A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL] promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.Ricin is the toxic lectin from Ricinus communis (castor bean) seeds. The ricin molecule is a 65,000-dalton (Da) heterodimer consisting of an A chain and a B chain linked by a disulfide bond (16). The B chain is a galatose-binding lectin which causes ricin to bind to mammalian cells. Once bound to cells, the ricin is internalized and the A chain translocates across endocytic vesicle or Golgi membranes to the cytosol. In the cytosol the ricin toxin A chain (RTA) catalytically inactivates protein synthesis by hydrolysis of the N-glycosidic bond of adenosine residue 4324 in the 28S rRNA of the eucaryotic 60S ribosomal subunit (5). This modification irreversibly inactivates the ribosome, possible by altering the binding site for elongation factors (D. Moazed, J. Robertson, and H. Noller, Nature [London], in press). A single molecule of RTA in the cytosol is sufficient to cause cell death (3).The detailed mechanism for the RNA N-glycosidase activity of RTA is unknown. X-ray crystallography shows a cleft in the second domain of RTA which resembles the active sites of other proteins that bind large polynucleotide substrates (14). Furthermore, a number of amino acid residues located in the second domain of RTA are conserved among a group of several additional toxins and RNA-binding proteins (19), suggesting that this region may be critical for catalytic activity.The genomic ricin gene encodes both the A and B proteins. The gene lacks introns and encodes a 24-amino-acid signal peptide, the A chain, a 12-amino-acid linker peptide, and a B chain (6). Cloned RTA prepared from both the genomic ricin DNA and a cDNA clone of ricin has been expressed in Escherichia coli (15,17). In each case a soluble biologically functional molecule was produced. However, procaryotic ribosomes are not a substrate for RTA (15), and no mutants with defects in enzymatic activity or other RTA functions have been described.We have been studying the expression of cloned RTA in the yeast Saccharomyces cerevisiae, whose ribosomes are sensitive to RTA. When RTA was expressed in yeast cells, growth was arrested. Plasmids encoding mutant RTA were readily identified by their failure to kill S. cerevisiae. These observations were the basis for a positive selection for mutations that inactivated RTA. By extracting both protein * Corresponding author. and nucleic acid from the yeast cells, we determined the DNA sequence of mutant RTA genes and the enzymatic properties of the proteins they encode. Our results indicate that in...

Immunotoxins are a new class of antitumor agents consisting of tumor-selective ligands (generally monoclonal antibodies [MoAbs]) linked to highly toxic protein molecules that have been modified to remove their normal tissue-binding domains. These immuno-conjugates combine the potency of the parent toxin with the specificity of the attached ligand. Toxins used in the construction of immunotoxins belong to a group of peptides that catalytically inhibit the elongation step of protein synthesis, and include ricin, abrin, pokeweed antiviral protein, gelonin, Pseudomonas exotoxin A, diptheria toxin, and alpha-sarcin. To synthesize immunotoxins, the normal cell-binding function must be removed by chemical cleavage or modification, or in the case of toxins that have been cloned, genetic engineering used to delete amino acids critical to cell binding. Covalent linkage of toxin to ligand generally involves a disulfide or thioether bond, though recently, recombinant toxin molecules with ligands that are genetically engineered into the protein have been made. The most successful clinical application of immunotoxins has been in the depletion of T cells from allogeneic bone marrow grafts to prevent graft-versus-host disease (GVHD). Clinical trials have been conducted using immunotoxins for the systemic treatment of chronic lymphocytic leukemia (CLL), GVHD, and selected solid tumors. With the possible exception of GVHD, responses have been limited. Obstacles have included rapid systemic clearance, poor delivery to extravascular tumor deposits, and humoral immune responses to the immunotoxin. Research to overcome these problems is in progress and should lead to a better definition of the role of immunotoxins in the therapy of malignancies.

No abstract