Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol

Ricin is a heterodimeric protein toxin. The ricin A chain is able to cross the membrane of intracellular compartments to reach the cytosol where it catalytically inactivates protein synthesis. It is linked via a disulfide bond to the B chain, a galactose-specific lectin, which allows ricin binding at the cell surface and endocytosis. To examine the potential of ricin A to carry proteins into the cytosol and the requirement for unfolding of the passenger protein, we connected mouse dihydrofolate reductase (DHFR) to ricin A by gene fusion via a spacer peptide. DHFR-ricin A expressed in Escherichia coli displayed the biological activities of the parent proteins and associated quantitatively with ricin B to form DHFR-ricin. The resulting toxin was highly cytotoxic to cells (4-8-fold less than recombinant ricin). DHFR-ricin cytotoxicity was inhibited by methotrexate, a DHFR inhibitor stabilizing DHFR-ricin A in a folded conformation. The DHFR moiety of DHFR ricin bound to the plasma membrane. Although methotrexate prevented this binding, it did not significantly affect DHFR-ricin endocytosis, which proceeded via ricin B chain. Intoxication kinetics data and a cell-free translocation assay demonstrated that protection of cells from DHFR-ricin cytotoxicity resulted from a selective inhibition by methotrexate of DHFR-ricin A translocation. We conclude that ricin A is a potential carrier of proteins to the cytosol, provided that the passenger protein is able to unfold for transmembrane transport.