Modification of ricin A chain, by addition of endoplasmic reticulum (KDEL) or Golgi (YQRL) retention sequences, enhances its cytotoxicity and translocation.
A pKK expression system in Escherichia coli was used to produce recombinant ricin A chain (rRTA) and rRTA modified by addition of organelle-specific amino acid retention sequences, including KDEL (an endoplasmic reticulum, ER, lumen retention signal), KKMP (an ER membrane retention signal), YQRL (a trans-Golgi network retention signal) and KFERQ (a lysosome-targeting signal) to the C terminus of rRTA. The toxicities of these RTA mutants were assessed in Jurkat cells following fluid-phase endocytosis. rRTA-KDEL and rRTA-YQRL were significantly more cytotoxic for Jurkat cells than rRTA, rRTA-KKMP or rRTA-KFERQ. This difference did not result from signal(KDEL or YQRL)-mediated binding of these RTA mutants to the cell surface. Reconstituted ER and Golgi vesicles have been employed to assess translocation of rRTA and mutant rRTA. RTA-KDEL and RTA-YQRL respectively exhibited 6.7-fold and 6.1-fold more protection against papain digestion in reconstituted ER vesicles and 2.2-fold and 1.8-fold more protection in reconstituted Golgi vesicles, than unmodified rRTA. These mutants were reassociated with ricin B chain to form holotoxins. The mutant RTA-KDEL and RTA-YQRL holotoxins were 3.8-fold and 1.5-fold more cytotoxic for target cells, respectively, than ricin produced using unmodified rRTA. Our results suggest that both ER and the trans-Golgi network may play important roles in the intracellular trafficking and translocation of ricin A chain.
Zhan
J
J
Department of Medicine, University of Washington, Seattle 98195, USA.
Stayton
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Press
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D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D012276:Ricin / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
D011994:Recombinant Proteins; D012276:Ricin