Diphtheria toxin and its ADP-ribosyltransferase-defective homologue CRM197 possess deoxyribonuclease activity.

Abstract

The cytotoxic mechanism of diphtheria toxin (DTx) is associated with its ability to inhibit protein synthesis by ADP-ribosylation of elongation factor 2. Although DTx intoxication leads to internucleosomal DNA cleavage and cell lysis, these events do not occur when protein synthesis is inhibited by alternative treatments (e.g., cycloheximide). Here we show that endonucleolytic degradation of DNA is an intrinsic activity of DTx and also of the crossreactive mutant protein CRM197. Assays using DNA-impregnated gels as well as linear and supercoiled DNA in solution revealed not only that CRM197 has nuclease activity but also that its specific activity is actually significantiy greater than that' of the wild-type molecule. Since CRM197 contains a single amino acid substitution that renders it incapable of ADP-ribosylation, we propose that the active sites for ADP-ribosyltrasferase and nuclease activities are distinct.Diphtheria toxin (DTx) is a well-characterized toxic protein (1, 2). It has two functional domains, A and B, which can be resolved by limited proteolytic digestion and reduction of an interchain disulfide bond. Intoxication of cells by DTx involves endocytosis of receptor-bound toxin. Once the toxin is sequestered in endosomes, acid-triggered conformational changes promote the translocation of fragment A to the cytosol (3-5). Fragment A then proceeds to catalyze the transfer ofthe ADP-ribosyl group ofNAD to the diphthamide residue of translational elongation factor 2 (EF-2). Recently we have discovered that DTx also exhibits a cationdependent nuclease activity (6). In this report we explore the relationship between the toxin's ADP-ribosyltransferase and nuclease activities. CRM197, a mutant DTx with a Gly -* Glu52 substitution in its A domain (7), is unable to ADPribosylate EF-2 in vitro because of its extremely low affinity for NAD (8,9 Nuclease Activity Assay in DNA-Containing Gels. Samples of cleaved DTx monomer, cleaved CRM197, and "intact" CRM197 were solubilized in SDS reducing buffer (containing 0.01% 2-mercaptoethanol) and electrophoresed in a SDS/ 12.5% polyacrylamide gel (10) prepared with 32P-labeled DNA [0.2 ng/ml; 800,000 cpm/ng of sonicated salmon sperm DNA radiolabeled by the primer-extension method (11, 12), as described (6)]. The stacking gel was omitted. The gels were pre-run at 170 V for 90 min. For nondenaturing gels, SDS was omitted and samples were not boiled. After electrophoresis, the DTx lanes were separated from the CRM197 lanes and each gel piece was washed three times (40 min per wash) with 100 ml of 40 mM Tris/0.04% NaN3, pH 7.6, at 22TC. The gel pieces were then incubated with the same buffer containing 2 mM CaCl2 and 2 mM MgCl2 for 24 hr at 300C (for SDS gels) or for 12 hr at 370C (for nondenaturing gels), prior to autoradiography (-850C, Kodak XAR film, DuPont Cronex screen).Cleavage of Supercoiled and Linear Plasmid DNA. CRM197, DNase I, and -DTx were incubated in 10 Al of 10 mM Tris, pH 7.6/2.5 mM CaCl2/2.5 mM MgCl2 with 0.2 ,Ag of linear (EcoRI-cleaved) pBluescr...

The cytotoxic effect of diphtheria toxin on the actin cytoskeleton

“…Cell lysates treated with diphtheria toxin were used to measure the enzymatic activity (FA~ 10 ng) for ADP-ribosylation of eEF2 (20 pmol). Deoxyribonuclease activity DT and FA have been repeatedly shown to possess deoxyribonuclease activity [23,24]. These findings were confirmed in the present study as assayed by changes in hyperchromicity.…”

Localization of diphtheria toxin nuclease activity to fragment A

“…Testing this hypothesis resulted in the discovery of a nuclease activity intrinsic to the DTx molecule (3). Detection of this activity is highly reproducible (10), optimal reaction conditions have been established (13), and an ADP ribosyltransferase (ADPrT)-defective form of DTx (called CRM197) exhibits greater nuclease activity than DTx itself (2). Moreover, nuclease activity was found to comigrate with the DTA portions of both DTx and CRM197 during electrophoresis in DNA-containing sodium dodecyl sulfate (SDS) gels (2,3,10) and with whole DTx and CRM197 during native gel electrophoresis (2), even though each of these forms of DTx migrates with a distinctive mobility in each gel system.…”

“…Detection of this activity is highly reproducible (10), optimal reaction conditions have been established (13), and an ADP ribosyltransferase (ADPrT)-defective form of DTx (called CRM197) exhibits greater nuclease activity than DTx itself (2). Moreover, nuclease activity was found to comigrate with the DTA portions of both DTx and CRM197 during electrophoresis in DNA-containing sodium dodecyl sulfate (SDS) gels (2,3,10) and with whole DTx and CRM197 during native gel electrophoresis (2), even though each of these forms of DTx migrates with a distinctive mobility in each gel system. We now report that the nuclease activity exhibited by DTx chromatography of DTx and trypsin-generated DTA (21); therefore, they proposed that the nuclease activity of their DTx preparations resides in an uncharacterized contaminating protein.…”

Isolation of temperature-sensitive diphtheria toxins in yeast and their effects on Drosophila cells

“…Indeed, DT-A 15 ™ was found to carry the same point mutation as CRM197. CRM197 has no cytotoxic effects (Uchida et al, 1973), exhibits structural and biochemical properties that distinguish it from the wild-type toxin (Bruce et al, 1990;Hu and Holmes, 1987;Bigio et al, 1987;Mekada and Uchida, 1985;Papini et al, 1987;Collins and Collier, 1985), does not cause ADP ribosylation of elongation factor-2 in vitro between 14°C and 37°C (B. J. Wisnieski, personal communication), and is much more sensitive to intracellular proteases (Yamaizumi et al, 1982). Interestingly, CRM197 shows high endonuclease activity in vitro (Bruce et al, 1990) between 14°C and 37°C (B. J. Wisnieski, personal communication).…”

“…The toxin's action is mediated by the inhibition of protein synthesis through ADP-ribosylation of elongation factor 2 (for review see Pappenheimer, 1977). In addition, recent but controversial evidence indicates that the toxin also acts as an endonuclease in vitro (Chang et al, 1989;Bruce et al, 1990;Nakamura and Wisnieski, 1990). A single molecule of the fully active toxin is sufficient to kill a mammalian cell (Yamaizumi et al, 1978).…”