Involvement of the amino acids outside the active‐site cleft in the catalysis of ricin A chain

Kitaoka, Yoshihisa

doi:10.1046/j.1432-1327.1998.2570255.x

Cited by 20 publications

(20 citation statements)

References 17 publications

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“…In previous studies, various random mutagenesis methods have been used to isolate nontoxic RTA mutants from yeast (1,10). Systematic deletion analysis has been used to identify amino acids critical for the activity of RTA in vitro (18,31). Our study is the first report which determines the precise effects of each mutation on cytotoxicity, ribosome depurination, and translation inhibition and provides evidence that cytotoxicity of RTA is not entirely due to ribosome depurination.…”

Section: Discussionmentioning

confidence: 89%

“…Since Gly83 is relatively distant from the active site, it is unlikely that Gly83 participates in the catalysis. Previous studies indicated that RTA lost its depurination activity when Gly83 was deleted (18,31). A point mutation in the corresponding Gly in PAP (G75D) led to a loss of depurination in vivo (15) and affected the binding of PAP to ribosomes (14).…”

Section: Discussionmentioning

confidence: 99%

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Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

Baricevic

Saidasan

et al. 2007

Infect Immun

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The plant toxin ricin is one of the most potent and lethal substances known. Ricin inhibits protein synthesis by removing a specific adenine from the highly conserved ␣-sarcin/ricin loop in the large rRNA. Very little is known about how ricin interacts with ribosomes and the molecular mechanism by which it kills cells. To gain insight to the mechanism of ricin-induced cell death, we set up yeast (Saccharomyces cerevisiae) as a simple and genetically tractable system to isolate mutants defective in cytotoxicity. Ribosomes were depurinated in yeast cells expressing the precursor form of the A chain of ricin (pre-RTA), and these cells displayed apoptotic markers such as nuclear fragmentation, chromatin condensation, and accumulation of reactive oxygen species. We conducted a large-scale mutagenesis of pre-RTA and isolated a panel of nontoxic RTA mutants based on their inability to kill yeast cells. Several nontoxic RTA mutants depurinated ribosomes and inhibited translation to the same extent as wild-type RTA in vivo. The mutant proteins isolated from yeast depurinated ribosomes in vitro, indicating that they were catalytically active. However, cells expressing these mutants did not display hallmarks of apoptosis. These results provide the first evidence that the ability to depurinate ribosomes and inhibit translation does not always correlate with ricin-mediated cell death, indicating that ribosome depurination and translation inhibition do not account entirely for the cytotoxicity of ricin.

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Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

Baricevic

Saidasan

et al. 2007

Infect Immun

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“…5B). Previous studies have demonstrated that there are four amino acids (Tyr-80, Tyr-123, Glu-177, and Arg-180) critical for the depurination activity of ricin (6,9,12,25). To investigate how the flexibility of the ␣-helix affects the enzyme activity of ricin, we performed a dihedral angle analysis.…”

Section: Rips With Very Low Similarity In Primary Structure Arementioning

confidence: 99%

Identification of a Novel Functional Domain of Ricin Responsible for Its Potent Toxicity

Dai

Lei

Yang

et al. 2011

Journal of Biological Chemistry

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Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved ␣-sarcin loop of large rRNAs. They have received attention in biological and biomedical research because of their unique biological activities toward animals and human cells as cell-killing agents. A better understanding of the depurination mechanism of RIPs could allow us to develop potent neutralizing antibodies and to design efficient immunotoxins for clinical use. Among these RIPs, ricin exhibited remarkable efficacy in depurination activity and highly conserved tertiary structure with other RIPs. It can be considered as a prototype to investigate the depurination mechanism of RIPs. In the present study, we successfully identified a novel functional domain responsible for controlling the depurination activity of ricin, which is located far from the enzymatic active site reported previously. Our study indicated that ricin A-chain mAbs binding to this domain (an ␣-helix comprising the residues 99 -106) exhibited an unusual potent neutralizing ability against ricin in vivo. To further investigate the potential role of the ␣-helix in regulating the catalytic activity of ricin, ricin A-chain variants with different flexibility of the ␣-helix were rationally designed. Our data clearly demonstrated that the flexibility of the ␣-helix is responsible for controlling the depurination activity of ricin and determining the extent of protein synthesis inhibition, suggesting that the conserved ␣-helix might be considered as a potential target for the prevention and treatment of RIP poisoning. Ribosome-inactivating proteins (RIPs)3 are depurinating rRNA N-glycosidases (E.C. 3.2.2.22) that cleave a single bond between a specific adenine and ribose of rRNA in eukaryotes (1). They are generally divided into two classes (1-3). Type I RIPs such as trichosanthin and gelonin are monomeric enzymes of ϳ30 kDa. Type II RIPs are heterodimeric proteins with an approximate molecular mass of 60 kDa, in which one polypeptide with RIP activity (A-chain) is linked by a disulfide bridge to a galactose-binding lectin (B-chain). The B-chain is able to bind to a galactose-containing receptor on the surface of sensitive cells and mediate transport of the A-chain through the secretory pathways into the cytoplasm.Ricin (a type II RIP), which has emerged as a powerful catalyst for mammalian ribosomes, is a good prototype to investigate the N-glycosidase mechanism of RIPs. Ricin A-chain (RTA) is the catalytic subunit of ricin, which catalyzes the depurination of an invariant adenosine residue, A 4324 , within the GA 4324 GA tetraloop motif of the highly conserved sarcinricin loop of eukaryotic 28 S rRNA (4). Most previous studies have focused on the role of the active-site residues that are crucial for catalytic activity of RIPs. Day et al. (5) have presented the crystal structure of ricin, indicating that RTA has a prominent cleft able to recognize the target rRNA stem-loop. Sitedirected mutagenesis, as well as analysis by systematic deletion ...

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“…The nature of the residue at the substrate-binding site is found to play a major role. Comparison of the residues that actively participate in the binding of the substrate in abrin, ricin, and APA-I reveal that Asn-200 of abrin-a, corresponding to Arg-213 of ricin A, is required for the binding to the substrate in the ␣-sarcin loop, a GpApApAp tetranucleotide sequence located at the 3Ј-terminal region of 28 S rRNA (45,46). In case of ricin, Arg-213 acts as a positively charged recognition group for the negatively charged substrate, possibly by forming a salt linkage with the phosphate backbone of RNA (47).…”

Section: Inhibition Of Protein Synthesis Bymentioning

confidence: 99%

Structure-Function Analysis and Insights into the Reduced Toxicity of Abrus precatorius Agglutinin I in Relation to Abrin

Bagaria

Surendranath

Ramagopal

et al. 2006

Journal of Biological Chemistry

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Abrin and agglutinin-I from the seeds of Abrus precatorius are type II ribosome-inactivating proteins that inhibit protein synthesis in eukaryotic cells. The two toxins share a high degree of sequence similarity; however, agglutinin-I is weaker in its activity. We compared the kinetics of protein synthesis inhibition by abrin and agglutinin-I in two different cell lines and found that ϳ200 -2000-fold higher concentration of agglutinin-I is needed for the same degree of inhibition. Like abrin, agglutinin-I also induced apoptosis in the cells by triggering the intrinsic mitochondrial pathway, although at higher concentrations as compared with abrin. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison with that of the reported structure of abrin. The overall protein folding of agglutinin-I is similar to that of abrin-a with a single disulfide bond holding the toxic A subunit and the lectin-like B-subunit together, constituting a heterodimer. However, there are significant differences in the secondary structural elements, mostly in the A chain. The substitution of Asn-200 in abrin-a with Pro-199 in agglutinin-I seems to be a major cause for the decreased toxicity of agglutinin-I. This perhaps is not a consequence of any kink formation by a proline residue in the helical segment, as reported by others earlier, but due to fewer interactions that proline can possibly have with the bound substrate.

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Involvement of the amino acids outside the active‐site cleft in the catalysis of ricin A chain

Cited by 20 publications

References 17 publications

Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

Identification of a Novel Functional Domain of Ricin Responsible for Its Potent Toxicity

Structure-Function Analysis and Insights into the Reduced Toxicity of Abrus precatorius Agglutinin I in Relation to Abrin

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