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Mechanism of Cell Entry and Toxicity of an Affinity-purified Lectin from Ricinus communis and Its Differential Effects on Normal and Virus-transformed Fibroblasts¹

Departments of Cancer Biology [G. L. N., M. L.] and Tumor Virology [T. R. H.]. The Salk Institute for Biological Studies, San Diego, California 92112

An affinity-purified plant lectin from Ricinus communis (RCA_II) was shown to exhibit differential toxicity toward SV40-transformed 3T3 fibroblasts grown in vitro. When macromolecular synthesis was examined in SV3T3 and 3T3 cells, RCA₁₁ suppressed cell protein synthesis in the transformed line at lower concentrations (1/50 to 1/100) compared to the 3T3 line, and these effects were blocked by the RCA_II inhibitors D-galactose or lactose. RNA and DNA synthesis and L-leucine transport were relatively unaffected by RCA_II concentrations (>1 µg/ml) that completely suppressed protein synthesis in both cell lines. The RCA_II-mediated inhibition of cell protein synthesis required incubation times longer than 60 min, but quantitative cell binding studies with ¹²⁵I-RCA_II indicated that the lectin binds to maximal levels in approximately 5 to 10 min, even at 4°. During 10-min labeling experiments with ¹²⁵I-RCA_II (1 µg/ml), it was demonstrated that the cell-bound lectin could be almost quantitatively removed from cells up to an additional 15 min after labeling without subsequent inhibition of protein synthesis. However, longer incubation times (> 30 min) after RCA_II cell labeling and washing resulted in incomplete removal of cell-bound lectin (less than 20 to 30% of cell-bound lectin could be removed after a 60-min incubation). The longer incubation times (>60 min) also resulted in almost complete inhibition of protein synthesis. Ferritin-conjugated RCA_II (ferritin-RCA_II) was used to follow the fate of the cell-bound lectin. Ferritin-RCA_II bound rapidly (<10 min) to SV3T3 cell surfaces and could be blocked from labeling with lactose. After a 10-min incubation at 4° in ferritin-RCA_II solutions, the ferritin label was exclusively located at the extracellular surface in a random distribution. After washing and incubation at 37°, the ferritin-RCA_II induced clustering of its receptors (15 to 30 min) and eventually induced endocytosis (30 to 60 min). Further incubation (>60 min) resulted in a predominantly intracellular localization of ferritin-RCA_II inside endocytotic vesicles and free in the cell cytoplasm. That RCA_II acts directly on protein synthesis after cell entry was confirmed with rabbit reticulocyte and mouse Krebs II ascites S30 cell-free protein synthesis systems. Concentrations of RCA_II (0.1 to 1 µg/ml) that inhibit cell protein synthesis in 90 min act within 1 to 3 min to suppress cell-free protein synthesis. The saccharide prevention of RCA_II inhibition of cell, but not cell-free, protein synthesis indicates that different sites may be involved in cell binding and inhibition of protein synthesis. These results suggest that RCA_II-mediated toxicity occurs by the following sequence of events: (a) cell surface binding; (b) lectin-induced surface clustering of RCA_II receptors; (c) endocytosis of RCA_II; (d) release of RCA_II from inside endocytotic vesicles into the cell cytoplasm; and (e) direct RCA_II interaction and inactivation of protein synthesis. The similarity between RCA_II, its mode of action, and the phytotoxic ricin is discussed.

¹ These studies were supported by NIH Grant CA-15122, National Cancer Institute Contract CB-33879 (Tumor Immunology Program), and National Science Foundation Grant GB-34178 (Human Cell Biology Program) and a grant from the Cancer Research Institute, Inc.

² To whom correspondence should be addressed.

³ Present address: Department of Biochemistry, Cambridge University, Tennis Court Road, Cambridge, England.

Received 6/14/74. Accepted 10/ 3/74.

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