A Collection of Single-Domain Antibodies that Crowd Ricin Toxin's Active Site.

Michael Rudolph,C. Middaugh,Chi Nguyen,Yinghui Rong,Siva Angalakurthi,David Volkin,Nicholas Mantis,David Vance,David Weis

doi:10.3390/antib7040045

Abstract

In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot (“cluster 3”) located in close proximity to RTA’s active site. HX-MS analysis revealed that the 21 VHHs recognized four distinct epitope subclusters (3.1–3.4). Sixteen of the 21 VHHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three VHHs grouped within subcluster 3.2, encompassing α-helices C and G, plus α-helix B. The single VHH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole VHH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 VHHs within subclusters 3.1–3.3 physically occlude RTA’s active site cleft, while the single antibody in subcluster 3.4 associates on the active site’s upper rim.

Highlights

Ricin is a member of the ribosome-inactivating protein (RIP) family of toxins and classified as a biothreat agent due to its high potential to induce morbidity and mortality after inhalation [1,2,3].The toxin is a ~65 kDa heterodimeric glycoprotein from the castor bean plant (Ricinus communis) consisting of a binding subunit (RTB) and an enzymatic subunit (RTA)
RiVax lacks high mannose residues normally found on ribosome-inactivating subunit (RTA), due to the fact that RiVax is expressed in E. coli
Manuscript in preparation) [23], we identified from different phage-displayed alpaca single chain libraries a total of 21 VH Hs whose binding to ricin toxin was partially or completely inhibited by IB2 in a capture ELISA (Figure 2)

Summary

Introduction

Ricin is a member of the ribosome-inactivating protein (RIP) family of toxins and classified as a biothreat agent due to its high potential to induce morbidity and mortality after inhalation [1,2,3]. The toxin is a ~65 kDa heterodimeric glycoprotein from the castor bean plant (Ricinus communis) consisting of a binding subunit (RTB) and an enzymatic subunit (RTA). RTA is an RNA N-glycosidase (EC 3.2.2.22) that depurinates a conserved adenosine within the sarcin-ricin loop (SRL) of 28S rRNA, thereby stalling ribosome translocation [5,6]. RTA is a globular protein with a total of 10 β-strands (A–J) and seven α-helices (A–G). RTA folds into three distinct domains: domain 1 (residues 1–117) is dominated by a six-stranded β-sheet, domain

Methods

Results

Conclusion