Structure and action of heteronemertine polypeptide toxins. Membrane penetration by Cerebratulus lacteus toxin A-III

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Abstract
The heteronemertine worm C. lacteus produces a family and 3 structurally homologous proteins that function as direct lytic factors for a variety of cells. The hemolytic activity of the most abundant variant, designated toxin A-III, was unaffected by extensive iodination or complete blockage of carboxylate groups by tyramine or glycine ethyl ester. Iodination of A-III with lactoperoxidase produced a derivative that was preferentially labeled at His-67 and to a lesser extent at Tyr-6. The ratio of labeling at these 2 positions was .apprx. 3:1. Iodinated A-III was completely insoluble in 10% Cl3CCOOH. Following treatment with trypsin-containing liposomes, 15% of the input counts were converted to a Cl3CCOOH-soluble form. Incubation with free trypsin in the presence of liposomes containing N.alpha.-tosyl-L-lysine chloromethyl ketone resulted in .apprx. 60% the input counts becoming Cl3CCOOH soluble. Free trypsin rendered toxin A-III 90% soluble in 10% ClCCOOH. Electrophoretic analysis of the labeled tryptic peptides generated in the presence of liposomes showed that internal trypsin hydrolyzed the Arg-13.sbd.Ser-14 bond, generating exclusively peptide T-1 (residues 1-13) while external trypsin produced peptide T-11 (residues 60-71) as the major radioactive product. These data were consistent with insertion of at least the amino-terminal 13 residues of A-III into the liposome and implied that membrane penetration by this protein may have been important for its cytolytic activity.