Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes.

Schlossman, David M.; Withers, Donald A.; Welsh, Philip; Alexander, Alice; Robertus, Jon D.; Frankel, Arthur E.

doi:10.1128/mcb.9.11.5012

Cited by 84 publications

(69 citation statements)

References 33 publications

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“…Based on x-ray crystal structure analysis and site-directed mutagenesis studies, Ready et al (18) proposed a mechanism for enzymatic activity of the ricin A chain in which Glu-177 is involved in transition-state stabilization. Consistent with this model is that conversion of negatively charged Glu-177 to Gln reduces activity almost 200-fold (19), while conversion to Ala (E177A) reduces activity 20-fold (20). Kinetic analysis showed that the E177A mutation affects kcat and not Km, consistent with the model that the carboxylate serves in transition-state stabilizaGenetics: Hur et aL tion (20).…”

Section: Discussionsupporting

confidence: 67%

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Hur

Hwang

Zoubenko

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP.The PAP cDNA was placed under control of the galactoseinducible GAL] promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast.The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.Pokeweed antiviral protein (PAP), a ribosome-inactivating protein (RIP) isolated from the leaves or seeds of Phytolacca americana, catalytically removes a specific adenine residue from a highly conserved stem-loop structure in the 28S rRNA of eukaryotic ribosomes (1, 2). This lesion interferes with elongation factor 2 binding and blocks protein synthesis. PAP was discovered when pokeweed extracts were found to inhibit the transmission of tobacco mosaic virus and was subsequently demonstrated to be equally effective against a number of other plant viruses (3). Studies that compare the relative antiviral properties of a number of RIPs showed that all of the RIPs tested had antiviral activity, but none was as effective as PAP (4). PAP has been shown to inhibit infection of both Vero and HeLa cells by herpes simplex virus (5) and to inhibit human immunodeficiency virus 1 (HIV-1) replication in T cells and macrophages infected in vitro at concentrations that do not inhibit cellular protein synthesis (6). A number of recent studies have shown that conjugating PAP with monoclonal antibodies dramatically increases its potency against cells infected with HIV and human cytomegalovirus (7). In addition, PAP has been used as the cytotoxic moiety of immunotoxins against acute lymphoblastic leukemia and PAPcontaining immunotoxins have shown significant antileukemic activity in clinical trials (7).Single-chain RIPs (type I RIPs), like PAP, are poorly characterized at the molecular level compared to the type II RIP, ricin, which consists of an enzymatically active...

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Section: Discussionsupporting

confidence: 67%

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Hur

Hwang

Zoubenko

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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“…Glu177, Arg180, Tyr80, Tyr123, Trp211 and Asn209) [16,17,20]. To date, the principle mechanism of RA action is thought to be as follows.…”

Section: Discussionmentioning

confidence: 99%

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

Kitaoka

1998

European Journal of Biochemistry

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The A chain of ricin (RA) is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine residue at position 4324 in 28S rRNA. Of the 267 amino acids in the protein, 231 could be deleted in one or another of 83 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA [Morris, K. N. & Wool, I. G. (1992) Proc. Natl Acad. Sci. USA 89, 4869Ϫ4873]. Expression of 29 selected deletion mutants of RA in prokaryotic cell-free coupled transcription-translation reactions was carried out and the activities of the mutants were assessed by monitoring depurination of reticulocyte ribosomes. Kinetic analysis of five deletion mutants which retained detectable activity was performed. Deletion of amino acids outside the putative active-site cleft in these mutants significantly affected the catalytic rate rather than the interaction with ribosomes. From these data, the amino acids far from the active-site cleft appeared to be involved in alignment of the key residues for catalysis and interaction with the target tetraloop structure of 28S rRNA.Keywords : ricin; N-glycosidase; cell-free coupled transcription-translation; ribosome; protein synthesis.Ricin is a cytotoxic RNA N-glycosidase that is found in the seeds of Ricinus communis. Proricin is processed by removal of a connecting peptide of 12 amino acids to form an A chain of 267 residues and a B chain of 262 residues linked by a disulfide bond [1]. The toxic ricin A chain (RA) inhibits protein synthesis by inactivating ribosomes ; the inhibition is the result of the hydrolysis of the bond between the base and the ribose of the adenosine at position 4324 (A4324) in 28S rRNA [2,3]. RA is extraordinarily toxic; and a single molecule will inactivate 1500 ribosomes min Ϫ1 ; indeed, a single molecule is sufficient to kill a cell [1] and this one covalent modification accounts entirely for the cytotoxicity.The value of an analysis of the mechanism of action of ribotoxins is that it directs attention to components of the ribosome where efforts to comprehend functional correlates of the structure are likely to be rewarded. That this one RA-catalyzed depurination inactivates the ribosome implies that this region of 28S rRNA is crucial for the function of the particle. There is evidence that the ricin domain is involved in elongation-factor-1-(or Tu)-dependent binding of aminoacyl-tRNA to the ribosomal A site and elongation-factor-2-(or G)-catalyzed GTP hydrolysis and translocation of peptidyl-tRNA to the P site [4].A substantial effort has been made to relate the structure of RA to its mechanism of action spurred in part by the use of the protein in the construction of immunotoxins for cancer therapy [5]. The amino acid sequences of ricin and of several homologous proteins have been determined [6Ϫ12] and an atomic structure of ricin has been obtained from X-ray diffraction of crystals [13Ϫ15]. In the three-dimensional structure, there is a prominent cleft that was proposed to be the active site of RA before the ...

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“…Additionally, as noted by Robertas and co-workers, RTA Glul77 to Gin 177 retains residual RIP activity and it appears to be far less well behaved than RTA in terms of expression levels (Schlossman et al, 1989;Ready et al, 1991).…”

Section: Comparison With Other Ricin Vaccine Candidatesmentioning

confidence: 99%

Finding a New Vaccine in the Ricin Protein Fold

Olson¹,

Carra²,

Roxas-Duncan³

et al. 2004

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Previous attempts to produce a vaccine for ricin toxin have been hampered by safety concerns arising from residual toxicity and the undesirable aggregation or precipitation caused by exposure of hydrophobic surfaces on the ricin A-chain (RTA) in the absence of its natural B-chain partner. We undertook a structure-based solution to this problem by reversing evolutionary selection on the 'ribosome inactivating protein' fold of RTA to arrive at a non-functional, compacted single-domain scaffold (sequence RTA1-198) for presentation of a specific protective epitope (RTA loop 95-110). An optimized protein based upon our modeling design (RTA1-33/44-198) showed greater resistance to thermal denaturation, less precipitation under physiological conditions and a reduction in toxic activity of at least three orders of magnitude compared with RTA. Most importantly, RTA1-198 or RTA1-33/44-198 protected 100% of vaccinated animals against supra-lethal challenge with aerosolized ricin. We conclude that comparative protein analysis and engineering yielded a superior vaccine by exploiting a component of the toxin that is inherently more stable than is the parent RTA molecule.

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Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes.

Cited by 84 publications

References 33 publications

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

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

Finding a New Vaccine in the Ricin Protein Fold

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