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Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi
E Tagge, J Chandler, BL Tang, W Hong, MC Willingham and A Frankel
Department of Surgery, Medical University of South Carolina, Charleston, USA.
DNAs encoding ricin toxin A chain (RTA), with or without a C-terminal endoplasmic reticulum retention signal KDEL, were subcloned into pGEX2T bacterial expression plasmid. After transformation of JM105 E. coli cells and induction with isopropylthio-beta-galactoside (IPTG), fusion proteins were bound to an immobilized glutathione matrix and recombinant ricin A chains released with thrombin. Both recombinant wild-type RTA and RTA with KDEL had immunological reactivity and catalytic activity indistinguishable from plant RTA. The bacterial RTA products reassociated with plant ricin B chain (RTB) similarly to plant RTA. Cell cytotoxicities were measured on seven cell lines for each A- chain and heterodimer. Although KDEL sequences enhanced cytotoxicity in most cases, significant variability was observed. In each case, addition of KDEL enhanced A-chain cytotoxicity more than holotoxin cytotoxicity. Three cell lines showed reduced KDEL enhancement of both RTA and ricin cytotoxicity. The concentration of KDEL receptor was examined on each cell line by immunofluorescence microscopy with an antireceptor monoclonal antibody. Differences in sensitivity to KDEL- containing toxins correlated with altered distribution of KDEL receptor between endoplasmic reticulum (ER) and Golgi compartments.
Volume 44,
Issue 2,
pp. 159-165,
02/01/1996
Copyright © 1996 by The Histochemical Society
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