α₂‐Macroglobulin Receptor Mediates Binding and Cytotoxicity of Plant Ribosome‐Inactivating Proteins

Abstract

It has been proposed that unconjugated type I ribosome-inactivating proteins (RIP) enter cells through passive mechanisms such as fluid-phase pinocytosis. However, some observations, such as the difference in sensitivity to type I RIP among different cell types, and the organ-specific toxicity of type I RIP, indicate a specific mechanism for the entry of these proteins into target cells. The a,-macroglobulin receptor (a,MRj is responsible for the binding and endocytosis of several ligands, including a,-macroglobulidproteinase complexes, plasminogen-activator-inhibitor complexes, apoE-enriched ,&very low density lipoproteins, and lipoprotein lipase. Here we demonstrate that saporin, a potent type I RIP, binds specifically to purified a,MR and the binding is prevented by some a,MR ligands. Moreover, the occupancy of specific ligand-binding sites on cell surface a,MR decreases the cytotoxicity of saporin. The A chain of ricin, a type I1 RIP, also interacts with a,MR. This, and the fact that saporin and ricin A chain both interact also with a,-macroglobulin, indicates a general mechanism of complex interactions between RIP and cellular membranes that is mediated by a,-macroglobulin and the a,MR system.

Characterization of a saporin isoform with lower ribosome-inhibiting activity

“…In addition, SAP-C also bound equally well to α2MR (Table 3). Binding could be inhibited by EDTA, heparin and both the competitors LpL and RAP, as previously shown for SAP-S [31]. This confirms the specificity of α2MR binding (Table 3).…”

“…Most of these studies have been carried out on the internalization pathway of the ricin holotoxin [30]. We previously demonstrated that saporin entry into the cells is not a passive mechanism as was generally believed, but is mediated by a large and widespread receptor, the α2MR [31]. α2MR is a multifunctional endocytic receptor bearing multiple binding sites [32].…”

Saporin as a novel suicide gene in anticancer gene therapy

“…RTA has a very low lysine content and a slightly acidic pI whereas 10% of the amino acids in SAP are lysine residues, which confer also an extremely high pI (almost 10) to this polypeptide. In addition, SAP can be internalized through members of the low-density lipoprotein-related receptor family 15,16 and has a much higher cell-free RIP activity, compared to RTA. 15,16 Acting catalytically, SAP has very high intrinsic activity as it has been established that only a few molecules of toxin reaching the cytosolic compartment are sufficient to inhibit protein synthesis and cause cell death.…”

“…In addition, SAP can be internalized through members of the low-density lipoprotein-related receptor family 15,16 and has a much higher cell-free RIP activity, compared to RTA. 15,16 Acting catalytically, SAP has very high intrinsic activity as it has been established that only a few molecules of toxin reaching the cytosolic compartment are sufficient to inhibit protein synthesis and cause cell death. 17 Seed-extracted SAP has been widely used to develop mouse models selectively impaired in central nervous system functions.…”

DNA‐nuclease activity of the single‐chain ribosome‐inactivating proteins dianthin 30, saporin 6 and gelonin

“…More recent results analyzed in 0.8% agarose and/or in 6% polyacrylamide gel electroobtained by others also suggest a possible penetration of the phoresis. DNA bands were visualized by staining with ethidium brosc-RIPs into cells [12,13]. In the light of our results and of mide [17].…”