Magnusson S, Kjeken R, Berg T
Department of Biology, University of Oslo, Norway.
Exp Cell Res. 1993 Mar;205(1):118-25. doi: 10.1006/excr.1993.1065.
We have studied the characteristics of internalization and intracellular transport of ricin via two distinct pathways in rat liver endothelial cells (EC), i.e., via binding to mannose receptors and surface galactosyl-residues, respectively. Treatments that inhibit endocytosis from coated pits, i.e., hyperosmolarity and acidification of the cytoplasm, decreased uptake via mannose receptors much more than uptake via galactosyl-residues, indicating that mannose receptors are largely internalized from coated pits, whereas internalization via galactosyl-residues is to a significant extent independent of coated pits. Uptake of ricin via mannose receptors was strongly inhibited by NH4Cl and monensin, and accordingly, NH4Cl protected the cells against ricin intoxication via mannose receptors. On the other hand, uptake via galactosyl-residues was not significantly inhibited by NH4Cl or monensin, and NH4Cl even sensitized the cells to intoxication via this pathway. Brefeldin A, which did not affect ricin uptake, protected the cells against ricin intoxication via either pathway. Protein synthesis in the EC was efficiently inhibited by ricin, even after very short periods of uptake at low ricin concentrations. The onset of protein synthesis inhibition was more rapid upon internalization of ricin via mannose receptors than via galactosyl-residues. Also, ricin internalized via mannose receptors was more efficiently transported from endosomes to lysosomes than ricin internalized via galactosyl-residues. Partial blocking of the galactosyl-binding sites of ricin caused a reduction in the extent of recycling of ricin from endosomes to the cell surface (retroendocytosis), indicating that binding of ricin to membrane galactosyl-residues, which is relatively stable at the slightly acidic pH of endosomes, is an important determinant of the intracellular handling of ricin. We suggest that the observed difference in the transport from endosomes to lysosomes between the two internalization pathways is related to the different stability of the two binding mechanisms at endosomal pH.
我们研究了蓖麻毒素在大鼠肝内皮细胞（EC）中通过两种不同途径进行内化和细胞内转运的特性，即分别通过与甘露糖受体和表面半乳糖基残基结合。抑制有被小窝内吞作用的处理，即高渗和细胞质酸化，通过甘露糖受体的摄取减少比通过半乳糖基残基的摄取更多，这表明甘露糖受体主要从有被小窝内化，而通过半乳糖基残基的内化在很大程度上独立于有被小窝。通过甘露糖受体摄取蓖麻毒素受到氯化铵和莫能菌素的强烈抑制，因此，氯化铵通过甘露糖受体保护细胞免受蓖麻毒素中毒。另一方面，氯化铵或莫能菌素对通过半乳糖基残基的摄取没有显著抑制作用，甚至氯化铵使细胞对通过该途径的中毒更敏感。布雷菲德菌素A不影响蓖麻毒素的摄取，但通过任何一种途径都能保护细胞免受蓖麻毒素中毒。即使在低蓖麻毒素浓度下摄取很短时间后，EC中的蛋白质合成也能被蓖麻毒素有效抑制。通过甘露糖受体内化蓖麻毒素后，蛋白质合成抑制的起始比通过半乳糖基残基内化更快。此外，通过甘露糖受体内化的蓖麻毒素比通过半乳糖基残基内化的蓖麻毒素更有效地从内体转运到溶酶体。部分阻断蓖麻毒素的半乳糖结合位点会导致蓖麻毒素从内体到细胞表面的循环（逆向内吞作用）程度降低，这表明蓖麻毒素与膜半乳糖基残基的结合在略酸性的内体pH下相对稳定，是蓖麻毒素细胞内处理的一个重要决定因素。我们认为，观察到的两种内化途径从内体到溶酶体转运的差异与两种结合机制在内体pH下的不同稳定性有关。
