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 (VHHs) that span the RTA-RTB interface, including four Tier 1 VHHs with IC50 values <1 nM. Crystal structures of each VHH 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). VHHs 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 VHHs 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.
Subject(s)
Antibodies, Neutralizing/pharmacology , Cytoplasm/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Ricin/chemistry , Animals , Chlorocebus aethiops , Crystallography, X-Ray , HeLa Cells , Humans , Models, Molecular , Protein Conformation , Ricin/immunology , Single-Domain Antibodies/pharmacology , THP-1 Cells , Vero CellsABSTRACT
Ricin toxin's B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin's ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, ß, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB's duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as "bait" in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB's two domains may contribute to distinct steps in the intoxication pathway.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Epitope Mapping/methods , Epitopes/immunology , Ricin/chemistry , Ricin/immunology , Toxins, Biological/immunology , Animals , Chlorocebus aethiops , Epitopes/genetics , HeLa Cells , Humans , Protein Conformation , Protein Subunits , Ricin/genetics , Vero CellsABSTRACT
As part of an effort to engineer ricin antitoxins and immunotherapies, we previously produced and characterized a collection of phage-displayed, heavy chain-only antibodies (VHHs) from alpacas that had been immunized with ricin antigens. In our initial screens, we identified nine VHHs directed against ricin toxin's binding subunit (RTB), but only one, JIZ-B7, had toxin-neutralizing activity. Linking JIZ-B7 to different VHHs against ricin's enzymatic subunit (RTA) resulted in several bispecific antibodies with potent toxin-neutralizing activity in vitro and in vivo. JIZ-B7 may therefore be an integral component of a future VHH-based neutralizing agent (VNA) for ricin toxin. In this study, we now localize, using competitive ELISA, JIZ-B7's epitope to a region of RTB's domain 2 sandwiched between the high-affinity galactose/N-acetylgalactosamine (Gal/GalNAc)-binding site and the boundary of a neutralizing hotspot on RTA known as cluster II. Analysis of additional RTB (n = 8)- and holotoxin (n = 4)-specific VHHs from a recent series of screens identified a "supercluster" of neutralizing epitopes at the RTA-RTB interface. Among the VHHs tested, toxin-neutralizing activity was most closely associated with epitope proximity to RTA, and not interference with RTB's ability to engage Gal/GalNAc receptors. We conclude that JIZ-B7 is representative of a larger group of potent toxin-neutralizing antibodies, possibly including many described in the literature dating back several decades, that recognize tertiary and possibly quaternary epitopes located at the RTA-RTB interface and that target a region of vulnerability on ricin toxin.
