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Biochemical and cytotoxic properties of conjugates of transferrin with equinatoxin II, a cytolysin from a sea anemone.

Transferrin, a serum glycoprotein, is a major regulator of cellular growth via its cellular receptor. Because transferrin receptors are absent from the plasma membranes of most normal adult resting cells, but are present on transformed, activated, and malignant cells, it can be used to address a toxin toward these cells. The cytolysin equinatoxin II, isolated from the sea anemone Actinia equina L., was coupled to human apo or diferric transferrin by using a heterobifunctional cross-linking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates were separated by column chromatography, and their composition was demonstrated by electrophoresis, antibody staining, and determination of the hemolytic activity in the absence or presence of a reducing agent. The average molar ratio of equinatoxin II to transferrin for the studied conjugates was found to be approximately 3.4. The activity of the conjugates against human erythrocytes and human tumor cells (Raji and Jurkat) was assessed. The conjugate is very active on tumor cells in vitro; however, the hybrid molecule maintains an unspecific hemolytic activity. This unspecific toxicity is due to the fact that transferrin-bound toxin partially retains its original ability to bind to the cell membrane directly. It could be strongly reduced (and even eliminated) by pretreating the conjugates with sphingomyelin, the natural ligand of sea anemone cytolysins. These conjugates were stable versus temperature (up to at least 40 degrees C), versus time (up to several weeks at 4 degrees C and at least 1 year at -80 degrees C), and versus repeated freeze-thaw cycles with liquid nitrogen (but not with -80 degrees C).