Toxin-Based Therapeutic Approaches

Shapira, Assaf; Benhar, Itai

Ricin, the prototype of type II RIPs, is a glycosylated heterodimer that binds through its lectin B-chain to galactose or N-acetylgalactosamine residues on glycoproteins and glycolipids which are present on the surface of most eukaryotic cells [221] [222] [223] [224] [225] [226] . In addition, certain cells like macrophages and rat liver endothelial cells that express surface mannose receptors were demonstrated to bind ricin also through its own oligosaccharide side chains [227] [228] [229] [230] [231] [232] . The cell surface-bound ricin is internalized by clathrin-dependent as well as clathrin-independent endocytosis and travels backward from the Golgi to the ER, where its disulfide linked chains are separated by protein disulfide isomerase. As in the case of PE, ricin is thought to subvert the ERAD pathway, exploiting it for the retrograde transport of the enzymatically active A chain (RTA) into the cell cytosol through the Sec61p translocon. Because of its paucity of lysines, RTA may escape, at least in part, degradation by the proteosome [167, 169, [233] [234] [235] [236] [237] [238] [239] [240] [241] [242] (Figure 5 ). Type I and type III RIPs lack the cell-binding lectin B chain and are thus generally much less toxic than type II RIPs. However, like ricin, some glycosylated type I RIPs may bind to carbohydrate receptors on the cell surface, and binding of type I RIPs to low density lipoprotein (LDL) receptor related protein (α2MR/LRP), (which also binds PE) has also been demonstrated [243] [244] [245] . In addition, coupling type I RIPs (which are equivalent to the enzymatically active A chain of type II RIPs) to a carrier that is capable of binding cells renders the conjugate highly cytotoxic [206, [246] [247] [248] [249] . The specific mechanism by which type I RIPs gain entry into the cell cytosol remains unclear, but is probably different from that of type II RIPs like ricin: Experiments with the type I RIP, saporin, indicated that it does not rely on Golgi-mediated retrograde transport and ERAD and may involve toxin translocation to the cytoplasm from the endosomes [242, 250] . Main entry route and mechanism of action of ricin. 1. Ricin toxin is translated as a single glycosylated polypeptide that is composed of a catalytic A domain and a lectin B domain (see 3D structure (PDB Entry: 2aai) in the left panel; the colors of the subunits correspond to those in the scheme). In the producing plant, a small peptide that links the A and B domains is removed, and the A and B chains remain associated via a single disulfide bond; 2. The toxin binds through the lectin B chain to cell-surface galactose or N-acetylgalactosamine residues on glycoproteins and glycolipids; 3. Cell-surface bound ricin is internalized by clathrin-dependent as well as clathrin-independent endocytosis and reaches the early endosome (EE); 4. The toxin travels backward through the Golgi to the endoplasmic reticulum (ER), where its' disulfide linked chains are separated; 5. The catalytic A chain (RTA) is retro-translocated via the Sec61p translocon into the cytoplasm; 6. The catalytically active RTA irreversibly damages ribosome by removing a specific adenine from a conserved 28S rRNA loop ("sarcin/ricin loop"-SRL), which causes translation inhibition and consequently cell death. Once in the cytosol, the enzymatic moiety of RIPs irreversibly damages ribosomes by removing a specific adenine (corresponding to residue A4324 in rat 28S rRNA) from a GAGA sequence in a