Localization of diphtheria toxin nuclease activity to fragment A

Abstract

). Here we show that (i) trypsinization of DTx does indeed produce nucleolytically active DTA, (ii) reduction of electroeluted, unreduced, cleaved DTx (58 kDa) yields nuclease-active DTA (24 kDa), and (iii) fractionation of DTx and DTA by anion-exchange chromatography leads to coelution of nuclease activity with both forms of the toxin, even though each form elutes at a distinct salt concentration. In addition, we show that Escherichia coli-derived DTA also expresses nuclease activity. These studies confirm our initial assertion that the nuclease activity observed in DTx preparations is intrinsic to the DTA portion of DTx.Diphtheria toxin (DTx) is synthesized as a single polypeptide chain that can be "nicked" by limited proteolysis to generate A and B subunits (DTA and DTB, respectively) that remain connected by a cystine bridge (7,14,16). DTB is involved in receptor binding; following sequestration in endosomes and subsequent acidification of the endosomal compartment, DTA is transported to the cytosol (8,14,22). DTA is involved in the ADP ribosylation of elongation factor 2, which leads to the inhibition of protein synthesis (7,14,16). We and others have noted that extensive toxin-induced translation inhibition is not correlated with cell death in human K562 cells (4,19); these cells are lysis resistant to doses of DTx as high as 7.5 ,ug/ml despite the rapid and complete abrogation of protein synthesis (4). In addition, extensive inhibition of protein synthesis by unrelated treatments does not lead to the prelytic intemucleosomal DNA cleavage that we have observed in DTx-intoxicated cells or to cell lysis (4). These and other findings (5) led us to propose that DTx triggers the programmed cell death pathway (4). 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. We now report that the nuclease activity exhibited by DTx is intrinsic to DTA. 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. Moreover, they showed that trypsin cleavage of intact DTx results in a severe decrease in total observed nuclease activity and a simultaneous increase in ADPrT activity (21). In contrast, we observed that endoproteinase ArgC-cleaved DTx remains fully active and that the amount of DTx-associated nuclease activity corresponds to the amount of DTA generated ...

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.…”

Characterization of a Cloned Temperature-Sensitive Construct of the Diphtheria Toxin A Domain

“…We found that for both CRM197 and DTx, Ca 2+ and Mg 2+ promote single-strand nicks, whereas Mn 2+ promotes doublestrand breaks ( Figure 7B). 4 These findings are similar to X-ray findings with bovine pancreatic DNase I:DNA cocrystals (23). With ssDNA, Mn 2+ leads to no increase in cleavage rate over that seen with Ca 2+ and Mg 2+ (data not shown).…”

“…4 Moreover, in a yeast expression system, the level of EF-2 available for ADP-ribosylation did not change with time, regardless of whether DTM was expressed at 18 or 30°C. 3 We conclude from these observations that DTM's temperature-sensitive cytotoxic effects in yeast and Drosophila are not the result of low-temperature-acquired ADPrT activity.…”

DNase test as a novel approach for the routine screening of Corynebacterium diphtheriae

“…The deoxyribonuclease activity in C. diphtheriae and C. ulcerans has been described in the literature for many decades (Messinova et al 1963;Barksdale 1970;Chang et al 1989;Lessnick et al 1992;Lee et al 2005). Toxigenic and nontoxigenic C. diphtheriae were found to be equally proficient at producing deoxyribonuclease (Barksdale 1970; Groman and Dean 1973).…”