , liposome , chemistry , virus , membrane , in vitro , virology , gene
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 N terminus of the HA2 hemagglutinin of influenza virus (pHA2), and a membrane‐acting peptide (pJVE). Chimeric dianthins (KFT25DIA, pHA2DIA and pJVEDIA) retained full enzymatic activity in cell‐free assays and showed increased ability to induce pH‐dependent calcein release from large unilamellar vesicles (LUVs). pHA2DIA and pJVEDIA also showed faster kinetics of interaction with LUVs, while KFT25DIA and pHA2DIA displayed a reduced cytotoxicity as compared to wild‐type dianthin. Conjugates made by chemically cross‐linking KFT25DIA or pJVEDIA and human transferrin (Tfn) showed greater cell‐killing efficiency than conjugates of Tfn and wild‐type dianthin. As a consequence, by fusion of membrane‐acting peptides to the dianthin sequence the specificity factor ( i.e., the ratio between non‐specific and specific toxicity) of Tfn‐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 enzymatic cytotoxins results in the acquisition of new physico‐chemical properties exploitable for designing new recombinant cytotoxins and to tackle cell‐intoxication mechanisms. Int. J. Cancer 86:582–589, 2000. © 2000 Wiley‐Liss, Inc.