Abstract
Proteolytic cleavage (nicking) of diphtheria toxin (DT) in the 14-amino acid loop subtended by the disulfide bond between Cys186 and Cys201 is required for the cytotoxic action of DT. The loop includes the consensus motif for cleavage by a membrane-anchored protease, furin. We found that a soluble form of furin cleaves intact DT between Arg103 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. When 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 intact DT. Bafilomycin A1, an inhibitor of intracellular vesicle acidification, did not inhibit cleavage of intact DT by LoVo/Fur1 or Vero cells, indicating that cleavage can proceed in a neutral environment. Inhibitors of endocytosis decreased DT cleavage but did not eliminate it. We also found a small amount of nicked DT in the culture medium. These results may indicate that intact DT is cleaved age by cell-associated furin on the cell surface as well as in endocytotic vesicles.
Highlights
(DT) in the 14-amino acildoop subtended by the disul- related fusion toxins showed that cleavage of intact DTin the fide bond between C~S'~'' anCdysa'' is required for the 14-amino acid loop subtended by the disulfide bond between cytotoxic action of Diphtheria toxin (DT).The loop includes the consensus CyslS and CysZo1is important for cytotoxicity (Williams et motif for cleavageby a membrane-anchored protease, al., 1990;Ariansen et al, 1993).both intactDT and furin
We found a small amount of nicked DT in the LoVocells, ahuman colon carcinoma cell line, do not culture medium
Furin Cleaves Intact D T to A- and B-fragments in VitroIntact DTwas treated with a soluble form of furin that lacks the COOH-terminal transmembraneregion (Hatsuzawa et al, 1992a, 1922b) for 60 min at 37 "C in the presence of bovine serum albumin, and thseamples were subjected to SDSPAGE
Summary
Kurume University, 2432-3 Aikawa,Kurume, Fukuoka 830, Japan. Fax: 81-942-37-6319. Inc. Prestained SDS-PAGE molecular weight standards, including phosphorylase b (M,106,000), bovine serum albumin (M,80,000), ovalbumin (M,49,5001, carbonic anhydrase (M,32,500), soybean trypsin inhibitor (M,27,500), and lysozyme (M,18,500), were purchased from Bio-Rad. Cells-Vero cells were grownin Eagle's minimum essential medium supplemented with nonessential amino acids, 100 units/ml penicillin G, 100pg/ml streptomycin, and 10%calf serum. Cleavageand Activation of Diphtheria Toxin by Furin twice with ice-cold phosphate-buffered saline (150 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.2), and then0.5 ml of Ham's F12 medium containing 0.5% bovine serum albumin or 0.5 ml of Ham's F-12 supplemented with 10% fetal calf serum was added. To evaluate nonspecific association of '*'I-DT to cells, a 100-fold excess of unlabeled toxin was added to the medium In this experiment, nonspecific association of DT was barely detected by SDS-PAGE
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