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 recomb inant ricin A chain (rRTA) and rRTA modified by addition of organelle- specific amino acid retention sequences, including KDEL (an endoplasmi c reticulum, ER, lumen retention signal), KKMP (an ER membrane retenti on 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-pha se endocytosis. rRTA-KDEL and rRTA-YQRL were significantly more cytoto xic for Jurkat cells than rRTA, rRTA-KKMP or rRTA-KFERQ. This differen ce did not result from signal(KDEL or YQRL)-mediated binding of these RTA mutants to the cell surface. Reconstituted ER and Golgi vesicles h ave 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 p rotection against papain digestion in reconstituted ER vesicles and 2. 2-fold and 1.8-fold more protection in reconstituted Golgi vesicles, t han unmodified rRTA. These mutants were reassociated with ricin B chai n 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 bo th ER and the trans-Golgi network may play important roles in the intr acellular trafficking and translocation of ricin A chain.