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A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast
Published on: December 15, 2017
Cell toxicity by ricin and elucidation of mechanism of Ricin inactivation
L C Meneguelli de Souza1, L P de Carvalho2, J S Araújo1
1Laboratory of Chemistry and Function of Proteins and Peptides - LQFPP, Center for Biosciences and Biotechnology - CBB, State University of Northern Fluminense Darcy Ribeiro (UENF), Av. Alberto Lamego, 2000, ZC, 28035-200 Campos dos Goytacazes, RJ, Brazil.
Abstract:
Castor cake is a by-product of the extraction of oil from from seeds of castor plants (Ricinus communis). This by-product contains high levels of proteins, but a toxic protein, ricin, limits its use as an animal feed. Ricin can be efficiently inactivated by treatment with calcium oxide (CaO), which can be evaluated by a cytotoxicity assay using LLC-MK2 cells. The mechanism by which the CaO treatment inactivates ricin, however, is unclear. We report the structural changes responsible for ricin inactivation. Purified ricin was treated with 0.6% CaO and then analyzed by mass spectrometry. This treatment degraded the ricin at preferential sites. The aqueous CaO solution had a pH >12, which preferentially cleaved asparagine residues, followed by glutamine, serine and glycine residues. The alkaline pH affected the tertiary structure of the ricin, cleaving its polypeptide chains and thereby eliminating its cytotoxic activity.
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