CHARACTERIZATION OF PROKARYOTIC RECOMBINANT ASPERGILLUS RIBOTOXIN ALPHA-SARCIN

The Aspergillus ribonuclease alpha-sarcin is toxic to intact mammalian cells but the mechanism by which it enters the cells to reach its rib osomal RNA substrate is unclear. Here we have compared the cytotoxicit y of alpha-sarcin to that of ricin, another catalytic toxin that targe ts the same rRNA sequence but whose mechanism of cell entry is better understood. Intact ricin binds to cell surface components and enters t he cells by receptor-mediated endocytosis, whereas the catalytic polyp eptide of ricin (the A chain or RTA) which, like alpha-sarcin, is unab le to bind to surface components directly and enters cells by fluid ph ase uptake. Recombinant alpha-sarcin was produced in Escherichia coli and purified to homogeneity. The protein was soluble, stable and its a bility to inhibit in vitro protein synthesis was indistinguishable fro m that of native alpha-sarcin. Further, recombinant alpha-sarcin had t he same in vitro protein synthesis inhibition activity as ricin A chai n. The cytotoxicity of alpha-sarcin and ricin A chain, to HeLa cells w as also the same. The cytotoxicity of alpha-sarcin was due to its RNAa se activity rather than to specific membrane effects at the cell surfa ce, since a mutant containing a single substitution at a putative key catalytic residue had reduced ribonuclease activity and an equivalent reduction in cytotoxicity. One interpretation of the data is that alph a-sarcin enters mammalian cells in the same way as free ricin A chain. (C) 1997 Elsevier Science B.V.