As a prerequisite for cell entry, toxin must bind to a cell surface component that thereby functions as a toxin receptor. Cells that do not contain surface receptors for a particular toxin are insensitive to that toxin. A variety of different plasma membrane components function as toxin receptors. Some toxins bind to specific proteins such as the heparin-binding EGF-like growth factor precursor (utilised by DT) (Naglich et al., 1992), or the a^-macroglobulin receptor (PE) (Kounnas et al., 1992). ST and the SLTs bind to a family of glycosphingolipids known as globotriosyl ceramides (Jacewiez et al., 1986) and the restricted distribution of this glycolipid limits the number of different cell types sensitive to these toxins. Ricin, on the other hand, can bind to virtually all cell types since its cell-binding B chain can interact with a variety of glycoproteins and glycolipids containing terminal galactose residues (Lord et al., 1994).
Surface bound toxin enters cells by endocytosis using both clathrin-dependent and clathrin-independent mechanisms. DT enters via clathrin-coated pits and vesicles. An EM study showed surface bound DT to be concentrated in coated pits (Morris et al., 1985). Treatments that inhibit clathrin-dependent endocytosis, such as potassium depletion (Moya et al., 1985) or acidification of the cytosol (Sandvig et al., 1987) protect sensitive cells against DT. Clathrin-dependent endocytosis is known to require the GTPase dynamin, which mediates the pinching off step that releases coated vesicles from the plasma membrane (Damke et al., 1994). Overexpressing trans dominant negative dynamin mutants inhibits clathrin-dependent endocytosis and protects cells completely from intoxication by DT (Simpson et al., 1998). PE and ST also enter mammalian cells by clathrin-dependent endocytosis. It is somewhat surprising that the glycolipid receptor for ST becomes concentrated in coated pits, but it is possible that such receptors can interact with proteins anchored within the pits. Ricin, which is able to bind a variety of surface galactosides, can enter cells by both clathrin-dependent and clathrin-independent endocytosis (Sandvig and van Deurs, 1996). Because of this, blocking the former by the treatments mentioned above does not protect cells against ricin intoxication. Little is known at present regarding the clathrin-independent endocytic pathway utilised by ricin for cell entry. Entry does not seem to be mediated by caveolae, however, since treating cells with cholesterol-binding compounds such as filipin or nystatin, which inhibit the formation of caveolae, does not prevent the clathrin-independent uptake of ricin (Simpson et al., 1998).
The clathrin-dependent and clathrin-independent entry pathways converge within endosomes (Sandvig and van Deurs, 1996). All endocytosed toxins therefore enter endosomal compartments, and respond to this in one of two ways; either they cross the endosomal membrane to enter the cytosol or they do not. DT translocates from endosomes, as do other bacterial toxins that do not act by directly inhibiting protein synthesis, including anthrax, botulinum and tetanus toxins.
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