The diphthamide modification on elongation factor-2 renders mammalian cells resistant to ricin

Abstract

Diphthamide is a post-translational derivative of histidine in protein synthesis elongation factor-2 (eEF-2) that is present in all eukaryotes with no known normal physiological role. Five proteins Dph1-Dph5 are required for the biosynthesis of diphthamide. Chinese hamster ovary (CHO) cells mutated in the biosynthetic genes lack diphthamide and are resistant to bacterial toxins such as diphtheria toxin. We found that diphthamide-deficient cultured cells were threefold more sensitive than their parental cells towards ricin, a ribosome-inactivating protein (RIP). RIPs bind to ribosomes at the same site as eEF-2 and cleave the large ribosomal RNA, inhibiting translation and causing cell death. We hypothesized that one role of diphthamide may be to protect ribosomes, and therefore all eukaryotic life forms, from RIPs, which are widely distributed in nature. A protective role of diphthamide against ricin was further demonstrated by complementation where dph mutant CHO cells transfected with the corresponding DPH gene acquired increased resistance to ricin in comparison with the control-transfected cells, and resembled the parental CHO cells in their response to the toxin. These data show that the presence of diphthamide in eEF-2 provides protection against ricin and suggest the hypothesis that diphthamide may have evolved to provide protection against RIPs.

Fig. 1. Diphthamide biosynthetic pathway and ADP-ribosylation

Dph1 to Dph5 are required for the biosynthesis of diphthamide. Dph1 to Dph4 are involved in the first step, the transfer of 3-amino-3-carboxypropyl to the His⁷¹⁵ (His⁶⁹⁹ in yeast) of eEF-2. Dph5 acts as methyl transferase in the next step, yielding diphthine. Amidation of diphthine is the last step of diphthamide biosynthesis. Arrowhead (N-1 of the histidine imidazole ring of diphthamide) indicates the site of ADP-ribosylation by ADP-ribosylating toxins. AdoMet, S-adenosyl methionine; Ado-S-Me, methylthioadenosine; Ado-Hcy, S-adenosylhomocysteine. In ADP-ribosyl diphthamide, A, adenine moiety; R, ribosyl moiety. The CHO cell mutants lacking corresponding functional Dph proteins used in this study are shown in parentheses.

Fig. 2. Toxin sensitivity of diphthamide-deficient CHO mutant cells compared to their parental cell lines

Cells were incubated with the indicated concentrations of FP59 + 500 ng/ml PA (a, c) or with ricin (b, d) for 48 h and MTT assay was done to measure cell viability. The A₅₄₀ values obtained for cells that received no toxin were considered as 100 % and used to calculate the percent viability of other data points. Insets show western blots for in vitro ADP-ribosylation assay with the cell lysates. In the inset, top panels show ADP-ribosylated eEF-2 while the bottom panel shows the blot using antibody against the carboxy terminus of eEF-2 of human origin. − and + denote the absence or addition of FP59 respectively.

Fig. 3. Toxin sensitivity of diphthamide-deficient CHO mutant cells complemented by transfection of the corresponding DPH genes

Cells transfected with corresponding DPH gene have been designated by adding “D” after the name of cell lines and are shown in blue, while control transfectants for same cell types have been shown by adding “C” and are shown in red. Each line is derived from an independent stable transformant cell line. Toxin sensitivities of representative individual clones were measured as in Fig. 2 for “PA + FP59” (a, c) and “ricin” (b, d, e). In panels b, d, and e the blue lines (“D” clones) are clustered to the right, indicating increased resistance to ricin. Insets show the western blot for in vitro ADP-ribosylation assays done with the cell lysates, as in Fig. 2. In the experiment of panel e, the “-C” clones are those transfected with an unrelated gene, VPS11, rather than empty vector. Also, in the experiment of panel e, both mutant and transfected cells show increased sensitivity towards ricin compared to other experiments (e.g., Fig. 2, 3b and 3d) due to use of a more potent batch of ricin, but the effect of complementation was again to increase resistance.