1. Structural Analysis of Toxin-Neutralizing, Single-Domain Antibodies that Bridge Ricin's A-B Subunit Interface.
- Author
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Rudolph MJ, Poon AY, Kavaliauskiene S, Myrann AG, Reynolds-Peterson C, Davis SA, Sandvig K, Vance DJ, and Mantis NJ
- Subjects
- Animals, Chlorocebus aethiops, Crystallography, X-Ray, HeLa Cells, Humans, Models, Molecular, Protein Conformation, Ricin immunology, Single-Domain Antibodies pharmacology, THP-1 Cells, Vero Cells, Antibodies, Neutralizing pharmacology, Cytoplasm metabolism, Endoplasmic Reticulum metabolism, Golgi Apparatus metabolism, Ricin chemistry
- Abstract
Ricin toxin kills mammalian cells with notorious efficiency. The toxin's B subunit (RTB) is a Gal/GalNAc-specific lectin that attaches to cell surfaces and promotes retrograde transport of ricin's A subunit (RTA) to the trans Golgi network (TGN) and endoplasmic reticulum (ER). RTA is liberated from RTB in the ER and translocated into the cell cytoplasm, where it functions as a ribosome-inactivating protein. While antibodies against ricin's individual subunits have been reported, we now describe seven alpaca-derived, single-domain antibodies (V
H Hs) that span the RTA-RTB interface, including four Tier 1 VH Hs with IC50 values <1 nM. Crystal structures of each VH H bound to native ricin holotoxin revealed three different binding modes, based on contact with RTA's F-G loop (mode 1), RTB's subdomain 2γ (mode 2) or both (mode 3). VH Hs in modes 2 and 3 were highly effective at blocking ricin attachment to HeLa cells and immobilized asialofetuin, due to framework residues (FR3) that occupied the 2γ Gal/GalNAc-binding pocket and mimic ligand. The four Tier 1 VH Hs also interfered with intracellular functions of RTB, as they neutralized ricin in a post-attachment cytotoxicity assay (e.g., the toxin was bound to cell surfaces before antibody addition) and reduced the efficiency of toxin transport to the TGN. We conclude that the RTA-RTB interface is a target of potent toxin-neutralizing antibodies that interfere with both extracellular and intracellular events in ricin's cytotoxic pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2021
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