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

Abstract

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...

An activity-dependent assay for ricin and related RNA N-glycosidases based on electrochemiluminescence

“…Nonetheless, RNase A caused degradation of the substrate, RNA GdAGA. Although ricin A chain has been reported to be unaffected by diethylpyrocarbonate [22], we observed inhibition of ricin (not shown) that limited the usable DEPC concentration such that treatment with this chemical rendered only a slight mitigation of the interference by RNase A (Table 4). Since RNases are known to be present in castor bean extracts [24] and perhaps in illicit weapons made therefrom, we investigated whether a polyclonal antiricin antibody linked to Dynabeads might be useful in removing ricin from potential interferences in solution.…”

“…DNases reportedly contaminate many preparations of RIPs, but DNase I had no effect on the assay (Table 4) [22]. The chelating agent EDTA was included in the toxin reaction (2 mM) and in the stop/detection reagents (0.7 mM) to avoid degradation of oligonucleotides due to Signal-to-Background Ratio Ricin (ng/ml in 5 μl sample) Fig.…”

Vinyldeoxyadenosine in a Sarcin−Ricin RNA Loop and Its Binding to Ricin Toxin A-Chain

“…PAP was heat treated to remove the non-specific glycosidase/nuclease activity while retaining the RNA N-glycosidase activity (49). Adenine release was detected on incubating A-14 with a commercial source wild type PAP at pH 7.8 (25 mM Tris/HCl buffer).…”

Crystal structure of PD‐L1, a ribosome inactivating protein from Phytolacca dioica L. Leaves with the property to induce DNA cleavage

“…19 The presumed novel enzymatic activities have been challenged, since concerns were formulated about contaminating nucleases in the RIP preparations. 20 However, various papers have shown that DNA cleavage activity is observed even for highly pure RIPs 7,[21][22][23][24][25][26][27] and that reduction in N-b-glycosidase activity results in a reduction of DNA cleaving activity, 21 reinforcing the concept that RIPs use the same catalytic site for their two-fold activity. 28 Leaves of Phytolacca dioica express four type 1 RIPs, named PD-L1, PD-L2, PDL3, and PD-L4.…”