Dataset Information

Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity.

ABSTRACT: Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal structure of A9 bound to RTA (2.6 Å resolution) revealed extensive antibody contact with RTA's ?-strand h (732 Å2 buried surface area; BSA), along with limited engagement with ?-helix D (90 Å2) and ?-helix C (138 Å2). Collectively, these contacts explain the overlap between epitope clusters I and III, as identified by competition ELISA. However, considerable binding affinity, and, consequently, toxin-neutralizing activity of A9 is mediated by an unusual CDR2 containing five consecutive Gly residues that interact with ?-helix B (82 Å2), a known neutralizing hotspot on RTA. Removal of a single Gly residue from the penta-glycine stretch in CDR2 reduced A9's binding affinity by 10-fold and eliminated toxin-neutralizing activity. Computational modeling indicates that removal of a Gly from CDR2 does not perturb contact with RTA per se, but results in the loss of an intramolecular hydrogen bond network involved in stabilizing CDR2 in the unbound state. These results reveal a novel configuration of a CDR2 element involved in neutralizing ricin toxin.

SUBMITTER: Rudolph MJ

PROVIDER: S-EPMC6277176 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity.

Rudolph Michael J MJ Vance David J DJ Kelow Simon S Angalakurthi Siva Krishna SK Nguyen Sophie S Davis Simon A SA Rong Yinghui Y Middaugh C Russell CR Weis David D DD Dunbrack Roland R Karanicolas John J Mantis Nicholas J NJ

Protein engineering, design & selection : PEDS 20180701 7-8

Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal str ...[more]

PMID: 30265352

Similar Datasets

Project description:Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody") specific for ricin's enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

Project description: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.

Dataset Information

Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity.

Publications

Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets