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.
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.
Lorenzetti
I
I
Section of Immunology, Department of Pathology, University of Verona, Verona, Italy.
Meneguzzi
A
A
Fracasso
G
G
Potrich
C
C
Costantini
L
L
Chiesa
E
E
Legname
G
G
Menestrina
G
G
Tridente
G
G
Colombatti
M
M
D006801:Humans; D006863:Hydrogen-Ion Concentration; D000922:Immunotoxins / Q000494:pharmacology*; D019169:Jurkat Cells; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D008985:Monensin / Q000494:pharmacology; D010940:Plant Proteins / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D054789:Ribosome Inactivating Proteins, Type 1; D014168:Transferrin / Q000494:pharmacology*
D000922:Immunotoxins; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D014168:Transferrin; D008985:Monensin