Quantification of Diphtheria Toxin–Mediated ADP-Ribosylation in a Solid-Phase Assay

Abstract

Background: Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADPribosylation by DT. Methods: Solid phase-bound his-tag eEF2 is ADPribosylated by toxins using biotinylated NAD؉ as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti-his-tag antibody using an LAS-1000 System. Results: The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase. Conclusions: The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.

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“…Ligand-toxin conjugates exert their anti-tumor effects by binding to their targets leading to the internalization of the toxin. Once internalized, the toxin ADP-ribosylates proteins involved in translation [86]. Toxins, such as a modified version of the Pseudomonas toxin, alpha PE-38, have been conjugated to TGF-alpha and have demonstrated efficacy in a glioblastoma xeongraft model [87].…”

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“…To assess in vitro ADP-ribosylation activity of the analyzed toxins, we adopted protocols for rapid detection and precise quantification of ribosylation, previously used for DT by Bachran et al [32]. The method for rapid detection is based on Western blot analysis using HRP-conjugated streptavidin, which detects biotin labeled ADP-ribose covalently bound to eEF2.…”

Reductive Methylation and Mutation of an Anthrax Toxin Fusion Protein Modulates its Stability and Cytotoxicity

“…The ADP-ribosylation assay to determine enzymatic activity of PEIII was performed with purified yeast eukaryotic elongation factor 2 (eEF2) as described before33. Active PEIII transfers biotinylated ADP-ribose from biotinylated nicotinamide adenine dinucleotide (NAD + ) to eEF2.…”