A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking.
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.
Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.|Department of Biomedical Sciences, University at Albany School of Public Health, Albany, New York, United States of America.
Cristina
C
Herrera
Department of Molecular Cell Biology and Centre for Cancer Biomedicine, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, Oslo, Norway.|Department of Biosciences, University of Oslo, Oslo, Norway.
Tove Irene
TI
Klokk
Department of Biomedical Sciences, University at Albany School of Public Health, Albany, New York, United States of America.|Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
Richard
R
Cole
Department of Molecular Cell Biology and Centre for Cancer Biomedicine, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, Oslo, Norway.|Department of Biosciences, University of Oslo, Oslo, Norway.
Kirsten
K
Sandvig
Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.|Department of Biomedical Sciences, University at Albany School of Public Health, Albany, New York, United States of America.
Nicholas J
http://orcid.org/0000-0002-5083-8640
NJ
Mantis
D000072283:A549 Cells; D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology / Q000494:pharmacology*; D000992:Antitoxins / Q000276:immunology / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D051379:Mice; D066329:Protein Aggregates / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects*; D012276:Ricin / Q000276:immunology / Q000378:metabolism*; D012278:Ricinus / Q000737:chemistry; D021601:trans-Golgi Network / Q000187:drug effects / Q000378:metabolism
D018033:Antibodies, Bispecific; D000992:Antitoxins; D066329:Protein Aggregates; D012276:Ricin