title: Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase.
abstract: Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.
authors.affiliation: Department of Pathology, Section of Immunology, University of Verona, Policlinico G.B. Rossi, L.go L.A. Scuro 10, I-37134 Verona, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Bellisola
authors.forename: Giulio
authors.initials: G
authors.lastname: Fracasso
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ippoliti
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Menestrina
authors.forename: Anders
authors.initials: A
authors.lastname: Rosén
authors.forename: Silvia
authors.initials: S
authors.lastname: Soldà
authors.forename: Silvia
authors.initials: S
authors.lastname: Udali
authors.forename: Rossella
authors.initials: R
authors.lastname: Tomazzolli
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Tridente
authors.forename: Marco
authors.initials: M
authors.lastname: Colombatti
mesh_terms: D001310:Auranofin / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000378:metabolism; D009249:NADP; D010084:Oxidation-Reduction / Q000187:drug effects; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D012276:Ricin / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*; D020298:U937 Cells
chemical_list: D000922:Immunotoxins; D001310:Auranofin; D009249:NADP; D012276:Ricin; D013880:Thioredoxin-Disulfide Reductase; D019704:Protein Disulfide-Isomerases