Does Diphtheria Toxin Have Nuclease Activity?

Wilson, Brenda A.; Blanke, Steven R.; Murphy, John R.; Pappenheimer, Alwin M.; Collier, R. J.

doi:10.1126/science.250.4982.834

Cited by 12 publications

(11 citation statements)

References 14 publications

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“…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. 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).…”

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confidence: 99%

Localization of diphtheria toxin nuclease activity to fragment A

et al. 1992

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). 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 ...

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Localization of diphtheria toxin nuclease activity to fragment A

et al. 1992

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“…Recombinant PAP was expressed and purified from E. coli BL21 (DE3) pLysS harbouring plasmid pET-PAPstop by cation-exchange chromatography, as described previously [26]. shows the relative activities of several RIPs (five type I and one type II), which are compared with the activity of a commercial preparation of diphtheria toxin (DT, Calbiochem) which is known to be contaminated at a low level with a DNase [18,19,27]. Of the RIPs tested here, gelonin and trichosanthin had the highest activity, D30, D32 and PAP had less activity and the type-II RIP ricin had the lowest activity.…”

Section: Methodsmentioning

confidence: 99%

“…The nuclease contaminating DT could be separated from DT by a number of chromatographic procedures [18,19]. Gel filtration of ricin over a Superose 6 FPLC column resulted in elution of a single protein peak, corresonding to ricin holotoxin, as visualised by SDS/PAGE (not shown).…”

Section: Pap Heat Treatment Of Pap Eliminates Dnase Activity But Notmentioning

confidence: 99%

“…Again, this activity had low specific activity when compared with bone fide DNase I. However, further purification resulted in complete separation of the DNase activity from the toxin [18,19] and, similarly, purifi-cation of toxin from a DNase-deficient strain of C. diphtheriae also resulted in protein lacking DNase activity. In this paper, we demonstrate that the DNase activity associated with a type-I RIP, pokeweed antiviral protein (PAP) and a type-II RIP, ricin is also due to contamination.…”

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The deoxyribonuclease activity attributed to ribosome‐inactivating proteins is due to contamination

Day

Lord

Roberts

1998

European Journal of Biochemistry

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The mode of action of ribosome-inactivating proteins (RIPs) has, for many years, been considered to be depurination of a specific adenyl residue of ribosomal RNA, resulting in inhibition of protein synthesis. Recently, this view has been challenged by the observation that many RIP preparations have significant DNase activity in addition to their N-glycosidase activity. In this study, we have investigated the putative DNase activity of two RIPs, ricin and pokeweed antiviral protein (PAP), and show that, in both cases, the DNase activity is due to the presence of contaminating nucleases. The N-glycosidase and DNase activities of PAP were separately and specifically inactivated by chemical modification and heat. Gel filtration of ricin allowed physical separation of the two activities. Furthermore, neither recombinant PAP nor recombinant ricin A-chain purified from Escherichia coli displayed DNase activity.Keywords : deoxyribonuclease; ribosome-inactivating protein; RNA N-glycosidase.Ribosome-inactivating proteins (RIPs) are a group of structurally related proteins which depurinate a specific adenyl residue of 28-S ribosomal RNA (A4324 in rat, [1]). Depurination of this residue prevents binding of elongation factors, thereby inhibiting protein synthesis and resulting in cell death. RIPs can be divided into two classes: type I, which consist of a single subunit possessing RNA N-glycosidase activity, and type II, which have a type I-like catalytic subunit (A chain) linked to a cell-binding subunit (B chain). The B chains of plant toxins, e.g. ricin, are typically galactose/N-acetylgalactosamine-specific lectins and are linked to the A chain via a disulphide bond. Bacterial toxins that catalyse the same depurination reaction as RIPs, e.g. Shiga toxin, have a non-covalently bound glycolipidbinding B-chain pentamer.Most type-II RIPs are potent cytotoxins due to their ability to bind to and enter mammalian cells. Once inside the cell, the toxins exploit the cellular retrograde transport system to reach the endoplasmic reticulum from where they translocate into the cytosol by an as yet undetermined mechanism [2]. As holotoxins show no catalytic activity against ribosomes, separation of the A chain and B chain following intoxication is essential in order to exhibit a toxic effect.Type-I RIPs are, by comparison, only weakly cytotoxic when added exogenously to cells. A number of roles have been proposed for type-I RIPs, including defence mechanisms and promotion of senescence. Those which are active on con-specific ribosomes are typically stored in non-cytosolic compartments or as inactive precursors. Such RIPs are believed to be released into the cytosol following cellular damage, such as that caused by viral infection, resulting in local cell suicide around infected areas. An antifungal role has been proposed for RIPs that are inactive against con-specific ribosomes. Barley RIP is co-expressed with a chitinase, and [1, 3] β glucanase, which, while having intrinsic antifungal activity themselves, also facilitate RIP entry i...

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Diphtheria Toxin and Pseudomonas aeruginosa Exotoxin A: Active-Site Structure and Enzymic Mechanism

Wilson

Collier

1992

Current Topics in Microbiology and Immunology

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Does Diphtheria Toxin Have Nuclease Activity?

Cited by 12 publications

References 14 publications

Localization of diphtheria toxin nuclease activity to fragment A

Localization of diphtheria toxin nuclease activity to fragment A

The deoxyribonuclease activity attributed to ribosome‐inactivating proteins is due to contamination

Diphtheria Toxin and Pseudomonas aeruginosa Exotoxin A: Active-Site Structure and Enzymic Mechanism

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