Mechanistic aspects of the deoxyribonuclease activity of diphtheria toxin

Abstract

Here we examined the intrinsic nuclease activity of diphtheria toxin (DTx) to determine the mechanism by which it catalyzes DNA degradation. Results show that DTx degrades double-stranded DNA (dsDNA) by non-processive, endonucleolytic attack, without apparent specificity for nucleotide sequence. Moreover, divalent cation composition determines whether supercoiled dsDNA is cleaved by the introduction of single-strand nicks or double-strand breaks. Circular single-stranded DNA (ssDNA) is also a substrate for endonucleolytic attack. Pre-incubation of DTx with a 2000-fold excess of NAD, the natural substrate for the toxin's ADP-ribosyltransferase (ADPrT) activity, inhibited the transfer of radiolabeled ADP-ribose to elongation factor 2 but had no effect on the degradation of radiolabeled DNA. Based on this result and the fact that compounds known to inhibit the ADPrT activity of DTx had no effect on its nuclease activity and pre-incubation of DTx with DNA had no effect on ADPrT activity, we conclude that the ADPrT and nuclease active sites of DTx are functionally and spatially distinct. Moreover, studies with an ADPrT-inactivated form of DTx indicate that nuclease activity alone can lead to target cell lysis.

Introduction

Diphtheria toxin (DTx) is a protein secreted by Corynebacterium diphtheriae lysogenized by β or closely related phage [1]. The toxin is well characterized as an inhibitor of eukaryotic protein synthesis [2], [3]. Cleavage and reduction of DTx yields domain A (21,164 Da), which possesses ADP-ribosyltransferase (ADPrT) activity, and domain B (37,194 Da), which is responsible for receptor-mediated binding to target cells and with domain A translocation. Domain A catalyzes the transfer of the ADP-ribose moiety from NAD to elongation factor 2 (EF-2), a modification that renders the factor inactive in translation [4], [5]. The consequent inhibition of protein synthesis has long been accepted as solely and directly responsible for cytotoxicity in toxin-sensitive cells [2], [3], [4], [5].

Demonstration of chromosomal damage in pre-lytic, DTx-treated target cells and a close correlation between cell lysis and DNA damage (rather than between cell lysis and the inhibition of protein synthesis) led us to propose [6] that DTx-induced target cell death might proceed in a manner akin to apoptosis, which is characterized by activation of an endogenous endonuclease [7]. Subsequently, however, we discovered a nuclease activity that was directly attributable to the A domain of the toxin molecule itself [8], [9], [10], [11], [12], [13]. Confirmation of co-migration of specific A chain-containing fragments and nuclease activity through a wide variety of gel types and columns was established by Western blotting with a battery of monoclonal antibodies and extensive sequencing of the protein bands. In addition to DTx, we discovered that CRM197, the ADPrT-deficient G52E-mutant form of DTx, possesses endonuclease activity [6], [8], [9], [10], [11], [12] and that its specific activity is four times higher than that of bovine pancreatic DNase I and 20 times higher than that of DTx [10]. No matter how CRM197 and DTx are purified or treated, all detectable nuclease-active peptides elute and migrate in accordance with the distinctive properties of the intact proteins, and of A domain-containing fragments found in preparations cleaved with endopeptidase argC, lysC, and trypsin, as well as in argC-nicked preparations of DTx and CRM197 that were subsequently cleaved with lysC, and vice versa. Independently obtained X-ray crystal grade preparations of CRM45, a mutant form of DTx that lacks a 17-kDa segment of the B domain, appropriately display nuclease activity at 45 kDa (intact CRM45) and at 24 kDa (the A chain) in a DNA-embedded gel assay [13]. Likewise, independently purified and provided preparations of E. coli-cloned DTA and E. coli-cloned E148S both renatured and expressed nuclease activity after electrophoresis in an SDS gel that contained DNA [11]. Once again, Western blotting and amino acid sequencing confirmed the identities of the nuclease-active bands. A summary of the evidence for toxin nuclease activity is presented in Table 1.

The goal of the current study was to characterize more fully the nuclease activity of DTx and to test the hypothesis that its ADPrT and nuclease active sites are distinct. DNA degradation assays were performed to ascertain whether inhibitors of the ADPrT reaction, including cold NAD, nicotinamide, ATP, ApUp, and adenine, have any effect on the nuclease activity of DTx. Conversely, ADPrT assays were performed to determine whether DNA has any effect on the ADPrT activity. Our findings not only confirm the lack of correlation between protein synthesis inhibition and cell lysis but also provide evidence that the ADPrT and nuclease active sites of DTx are indeed functionally and spatially distinct. Furthermore, we also reveal functional similarities between DTx and pancreatic DNase I.