Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins

Abstract

During ricin intoxication in mammalian cells, ricin's enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin–ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (VHHs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such VHHs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these VHHs block RTA–P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as “intrabodies,” these VHHs rendered cells resistant to ricin intoxication. One VHH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation. During ricin intoxication in mammalian cells, ricin's enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin–ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (VHHs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such VHHs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these VHHs block RTA–P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as “intrabodies,” these VHHs rendered cells resistant to ricin intoxication. One VHH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation. Ricin toxin is the archetype of the large and diverse family of medically important plant and bacterial ribosome-inactivating proteins (RIPs). A byproduct of castor beans (Ricinus communis), ricin toxin consists of two equal sized subunits, ricin toxin A subunit (RTA) and ricin toxin B subunit (RTB), that associate through electrostatic and covalent interactions (1Montfort W. Villafranca J.E. Monzingo A.F. Ernst S.R. Katzin B. Rutenber E. Xuong N.H. Hamlin R. Robertus J.D. The three-dimensional structure of ricin at 2.8 A.J. Biol. Chem. 1987; 262: 5398-5403Abstract Full Text PDF PubMed Google Scholar, 2Olsnes S. Refsnes K. Pihl A. Mechanism of action of the toxic lectins abrin and ricin.Nature. 1974; 249: 627-631Crossref PubMed Scopus (348) Google Scholar). RTB is a bivalent Gal/GalNAc-specific lectin that traffics RTA from the plasma membrane to the endoplasmic reticulum (ER) (3Sowa-Rogozinska N. Sominka H. Nowakowska-Golacka J. Sandvig K. Slominska-Wojewodzka M. Intracellular transport and cytotoxicity of the protein toxin ricin.Toxins (Basel). 2019; 11: 350Crossref Scopus (29) Google Scholar). Within the ER, the interchain disulfide bond linking RTA and RTB is reduced by protein disulfide isomerase, and RTA is then retrotranslocated in an the membrane and into the cytoplasm R. A. S. S. R. M. of ricin and ricin by protein disulfide and PubMed Scopus Google Scholar, J. Sominka H. N. M. the endoplasmic protein in intoxication J. 2019; Scopus Google Scholar, ricin to its A and B in the endoplasmic J. PubMed Scopus Google Scholar, J. A. S. of ricin on of the toxin from the endoplasmic reticulum to the Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that RTA functions as a the and K. M. K. The of action of ricin and toxic lectins on The and the of the in ribosomal by the Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar, K. activity of ricin Mechanism of action of the toxic lectin ricin on Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar, of the protein by of the and by to the loop in the 28S Biol. PubMed Google Scholar, S. is a PubMed Scopus Google Scholar). The of action of RTA in mammalian ribosomes is RTA the of the bond of a within the sarcin–ricin loop (SRL) of the 28S rRNA, a conserved structure that with and ribosome (1Montfort W. Villafranca J.E. Monzingo A.F. Ernst S.R. Katzin B. Rutenber E. Xuong N.H. Hamlin R. Robertus J.D. The three-dimensional structure of ricin at 2.8 A.J. Biol. Chem. 1987; 262: 5398-5403Abstract Full Text PDF PubMed Google Scholar, 2Olsnes S. Refsnes K. Pihl A. Mechanism of action of the toxic lectins abrin and ricin.Nature. 1974; 249: 627-631Crossref PubMed Scopus (348) Google Scholar, K. activity of ricin Mechanism of action of the toxic lectin ricin on Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar, ribosomal with and PubMed Scopus Google Scholar, K. The activity of ricin The of the enzymatic activity of ricin with ribosomes and with Biol. Chem. Full Text PDF PubMed Google Scholar, Robertus J.D. of ricin and for the of PubMed Scopus Google Scholar, of and with a conserved loop in PubMed Scopus Google Scholar, A.F. Robertus J.D. of in the ricin Biol. PubMed Scopus Google Scholar). The of RTA consists of a large on of the that through a and A.F. Robertus J.D. of in the ricin Biol. PubMed Scopus Google Scholar, Monzingo A.F. Katzin Ernst S. Robertus J.D. of ricin A at PubMed Scopus Google Scholar). has the within the that are with activity J.D. Ernst S. Monzingo A. S. M. of ricin and for PubMed Scopus Google Scholar). and the a is in of the in the of the is evidence that the to SRL are that RTA are to is a of evidence that SRL is the event in a interaction RTA and the ribosome M. M. ricin the (Basel). 2019; 11: Scopus Google Scholar, ricin to the PubMed Scopus Google Scholar). to the RTA with the of the P-stalk proteins and and of the ribosome to the SRL ricin to the PubMed Scopus Google Scholar, M. A binding proposed for the electrostatic interactions of ricin a with PubMed Scopus Google Scholar, M. The proteins of the ribosomal are for ribosome binding and by ricin in J. PubMed Scopus Google Scholar). the of RTA normally by RTB as critical for ribosomal with the important A of ribosome by of ricin toxin A protein and in mammalian J. Biol. PubMed Scopus Google Scholar, Ricin as an residue to to of the ribosomal PubMed Scopus Google Scholar). The structures of RTA in complex with C-terminal peptides of the protein and M. into the interaction of the ribosomal protein with a ribosome-inactivating protein PubMed Scopus Google Scholar, and ribosomal interaction of ribosome-inactivating Scopus Google Scholar). The structures revealed a pocket on RTA by and as the of the peptides to the RTA with and RTA of with the of the ribosomal the activity of ricin A by ribosome (Basel). Scopus Google Scholar). the large of RTA normally by RTB is in the cytoplasm to in recruitment to the In this we the and of single-domain antibodies (VHHs) that epitopes on the of RTA normally by the structures of VHHs (V9E1, V9F9, and to that three of the antibodies not only the ribosomal P-stalk binding pocket on RTA but also P-stalk peptide The an epitope adjacent to but not the ribosomal P-stalk binding the VHHs that the ribosomal P-stalk binding pocket RTA in assays and peptide we that of and VHHs as antibodies cells resistant to ricin toxin to to that with VHHs the of Intracellular of ricin toxin by single-domain antibodies the Biol. PubMed Scopus Google Scholar). findings a in the interaction of RTA with ribosomal P-stalk proteins is a to SRL and the of that two on RTA. of a VHH diverse epitopes on ricin A and B subunits Intracellular of ricin toxin by single-domain antibodies the Biol. PubMed Scopus Google Scholar, A of epitopes at the of ricin's enzymatic (RTA) and binding (RTB) (Basel). Scopus Google Scholar, S. Sandvig K. of single-domain antibodies that ricin's subunit Biol. 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Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that the VHHs in a cytotoxicity to the epitopes on RTA by VHH family this the structures of VHHs (V9E1, V9F9, and with RTA at from to and and VHH a that of in two of and with the of as the in a loop of a and on of VHH and VHH or In to with the interaction was by VHHs and the disulfide bond and and in linking and S. of PubMed Scopus Google Scholar). The disulfide bond was in the of the requisite and a disulfide with and in and and in The structure of RTA in complex of to three and RTA structure are in for binding and determined in in vitro determined in in vitro complex in complex in determined in in vitro complex in in a the structures and to as by an of an of with and an of The structure was as with a structurally RTA in RTA when by as RTA in of to when RTA on RTA of the VHHs (V9E1, and and epitopes that overlap with the ribosomal P-stalk binding pocket of RTA and A and and RTA and and loop and and as as in loop on RTA The three VHHs the P-stalk binding pocket, with RTA A and loop loop loop and and in and immediately C-terminal to in not the P-stalk protein binding and The epitope of the C-terminal of A loop loop and two in as as immediately to a with the epitopes of and V9F9, RTA A loop loop and to within with residue immediately C-terminal to The and RTA and consists of two and The two the of in and in with the from in and of RTA. The and RTA with RTA with RTA and with with residue also a interaction RTA and interaction the interaction that RTA and with in the ribosomal P-stalk peptide as revealed in the RTA–P-stalk peptide crystal structure and structure of ribosome-inactivating protein ricin A in complex with the C-terminal peptide of the ribosomal protein (Basel). Scopus Google Scholar). residue that into a on RTA by and in a to within the P-stalk peptide in complex with RTA and has a of with interactions with the P-stalk binding of the of RTA in complex with VHH with the P-stalk and the of to P-stalk binding on RTA. VHH and RTA are as and to with and The P-stalk peptide is as with and and are as ricin toxin subunit The and RTA and and are and with RTA and residue and residue two with RTA and and are in that with the C-terminal residue in the P-stalk protein when to RTA also residue RTA and to P-stalk peptide residue has an of with RTA. 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PubMed Scopus Google Scholar). and with of RTA and then to a In the of the the of RTA in a in as by in activity The to the to on the activity of RTA. In the of in a of as with RTA In activity was to that of a VHH that the of RTA not Intracellular of ricin toxin by single-domain antibodies the Biol. PubMed Scopus Google Scholar). that neutralize the activity in vitro of RTA. with the ability of RTA to associate with ribosome P-stalk proteins, we a binding in with a and then with RTA in the or the of The of in a of RTA to In to a RTA binding to was at a In on the ability of RTA to interact with when the was in These are consistent with from the crystal structures in that of RTA to the ribosomal P-stalk proteins, whose epitope is a the ribosomal P-stalk binding on has ricin toxin activity when to cells we that it protect cells expressed within the cytoplasm it RTA to with the In a we a to VHHs as antibodies or in cells Intracellular of ricin toxin by single-domain antibodies the Biol. 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PubMed Scopus Google Scholar). that the and into the mammalian and into cells by and in at as determined an the of the and on the of cells to ricin cells with of ricin toxin for and then for A was as a for these J. domain antibodies (VHHs) as binding and of PubMed Scopus Google Scholar). cells with resistant to as with cells with In cells with as to ricin as cells that the ribosomal P-stalk binding of RTA is to protect the ribosomes from to the VHH family and and of the ribosome P-stalk binding of RTA but RTA with and and RTA from binding to in a VHHs from family also a of family not in the was a of RTA with and and VHHs in family the by and and In was at RTA in the These that and not the ribosomal recruitment of RTA is to block binding and to neutralize RTA these we when expressed as and and three VHHs in family be at cells from ricin intoxication. this cells with and VHHs V9F9, and and then with ricin toxin of the VHHs rendered cells resistant to ricin the cells, In the that was to VHHs in and not VHHs in and from in that are to a disulfide bond that and protein S. of PubMed Scopus Google Scholar). this cells as and then to RTA was expressed at as with VHHs in and VHHs with a disulfide bond expressed at cells resistant to as with cells, that of has the to neutralize ricin we and as a disulfide expressed in cells cells resistant to ricin with cells in from the toxin the of the P-stalk binding and that P-stalk binding the enzymatic activity of is in RTA that P-stalk binding B. 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Scopus Google Scholar). by an epitope immediately adjacent to but not the P-stalk binding The in epitope in of with RTA ribosomes from RTA in to ricin toxin when expressed as an in cells, These that occlusion of the ribosomal P-stalk peptide binding is to neutralize the activity of RTA within the Moreover, that ribosomal P-stalk protein interactions are to a on RTA by the of Moreover, to the P-stalk binding pocket of RTA the activity of RTA to a to that by with the of RTA. is on the of with is of VHHs that the pocket of RTA Intracellular of ricin toxin by single-domain antibodies the Biol. PubMed Scopus Google Scholar). The binding of for RTA is to The two antibodies and In the and RTA when of in of to ricin toxin when expressed as in In with the ribosomal P-stalk proteins is a requisite in the and as as important as the event M. A binding proposed for the electrostatic interactions of ricin a with PubMed Scopus Google Scholar, M. 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PubMed Scopus Google Scholar). at with the in epitope on ricin toxin by PubMed Scopus Google Scholar). and for at RTA in was to for was in and to for and was for and was for to by a with at an at VHH protein with a C-terminal was as a binding for in and in block VHHs at a of in in in and with of RTA in from to to a of VHH and to RTA. was to an and The was for at and then on for to of an with an equal of at was immediately a was determined as a of RTA for RTA with and VHH was cells in with and at and cells in and as Intracellular of ricin toxin by single-domain antibodies the Biol. PubMed Scopus Google Scholar). cytotoxicity cells in and with ricin as Intracellular of ricin toxin by single-domain antibodies the Biol. 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