Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol
UNSPECIFIED. (1997) Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol. JOURNAL OF BIOLOGICAL CHEMISTRY, 272 (35). pp. 22097-22102. ISSN 0021-9258Full text not available from this repository.
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.
|Item Type:||Journal Article|
|Subjects:||Q Science > QD Chemistry|
|Journal or Publication Title:||JOURNAL OF BIOLOGICAL CHEMISTRY|
|Publisher:||AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC|
|Date:||29 August 1997|
|Number of Pages:||6|
|Page Range:||pp. 22097-22102|
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