Ligand-toxin Hybrids Directed to the α2-Macroglobulin Receptor/Low Density Lipoprotein Receptor-related Protein Exhibit Lower Toxicity than Native Pseudomonas Exotoxin

Abstract

Pseudomonas exotoxin (PE) binds the heavy chain of the ␣ 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). To understand the significance of this interaction, novel toxin-derived gene fusions were constructed with two ligands that also bind this receptor. A 39-kDa cellular protein, termed RAP, binds LRP with high affinity and often co-purifies with it. Two RAP toxins were constructed, one with PE and one with diphtheria toxin (DT). RAP, which replaced the toxins binding domains, was combined with each of the corresponding translocating and ADP-ribosylating domains. Both RAP-toxins bound LRP with an apparent higher affinity than native PE. Despite this, RAP-PE and DT-RAP were less toxic than native PE. Apparently, RAP-toxin molecules bound and entered cells but used a pathway that afforded only low efficiency of toxin transport to the cytosol. This was evident because co-internalization with adenovirus increased the toxicity of RAP-toxins by 10-fold. We speculate that the high affinity of RAP binding may not allow the toxin's translocating and ADP-ribosylating domains to reach the cytosol but rather causes the toxin to take another pathway, possibly one that leads to lysosomes. To test this hypothesis, additional RAP-PE fusions were constructed. Nterminal or C-terminal fragments of RAP were joined to PE to produce two novel fusion proteins which were likely to have reduced affinity for LRP. Both of these shorter fusion proteins exhibited greater toxicity than full-length RAP-PE. A second ligand-toxin gene fusion was constructed between plasminogen activator inhibitor type 1 and DT. DT-plasminogen activator inhibitor type 1 formed a complex with tissue-type plasminogen activator and inhibited its proteolytic activity. However, like the RAP-toxins, this hybrid was less toxic for cells than native PE.

Regulation of tau internalization, degradation, and seeding by LRP1 reveals multiple pathways for tau catabolism

“…The weaker affinity of these pathologic forms of tau for LRP1 may have important consequences on trafficking of tau by allowing modified tau to dissociate from LRP1 within early endosomes to facilitate endosomal escape. It is interesting to highlight in this regard that experiments employing hybrid constructs of Pseudomonas exotoxin A noted an inverse correlation between LRP1 binding affinity and toxicity (62), which requires endosomal escape of the toxin. Thus, a hybrid toxin containing a domain with high affinity for LRP1 was much less toxic to cells, likely due to more effective delivery to lysosomal compartments for degradation.…”

LRP1 and SORL1 regulate tau internalization and degradation and enhance tau seeding

“…The weaker affinity of these pathologic forms of tau for LRP1 may have important consequences on trafficking of tau and may allow modified tau to dissociate from LRP1 within early endosomes to facilitate endosomal escape. It is interesting to highlight in this regard that experiments employing hybrid constructs of Pseudomonas exotoxin A noted an inverse correlation between LRP1 binding affinity and toxicity (Zdanovsky et al, 1996), which requires endosomal escape of the toxin. For example, replacement of the receptor binding domain on the toxin with RAP generated a hybrid toxin with substantial increase in affinity for LRP1 but was much less toxic to cells.…”

Development of a prolactin receptor-targeting fusion toxin using a prolactin antagonist and a recombinant form of Pseudomonas exotoxin A

“…Domain I is responsible for cell recognition, domain II for translocation, and domain III for inhibition of protein synthesis. PE binds to the membraneassociated heavy chain of α 2 -macroglobulin receptor and undergoes receptor-mediated endocytosis [21,22,23]. Within the endosome PE is acidified and processed [24,25,26,27]; the low pH induces conformational changes in PE necessary for translocation [28] and for processing by furan [29,30], which cleaves PE between arginine 279 and glycine 280 [31].…”