EVIDENCE FOR INVOLVEMENT OF FURIN IN CLEAVAGE AND ACTIVATION OF DIPHTHERIA-TOXIN

Proteolytic cleavage (nicking) of diphtheria toxin (DT) in the 14-amin o acid loop subtended by the disulfide bond between Cys186 and Cys201 is required for the cytotoxic action of DT. The loop includes the cons ensus motif for cleavage by a membrane-anchored protease, furin. We fo und that a soluble form of furin cleaves intact DT between Arg198 and Ser194 in vitro. LoVo cells, a human colon carcinoma cell line, do not produce functional furin. We show here that intact DT is not cleaved by LoVo cells. The cells are resistant to intact DT, although they are sensitive to DT nicked by furin before it is added to the medium. Whe n intact DT is added to LoVo/Fur1 cells, a stable transfectant of LoVo cells expressing mouse furin, nicked DT associated with the cells is observed. LoVo/Fur1 cells are sensitive to both intact and nicked DT. These results indicate that furin is involved in the toxicity of intac t DT. Bafilomycin A1, an inhibitor of intracellular vesicle acidificat ion, did not inhibit cleavage of intact DT by LoVo/Fur1 or Vero cells, indicating that cleavage can proceed in a neutral environment. Inhibi tors of endocytosis decreased DT cleavage but did not eliminate it. We also found a small amount of nicked DT in the culture medium. These r esults may indicate that intact DT is cleaved by cell-associated furin on the cell surface as well as in endocytotic vesicles.