PE binds and enters mammalian cells and is then processed to an active C- terminal fragment which translocates to the cell cytoplasm and inhibits protein synthesis by ADP-ribosylating EF-2. At the cell surface, PE binds to a large MW glycoprotein which has been identified as the alpha2- macroglobulin receptor (alpha2M-R). In an ELISA assay PE but not PEGlu57 bound to affinity purified alpha2M-R. A 39 kD receptor associated protein (RAP) blocked PE binding to intact cells and pretreatment of cells with RAP prevented toxin-mediated inhibition of protein synthesis. Once delivered to the endosomal compartment by the alpha2M-R, PE is cleaved by an membrane-associated protease (MAP). Purification of this protease has begun using beef liver as a source of the enzyme. Crude membranes are treated with papain to release the MAP in a soluble form. Column chromatography is used to complete the purification of the soluble enzyme. In the endosome PE is cleaved between arginine 279 and glycine 280 and then reduced to generate a 37 kD C-terminal fragment which translocates to the cytosol. In the presence of excess PE553D (a mutant form of PE lacking ADP-ribosylating activity), the toxicity of native PE is greatly reduced. However, excess PE553D does not compete strongly for the binding of native PE to its surface receptor, rather it competes within cells for a step taken by the 37 kD fragment en route to the cytosol. Mutant forms of PE553D with substitutions of other amino acids for Trp281 compete dramatically less well than PE553D for this step. PE553D with an alanine in place of tryptophan 281 competes 100-fold less well than PE553D, despite the fact that PE553DAla281 binds to cells, is internalized, and is cleaved appropriately by the cellular protease. In PE, glycine 280 can be changed to methionine without loss of biological activity. This allows the production of recombinant forms of PE that begin at residue 280 (with methionine in this position) and do not need to be processed within cells. PE37-Transforming Growth Factor-alpha (TGFalpha) which was constructed by cloning TGFalpha into the C-terminal end of PE37 was found to be more active than TGFalpha-PE40, possibly because there was no need for cellular processing.
