Abstract
Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin’s enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin’s egress from EEA-1+ and Rab7+ vesicles and enhanced toxin accumulation in LAMP-1+ vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA.
Highlights
® time points (30 min, 90 min and 4 hr) the cells were fixed, stained with DyLight 549-labeled secondary Ab and imaged by confocal microscopy
In the current study we demonstrate using a combination of confocal microscopy and TGN-specific labeling methods that R70 and SyH7, as well as three other toxin-neutralizing RTA-specific monoclonal antibodies (mAbs) impair retrograde trafficking of ricin to the TGN
At the 30 min time point, toxin-mAb complexes were situated within vesicles that were distributed throughout the cytoplasm
Summary
® time points (30 min, 90 min and 4 hr) the cells were fixed, stained with DyLight 549-labeled secondary Ab and imaged by confocal microscopy. We recently described three other SyH7-like mAbs, each with the capacity to passively protect mice against ricin toxin challenge[9]. Pincus and colleagues suggested that certain toxin-neutralizing, RTA-specific murine mAbs delay toxin internalization and/or interfere with intracellular trafficking to the ER12. We concur with this model and, based on numerous studies from our group, would argue that ricin RTA-specific mAbs likely influence very upstream events in the retrograde trafficking pathway, impairing delivery of ricin to the TGN13–16. In the current study we demonstrate using a combination of confocal microscopy and TGN-specific labeling methods that R70 and SyH7, as well as three other toxin-neutralizing RTA-specific mAbs impair retrograde trafficking of ricin to the TGN
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