Abstract
JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin’s enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin’s bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells.
Highlights
Ricin, a member of the A-B family of bacterial and plant proteins toxins, is classified by the Centers for Disease Control and Prevention (CDC) as a potential biothreat agent [1, 2]
THP-1 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and were grown in RPMI supplemented with 10% fetal bovine serum (FBS)
These results were intriguing in light of the fact that JJX12 promotes the formation of higher order toxin-antibody complexes in solution, while JNA6 associated with ricin in 1:1 and 2:1 ricin:antibody complexes [25, 26]
Summary
A member of the A-B family of bacterial and plant proteins toxins, is classified by the Centers for Disease Control and Prevention (CDC) as a potential biothreat agent [1, 2]. Ricin’s B subunit (RTB) is a galactose- and N-acetylgalactosamine (Gal/GalNAc)-specific lectin that promotes ricin clathrin-dependent and independent endocytosis into mammalian cells, including epithelial cells that line the respiratory tract [5, 6]. Once within the ER, the single disulfide bond that links RTA to RTB is reduced by protein disulfide isomerase and RTA is retro-translocated (dislocated) into the cell cytoplasm where it promotes ribosome inactivation and cell death [7, 8]. Of particular interest are antitoxin agents based on toxin-specific camelid heavy-chain only VH domains (VHHs) antibodies [13]. The prophylactic and therapeutic potential of these so-called VHH-based neutralizing agents (VNAs) is even more remarkable considering that they have been successfully engineered and administered to mice and piglets via a non-replicating adenovirus vector [14, 21, 22]
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