Abstract
Ricin belongs to the type II ribosome-inactivating proteins that depurinate the universally conserved α-sarcin loop of rRNA. The RNA N-glycosidase activity of ricin also largely depends on the ribosomal proteins that play an important role during the process of rRNA depurination. Therefore, the study of the interaction between ricin and the ribosomal elements will be better to understand the catalysis mechanism of ricin. The antibody 6C2 is a mouse monoclonal antibody exhibiting unusually potent neutralizing ability against ricin, but the neutralization mechanism remains unknown. Here, we report the 2.8 Å crystal structure of 6C2 Fab in complex with the A-chain of ricin (RTA), which reveals an extensive antigen-antibody interface that contains both hydrogen bonds and van der Waals contacts. The complementarity-determining region loops H1, H2, H3, and L3 form a pocket to accommodate the epitope on the RTA (residues Asp(96)-Thr(116)). ELISA results show that Gln(98), Glu(99), Glu(102), and Thr(105) (RTA) are the key residues that play an important role in recognizing 6C2. With the perturbation of the 6C2 Fab-RTA interface, 6C2 loses its neutralization ability, measured based on the inhibition of protein synthesis in a cell-free system. Finally, we propose that the neutralization mechanism of 6C2 against ricin is that the binding of 6C2 hinders the interaction between RTA and the ribosome and the surface plasmon resonance and pulldown results confirm our hypothesis. In short, our data explain the neutralization mechanism of mAb 6C2 against ricin and provide a structural basis for the development of improved antibody drugs with better specificity and higher affinity.
Highlights
The antibody 6C2 exhibited an unusually potent neutralizing ability against ricin
The results showed that WT-RTA and WT-RTAϩBSA could bind 80S ribosomes normally, but RTA was not detected after preincubating with 6C2 Fab (Fig. 4D)
We determined the interface between 6C2 Fab and RTA, and we further mapped the residues on RTA that played an important role in interacting with 6C2 Fab by an ELISA experiment
Summary
The antibody 6C2 exhibited an unusually potent neutralizing ability against ricin. Results: We determined the crystal structure of 6C2 Fab in complex with RTA and mapped the epitope on RTA. Scientists have used a portion of the ricin toxin to develop a new class of therapeutics called “immunotoxins.” Over the past few decades, immunotoxins have been taken from discovery to clinical trials in patients with a variety of cancers From another perspective, ricin is a good prototype to investigate the N-glycosidase mechanism of RIPs because of its powerful catalytic ability for mammalian ribosomes. Exploiting the extraordinary capacity of ricin to provoke an immune response, Erhlich (40) and others (8) were the first to demonstrate the potential of Abs to completely inactivate ricin in the late 1880s Since those early studies, dozens of polyclonal antibodies derived from different animal species (e.g. mouse and rabbit) have been tested on a diversity of cell types (e.g. human, non-human primate, and mouse) and animal models (e.g. mice, rats, and rabbits) (9 –19). Combining the surface plasmon resonance (SPR) and pulldown results, we propose that the binding of 6C2 hinders the interaction between RTA and the ribosome, inhibiting the activities of RTA
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