Studies on the entry of ricin subunits into cells

The potent cytotoxin ricin, obtained from the seeds of the castor oil plant Ricinus communis, is composed of two polypeptide subunits linked by a single disulphide bond. The binding of this molecule to the surface of eukaryotic cells is mediated via the sugar-binding activity of the B subunit. The exact nature of the ricin receptor(s) on the cell surface is unclear, but is most probably some glycoprotein or glycolipid containing exposed galactosyl residues. The ricin molecule becomes internalized by the cell and, by an unclear mechanism, ricin A chain escapes its endocytic vesicle entering the cell cytoplasm where it enzymatically and irreversibly inhibits eukaryotic ribosomes, bringing about the cessation of protein synthesis. The toxicity of ricin A chain-containing immunotoxins can in many model systems be enhanced by the subsequent addition of ricin B chain. This apparent ricin B chain-mediated potentiation of cytotoxicity suggests that this subunit has some role in the mechanism(s) facilitating the entry of ricin A chain into the cell cytosol. The work presented in this thesis has attempted to examine the potential of ricin B chain as a carrier of proteins into cells other than ricin A chain. In this example ricin A chain has been replaced with the type I ribosome inactivating protein, gelonin. Further to these studies, preliminary work considering the possible Importance of the hydrophobic C-terminus of ricin A chain in the translocation events, has been presented. To date this work has demonstrated that it is possible to delete at least 30 amino acid residues from the C-terminus of the A chain and retain full ribosomal inactivation activity as judged by in vitro analysis. This truncated form of ricin A chain has been expressed in an E. coli expression system and a soluble and active recombinant protein has been partially purified. The implications of this work and possible future analysis of this mutant polypeptide have also been considered