Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome

Abstract

Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarly to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication.

Functional Assays for Measuring the Catalytic Activity of Ribosome Inactivating Proteins

“…The base change at the depurination site was detected using a reverse primer with adenine at the 3′ terminus and the specificity for detection was enhanced by the inclusion of a secondary mismatch two base pairs 5′ to the adenine. The modified qRT-PCR assay provided a sensitive and highly reproducible measure of the extent of depurination by ricin and Stxs in mammalian cells [ 31 , 32 , 37 , 40 ] and in yeast [ 31 , 34 , 36 , 37 ] in vivo.…”

“…RTA expression limited the expression of GFP in 70–80% of cells [ 75 ]. Jetzt et al used a slightly modified enhanced green fluorescent protein (EGFP) transfection assay to study ricin mutants in mammalian cells [ 39 , 40 ]. Cells were cotransfected with an EGFP reporter plasmid and RTA mutants and EGFP fluorescent signal was quantified using a plate reader [ 39 , 40 ].…”

“…The results correlated well with ribosome depurination measured for the mutants by qRT-PCR. Jetzt et al found that apoptosis could be triggered by a low level of ribosome depurination [ 39 ] and showed that ribosome interactions were critical for RTA toxicity in mammalian cells [ 40 ]. The GFP transfection assay [ 40 ] was sensitive and useful for examining variations of enzymatic activity among the mutant A1 subunits of Stxs [ 31 ].…”

“…Jetzt et al found that apoptosis could be triggered by a low level of ribosome depurination [ 39 ] and showed that ribosome interactions were critical for RTA toxicity in mammalian cells [ 40 ]. The GFP transfection assay [ 40 ] was sensitive and useful for examining variations of enzymatic activity among the mutant A1 subunits of Stxs [ 31 ]. A stably transfected cell line expressing GFP [ 76 , 77 ] was used for cholera toxin and other type II RIPs in related studies [ 77 , 78 , 79 ].…”

“…Assays that directly measure depurination activity for toxin detection and mitigation procedures were recently reviewed [ 29 , 30 ]. Here, we specifically review the assays, which identified differences in the activity of Stxs [ 31 ], differences in ribosome interactions of RTA [ 22 , 32 , 33 , 34 , 35 , 36 ], Stxs [ 21 , 22 , 37 ], TCS [ 23 , 26 ], and PAP [ 24 , 25 ], provided information about the downstream consequences of depurination, such as the ribotoxic stress response and apoptosis [ 38 , 39 , 40 ], allowed the use of RIPs as tools to probe the structure and function of the ribosomal stalk [ 41 ], provided information about the functional divergence of the ribosomal stalk P1-P2 dimers [ 42 ] and were useful in the search for RIP inhibitors [ 43 , 44 , 45 , 46 , 47 , 48 ]. We discuss the advantages and limitations of each method and explain how more sensitive assays can distinguish the differences in depurination activity and provide information about the kinetics and the mechanism of the depurination reaction.…”