Ricin is a member of the ubiquitous family of plant and bacterial AB toxins that gain entry into the cytosol of host cells through receptor-mediated endocytosis and retrograde traffic through the trans-Golgi network (TGN) and endoplasmic reticulum (ER). While a few
ricin toxin-specific neutralizing
monoclonal antibodies (MAbs) have been identified, the mechanisms by which these
antibodies prevent toxin-induced cell death are largely unknown. Using immunofluorescence confocal microscopy and a TGN-specific sulfation assay, we demonstrate that 24B11, a MAb against
ricin's binding subunit (RTB), associates with
ricin in
solution or when prebound to cell surfaces and then markedly enhances toxin uptake into host cells. Following endocytosis, however, toxin-antibody complexes failed to reach the TGN; instead, they were shunted to Rab7-positive late endosomes and LAMP-1-positive lysosomes. Monovalent 24B11
Fab fragments also interfered with toxin retrograde transport, indicating that neither cross-linking of
membrane glycoproteins/
glycolipids nor the recently identified intracellular
Fc receptor is required to derail
ricin en route to the TGN. Identification of the mechanism(s) by which
antibodies like 24B11 neutralize
ricin will advance our fundamental understanding of protein trafficking in mammalian cells and may lead to the discovery of new classes of toxin inhibitors and
therapeutics for biodefense and
emerging infectious diseases. IMPORTANCE
Ricin is the prototypic member of the AB family of medically important plant and
bacterial toxins that includes
cholera and
Shiga toxins.
Ricin is also a category B biothreat agent. Despite ongoing efforts to develop
vaccines and antibody-based
therapeutics against
ricin, very little is known about the mechanisms by which
antibodies neutralize this toxin. In general, it is thought that
antibodies simply prevent toxins from attaching to
cell surface receptors or promote their clearance through
Fc receptor (FcR)-mediated uptake. In this report, however, we describe a neutralizing
monoclonal antibody (MAb) against
ricin's binding subunit (RTB) that not only associates with
ricin after the toxin has bound to the cell's surface but actually enhances toxin uptake into host cells. Following endocytosis, the antibody-toxin complexes are then routed for degradation. The results of this study are important because they reveal a previously unappreciated role for B-subunit-specific
antibodies in intracellular neutralization of
ricin toxin.