ABSTRACT
A single-chain fusion protein that directed the cytolytic activity of α-sarcin to A33 tumor antigen expressing cells was constructed and shown to effectively kill targeted cells. Glycoprotein A33 (GPA33) is a well-known colon cancer marker and a humanized antibody against it was used to target the α-sarcin. The fungal ribotoxin α-sarcin is one of the most potent and specific toxins known. It is small, protease resistant, thermostable and highly efficient towards the inactivation of ribosomes. This work describes the production and characterization of an immunotoxin resulting from fusing the single-chain variable fragment (scFv) of the monoclonal antibody that targets GPA33 to fungal α-sarcin. This chimeric protein (scFvA33αsarcin), produced in Pichia pastoris and purified in high yield was proven to be properly folded, active, specific and stable. It showed high specific toxicity against GPA33-positive tumoral cell lines providing scientific evidence to sustain that scFvA33αsarcin is a good immunotherapeutic candidate against GPA33-positive colon carcinomas.
Subject(s)
Colonic Neoplasms/metabolism , Endoribonucleases/chemistry , Fungal Proteins/chemistry , Immunotoxins/chemistry , Membrane Glycoproteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Single-Chain Antibodies/chemistry , Cell Line, Tumor , Colonic Neoplasms/drug therapy , Colonic Neoplasms/immunology , Endoribonucleases/genetics , Endoribonucleases/metabolism , Flow Cytometry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Immunotoxins/genetics , Immunotoxins/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Microscopy, Fluorescence , Pichia/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolismABSTRACT
Ribotoxins constitute a family of toxic extracellular fungal RNases that exert a highly specific activity on a conserved region of the larger molecule of rRNA, known as the sarcin-ricin loop. This cleavage of a single phosphodiester bond inactivates the ribosome and leads to protein synthesis inhibition and cell death. In addition to this ribonucleolytic activity, ribotoxins can cross lipid membranes in the absence of any known protein receptor. This ability is due to their capacity to interact with acid phospholipid-containing membranes. Both activities together explain their cytotoxic character, being rather specific when assayed against some transformed cell lines. The determination of high-resolution structures of some ribotoxins, the characterization of a large number of mutants, and the use of lipid model vesicles and transformed cell lines have been the tools used for the study of their mechanism of action at the molecular level. The present knowledge suggests that wild-type ribotoxins or some modified variants might be used in human therapies. Production of hypoallergenic mutants and immunotoxins designed against specific tumors stand out as feasible alternatives to treat some human pathology in the mid-term future.
Subject(s)
Fungal Proteins/therapeutic use , Fungi/enzymology , Immunologic Factors/therapeutic use , Protein Synthesis Inhibitors/therapeutic use , Ribonucleases/therapeutic use , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Fungal Proteins/chemistry , Fungal Proteins/immunology , Fungal Proteins/toxicity , Humans , Hypersensitivity/drug therapy , Immunologic Factors/chemistry , Immunologic Factors/immunology , Immunologic Factors/toxicity , Models, Molecular , Neoplasms/drug therapy , Phospholipids/metabolism , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/immunology , Protein Synthesis Inhibitors/toxicity , Ribonucleases/chemistry , Ribonucleases/immunology , Ribonucleases/toxicityABSTRACT
RNase T1 is the best known representative of a large family of ribonucleolytic proteins secreted by fungi, mostly Aspergillus and Penicillium species. Ribotoxins stand out among them by their cytotoxic character. They exert their toxic action by first entering the cells and then cleaving a single phosphodiester bond located within a universally conserved sequence of the large rRNA gene, known as the sarcin-ricin loop. This cleavage leads to inhibition of protein biosynthesis, followed by cellular death by apoptosis. Although no protein receptor has been found for ribotoxins, they preferentially kill cells showing altered membrane permeability, such as those that are infected with virus or transformed. Many steps of the cytotoxic process have been elucidated at the molecular level by means of a variety of methodological approaches and the construction and purification of different mutant versions of these ribotoxins. Ribotoxins have been used for the construction of immunotoxins, because of their cytotoxicity. Besides this activity, Aspf1, a ribotoxin produced by Aspergillus fumigatus, has been shown to be one of the major allergens involved in allergic aspergillosis-related pathologies. Protein engineering and peptide synthesis have been used in order to understand the basis of these pathogenic mechanisms as well as to produce hypoallergenic proteins with potential diagnostic and immunotherapeutic applications.
