Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol

被引:19
作者
Beaumelle, B [1 ]
Taupiac, MP [1 ]
Lord, JM [1 ]
Roberts, LM [1 ]
机构
[1] UNIV WARWICK,DEPT BIOL SCI,COVENTRY CV4 7AL,W MIDLANDS,ENGLAND
关键词
D O I
10.1074/jbc.272.35.22097
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:22097 / 22102
页数:6
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