Use of ribosome-inactivating proteins from Sambucus for the construction of immunotoxins and conjugates for cancer therapy

Abstract

The type 2 ribosome-inactivating proteins (RIPs) isolated from some species belonging to the Sambucus genus, have the characteristic that although being even more active than ricin inhibiting protein synthesis in cell-free extracts, they lack the high toxicity of ricin and related type 2 RIPs to intact cells and animals. This is due to the fact that after internalization, they follow a different intracellular pathway that does not allow them to reach the cytosolic ribosomes. The lack of toxicity of type 2 RIPs from Sambucus make them good candidates as toxic moieties in the construction of immunotoxins and conjugates directed against specific targets. Up to now they have been conjugated with either transferrin or anti-CD105 to target either transferrin receptor- or endoglin-overexpressing cells, respectively.

Keywords: ribosome-inactivating proteins; Sambucus; endoglin (CD105); immunotoxin; transferrin.

Figure 1

Three-dimensional models of ebulin l and ricin. The disulphide bridge between the A and B chains is indicated. The active sites (balls and sticks) of ebulin l and ricin bound to the substrate analog pteroic acid (CPK) and the sugar-binding sites 1 alpha and 2 gamma (balls and sticks) bound to D-galactose (CPK) are emphasized. The key residues in ebulin l and the corresponding residues of ricin are indicated.

Figure 2

Intracellular trafficking of nigrin b and ricin. Ricin (at pM concentrations) binds to glycoproteins of the plasma membrane and internalize into the cell. Some protein molecules are recycled back to the plasma membrane, others undergo degradation in the lysosomes, and a small number are transported first to the Golgi network and then to the endoplasmic reticulum. In the endoplasmic reticulum, the disulphide bridge is reduced and the A chain translocates to the cytosol by the endoplasmic reticulum-associated degradation (ERAD) pathway. In the cytosol, the A chain inactivates the ribosomes, inhibiting protein synthesis and causing cell death. This pathway is sensitive to low temperature and brefeldin A. Nigrin b (at pM concentrations) can bind to different glycoproteins of the plasma membrane than ricin and internalize into the cell. All the protein molecules are either recycled back to the plasma membrane or transported to lysosomes for degradation. This pathway is not sensitive to low temperature and brefeldin A. However, at much higher extracellular concentration (40,000 folds), the saturation of the endosome with nigrin b can lead to a spontaneous release of nigrin b into the cytosol, causing ribosome inactivation.