Figure 4

Fucosylation controls ricin toxicity via intracellular trafficking. (A) A modified version of the ricin toxin that harbors one specific sulfation (RS1) and three mannosylation (RS2) sites in the catalytically active RTA subunit allows for quantification of the amount of intracellular toxin trafficking to the Golgi and the ER, using radioactively-labeled isotopes. (B, C) Slc35c1 WT and KO mESCs were incubated with engineered RTA (see scheme in A) to quantify ricin trafficking. The total amount of ricin (total RTA), as well as the amount of the sulfated, or sulfated and mannosylated form, was determined by western blot or autoradiography, respectively (C), and quantified (B). Data are shown as mean ± SD (n = 3). Each data set is representative of two independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (Student's t-test). (D) The rate of protein synthesis was determined after 3 h of ricin exposure in Slc35c1 control (WT) and Slc35c1 mutant (KO) sister mESCs, using radioactively-labeled isotopes. Data are shown as mean ± SD of triplicate cultures. (E) Addition of galactose (200 μM) or Lewis X (200 μM), but not fucose (200 μM) increased viability of ricin-treated cells. Experiments were repeated three times; representative data are shown. NS, not significant; RTA, ricin toxin A subunit; RTB, ricin toxin B subunit.