A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ

A new enzyme, which we named ribosomal RNA apurinic site specific lyase (RALyase), is described. The protein was found in wheat embryos and has a molecular weight of 50 625 Da. The enzyme specifically cleaves the phosphodiester bond at the 3' side of the apurinic site introduced by ribosome-inactivating proteins into the sarcin/ricin domain of 28S rRNA. The 3' and 5' ends of wheat 28S rRNA at the cleavage site are 5'-GUACG-alpha-hydroxy-alpha, beta-unsaturated aldehyde and pGAGGA-3', demonstrating that the enzyme catalyzes a beta-elimination reaction. The substrate specificity of the enzyme is extremely high: it acts only at the apurinic site in the sarcin/ricin domain of intact ribosomes, not on deproteinized rRNA or DNA containing apurinic sites. The amino acid sequences of five endopeptidase LysC-liberated peptides from the purified enzyme were determined and used to obtain a cDNA sequence. The open reading frame encodes a protein of 456 amino acids, and a homology search revealed a related rice protein. Similar enzyme activities were also found in other plants that express ribosome-inactivating proteins. We believe that RALyase is part of a complex self-defense mechanism.     

Protein toxin inhibitors of protein synthesis.

Two classes of extremely toxic proteins kill eukaryotic cells by covalently modifying unique structural features of components that are essential for protein synthesis. Intoxication by these proteins results from the entry of a catalytic fragment into the cytoplasm. One class is typified by diphtheria toxin and Pseudomonas exotoxin A. The catalytic component of these toxins ADP-ribosylates and inactivates elongation factor 2 which is an essential participant in protein synthesis. This modification occurs at a unique post-translational histidine derivative, diphthamide, that is present in the ribosomal binding site of the elongation factor. The two toxins differ in their molecular organization but appear to possess identical reaction mechanisms and very similar active sites. The other class contains two types of toxins typified, respectively, by alpha-sarcin, a member of a family of fungal toxins, and ricin, a member of a group of closely related plant proteins collectively termed ribosome-inactivating proteins. The catalytic components of the two types of toxins in this second class inactivate the large ribosomal subunit through two different hydrolytic alterations of 23-28S RNA. alpha-Sarcin and its congeners act as a specific endonuclease whereas ricin and its congeners act as a specific N-glycosidase. These hydrolytic cleavages occur at a pair of adjacent nucleotides within a highly conserved sequence near the 3' terminus of 23-28S RNA. The covalent integrity of this region of RNA is essential to elongation factor-dependent ribosomal functions and is located within the ribosomal binding domain of these factors. Both of these classes of toxins are being employed as 'magic bullets' to eliminate pathological cells. By combining the catalytic component of these toxins with various cell targeting components, useful and specific anticancer and immunomodulatory agents have been created.     

The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes: the RNA N-glycosidase activity of the proteins

The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes was studied. Treatment of ribosomes with any one of these proteins caused the 28S rRNA extracted from the inactivated ribosomes to become sensitive to treatment with aniline. A fragment containing about 450 nucleotides was released from the 28S rRNA. Further analysis of the nucleotide sequences of the 450-nucleotide fragments revealed that the aniline-sensitive phosphodiester bond was between A-4324 and G-4325 of the 28S rRNA. These results indicate that all six ribosome-inactivating proteins damage eukaryotic ribosomes by cleaving the N-glycosidic bond at A-4324 of the 28S rRNA of the ribosomes, as does ricin A-chain.     

Ribonuclease activity associated with the 60S ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin

All purified preparations of the ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin were shown to exhibit ribonuclease activity with 5S or 5.8S rRNA substrates. These toxin species generated reproducible patterns of RNA fragments distinct for each toxin species while multiple preparations of a single toxin species yielded similar RNA fragment patterns. The heat inactivation profile of Shiga toxin was identical for its RNase and protein synthesis inhibitory activities. These data are the first to indicate that the ribosome-inactivating catalytic toxins, in addition to alpha-sarcin, exhibit RNase activity. These results suggest RNase activity may be responsible for ribosome-inactivation catalyzed by ricin, phytolaccin and Shiga toxin proteins.     

Isolation and enzymatic characterization of lamjapin, the first ribosome-inactivating protein from cryptogamic algal plant (Laminaria japonica A).

Lamjapin, a novel type Iota ribosome-inactivating protein, has been isolated from kelp (Laminaria japonica A), a marine alga. This protein has been extensively purified through multiple chromatography columns. With a molecular mass of approximately 36 kDa, lamjapin is slightly larger than the other known single-chain ribosome-inactivating proteins from the higher plants. Lamjapin can inhibit protein synthesis in rabbit reticulocyte lysate with an IC50 of 0.69 nm. It can depurinate at multiple sites of RNA in rat ribosome and produce the diagnostic R-fragment and three additional larger fragments after the aniline reaction. Lamjapin can deadenylate specifically at the site A20 of the synthetic oligoribonucleotide (35-mer) substrate that mimics the sarcin/ricin domain (SRD) of rat ribosomal 28S RNA. However, it cannot hydrolyze the N-C glycosidic bond of guanosine, cytidine or uridine at the corresponding site of the A20 of three mutant SRD RNAs. Lamjapin exhibits the same base and position requirement as the ribosome-inactivating proteins from higher plants. We conclude that lamjapin is an RNA N-glycosidase that belongs to the ribosome-inactivating protein family. This study reports for the first time that ribosome-inactivating protein exists in the lower cryptogamic algal plant.     

Non-specific deadenylation and deguanylation of naked RNA catalyzed by ricin under acidic condition.

Ricin A-chain catalyzes the hydrolysis of the N-glycosidic bond of a conserved adenosine residue at position 4324 in the sarcin/ricin domain of 28S RNA of rat ribosome. The GAGA tetraloop closed by C-G pairs is required for recognition of the cleavage site on 28S ribosomal RNA by ricin A-chain. In this study, ricin A-chain (reduced ricin) exhibits specific depurination on a synthetic oligoribonucleotide (named SRD RNA) mimic of the sarcin/ricin domain of rat 28S ribosomal RNA under neutral and weak acidic conditions. Furthermore, the activity of intact ricin is also similar to that of ricin A-chain. However, under more acidic conditions, both enzymes lose their site specificity. The alteration in specificity of depurination is not dependent on the GAGA tetraloop of SRD RNA. A higher concentration of KCl inhibits the non-specific N-glycosidase activity much more than the specific activity of ricin A-chain. In addition, characterization of depurination sites by RNA sequencing reveals that under acidic conditions ricin A-chain can release not only adenines, but also guanines from SRD RNA or 5S ribosomal RNA. This is the first report of the non-specific deadenylation and deguanylation activity of ricin A-chain to the naked RNA under acidic conditions.     

The mechanism of action of barley toxin: a type 1 ribosome-inactivating protein with RNA N-glycosidase activity

In a previous report (Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130) it was shown that the RNA N-glycosidase activity of ricin A-chain was responsible for the ability of this protein to inactivate eukaryotic ribosomes. The objective of the present study was to determine whether a similar mechanism was used by a ribosome-inactivating protein from pearled barley (barley toxin). Rat liver ribosomes were incubated either with ricin A-chain or barley toxin, and the rRNA was extracted and treated with acidic aniline to hydrolyze phosphodiester bonds rendered susceptible by removal of a purine or pyrimidine base. Evaluation of the rRNA by polyacrylamide/agarose electrophoresis disclosed two 28 S rRNA-derived fragments which differed in size from those generated by untreated (control) ribosomes. Sequencing of the smaller of these fragments confirmed that - as is the case for ricin A-chain - the aniline-sensitive site in barley toxin-treated ribosomes was between A and G in 28 S rRNA. We conclude that barley toxin inactivates ribosomes via a mechanism identical to that of ricin A-chain: enzymatic hydrolysis of the N-glycosidic bond at A of 28 S rRNA.     

Toxicity of two type II ribosome-inactivating proteins (cinnamomin and ricin) to domestic silkworm larvae

Cinnamomin and ricin are two type II ribosome-inactivating proteins. They exhibited a different toxicity to domestic silkworm (Bombyx mori) larvae by oral feeding bioassay. The LC50 of ricin to the silkworm larvae at third instar was much lower than that of cinnamomin. When the isolated 80S ribosome from domestic silkworm pupae was treated separately with the reduced cinnamomin or the reduced ricin, a specific RNA fragment (R-fragment) was produced as characterized by 8 M urea-denatured polyacrylamide gel (3.5%) electrophoresis. The purified A-chains of both cinnamomin and ricin showed a slightly different RNA N-glycosidase activity to the domestic silkworm pupal ribosome. It was proposed that the difference of their toxicity to domestic silkworm larvae was not related to their A-chains but to the properties of their B-chains. It was also found that the vomit obtained from the midgut of domestic silkworm larvae could hydrolyze these two proteins apparently to a similar extent.     

Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system.     

Non-specific depurination activity of saporin-S6, a ribosome-inactivating protein, under acidic conditions

Among five ribosome-inactivating proteins tested only saporin-S6 could efficiently release the adenine from adenosine 20 of the synthetic oligoribonucleotide (SRD RNA) mimic of the sarcin/ricin domain of rat 28S rRNA with a Km of 9 microM and a kcat of approximately 0.4 min(-1) at pH 7.6. The optimal pH for the depurination activity of saporin-S6 is 5.0. However, saporin-S6 lost its site-specificity of depurination on SRD RNA around the optimal pH. The non-specific depurination activity of saporin-S6 was dependent on the enzyme concentration and pH conditions. These results are valuable to understand the diversity and the depurination mechanism of ribosome-inactivating proteins.     

The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins

Ricin is a potent cytotoxic protein derived from the higher plant Ricinus communis that inactivates eukaryotic ribosomes. In this paper we have studied the mechanism of action of ricin A-chain on rat liver ribosomes in vitro. Our findings indicate that the toxin inactivates the ribosomes by modifying both or either of two nucleoside residues, G4323 and A4324, in 28 S rRNA. These nucleotides are located close to the alpha-sarcin cleavage site and become resistant to all ribonucleases tested. The examination of the lability of phosphodiester bonds of these nucleotides to both mild alkaline digestion and aniline treatment at acidic pH suggests that the base of A4324 is removed by the toxin. This unique activity of ricin A-chain was also observed when naked 28 S rRNA is used as a substrate, indicating that the toxin directly acts on the RNA. Similar activity on 28 S rRNA is also exhibited by abrin and modeccin, ricin-related toxins, suggesting a general mechanistic pathway for ribosome inactivation by lectin toxins.     

Cytotoxic ribosome-inactivating lectins from plants

A class of heterodimeric plant proteins consisting of a carbohydrate-binding B-chain and an enzymatic A-chain which act on ribosomes to inhibit protein synthesis are amongst the most toxic substances known. The best known example of such a toxic lectin is ricin, produced by the seeds of the castor oil plant, Ricinnus communis. For ricin to reach its substrate in the cytosol, it must be endocytosed, transported through the endomembrane system to reach the compartment from which it is translocated into the cytosol, and there avoid degradation making it possible for a few molecules to inactivate a large proportion of the ribosomes and hence kill the cell. Cell entry by ricin involves the following steps: (i) binding to cell-surface glycolipids and glycoproteins bearing beta-1,4-linked galactose residues through the lectin activity of the B-chain (RTB); (ii) uptake by endocytosis and entry into early endosomes; (iii) transfer by vesicular transport to the trans-Golgi network; (iv) retrograde vesicular transport through the Golgi complex and into the endoplasmic reticulum (ER); (v) reduction of the disulfide bond connecting the A- and B-chains; (vi) a partial unfolding of the A-chain (RTA) to enable it to translocate across the ER membrane via the Sec61p translocon using the pathway normally followed by misfolded ER proteins for targeting to the ER-associated degradation (ERAD) machinery; (vi) refolding in the cytosol into a protease-resistant, enzymatically active structure; (vii) interaction with the sarcin-ricin domain (SRD) of the large ribosome subunit RNA followed by cleavage of a single N-glycosidic bond in the RNA to generate a depurinated, inactive ribosome. In addition to the highly specific action on ribosomes, ricin and related ribosome-inactivating proteins (RIPs) have a less specific action in vitro on DNA and RNA substrates releasing multiple adenine, and in some instances, guanine residues. This polynucleotide:adenosine glycosidase activity has been implicated in the general antiviral, and specifically, the anti HIV-1 activity of several single-chain RIPs which are homologous to the A-chains of the heterodimeric lectins. However, in the absence of clear cause and effect evidence in vivo, such claims should be regarded with caution.     

Mechanism and site of action of a ribosome-inactivating protein type 1 from Dianthus barbatus which inactivates Escherichia coli ribosomes.

A single chain ribosome-inactivating protein with RNA N-glycosidase activity, here named Dianthin 29, was isolated from leaves of Dianthus barbatus L. Incubation of intact Escherichia coli ribosomes with Dianthin 29 and subsequent aniline treatment of the isolated rRNA releases a rRNA fragment of 243 nucleotides from 23 S rRNA. Nucleotide sequence studies showed that the site of N-glycosidic bond cleavage is at A-2660 within the universally conserved sequence 5'-AGUACGAGAGGA-3' near the 3'-end of 23/28 S rRNAs. To our knowledge, Dianthin 29 is the first ribosome-inactivating protein which is shown to inactivate intact prokaryotic ribosomes in the same manner as eukaryotic ribosomes.     

RIP and RALyase cleave the sarcin/ricin domain, a critical domain for ribosome function, during senescence of wheat coleoptiles

Type-I ribosome-inactivating protein (RIP), which is found in many plants, catalyzes depurination of a specific adenine in the sarcin/ricin domain (SRD) of the large rRNA causing loss of ribosomal activity. Previously, we found a RNA apurinic site-specific lyase (RALyase) that catalytically cleaved the phosphodiester bond at the RIP-dependent depurination site by β-elimination reaction. Here we show that both the RIP activity and RIP-RALyase-mediated cleavage of SRD in the cytoplasmic ribosome were induced at the late stage of senescence of wheat coleoptiles. Following this process, tissue death was observed. Furthermore, transgenic tobacco plants expressing glucocorticoid-induced RIP developed senescence-like phenotype. Our results suggest that ribosome inactivation due to the cleavage of SRD by the inducible RIP and constitutively expressed RALyase may be a unique plant system that mediates programmed cell death at the late senescent stage.     

Isolation of Chinese hamster ovary ribosomal mutants differentially resistant to ricin, abrin, and modeccin

The molecular action of ricin A chain involves cleavage of the N-glycosidic bond between ribose and the adenine 4324 nucleotides from the 5' end of mammalian 28 S rRNA (Endo, Y., and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130). In this paper, four ricin- and abrin-resistant Chinese hamster ovary cell mutants that possess ribosomes resistant to this N-glycosidase action are described. Three of the mutant phenotypes, Lec26, Lec27, and Lec28, were recessive in somatic cell hybrids and define at least two new lectin-resistant complementation groups. The most extensively characterized mutant type, LEC17, was dominant in such hybrids. None of the mutants were cross-resistant to modeccin. Post-mitochondrial supernatants from each of the four mutants were resistant to inhibition of cell-free protein synthesis by ricin, ricin A chain, and abrin. In addition, polysomes isolated from mutant cells were resistant to cleavage of the adenine-ribose N-glycosidic bond by ricin A chain or abrin, as assayed by the release of an approximately 470-nucleotide fragment following aniline treatment of ribosomal RNA extracted from toxin-treated polysomes. The unique lectin-resistance properties of the different mutants suggests that the accessibility of adenine 4324 to each toxin differs. It seems likely that the recessive Chinese hamster ovary ribosomal mutants reflect structural changes in different ribosomal proteins while the dominant phenotype may be due to the modification of protein(s) or rRNA involved in toxin-ribosome interaction. Further analysis of these cell lines should provide new insights into the structure/function relationships of eukaryotic ribosomes.     

Features of the modulating effect of complexes of ribosome-inactivating proteins type II with sugars on respiratory burst of neturophils, caused by a chemotactic peptide

It was shown that agents inducing phagocytosis (zymosan, lectins) cause changes in the number of receptors responsible for fast neutrophil reaction (chemotaxis or respiratory burst) or inhibit the binding of the agonist to its receptor. Among lectins are ribosome-inactivating proteins of type II ricin and agglutinin ricin, which penetrate the cell by binding to mannose and galactose receptors. It was shown that ribosome-inactivating proteins of type II can exhibit the properties of the antagonist of the receptor N-formylmethionylleucylphenylalanine. Ricin is more effective in modulating the respiratory burst induced by the chemotactic peptide than agglutinin ricin. The modulating effect of ribosome-inactivating proteins of type II on neutrophils is likely to be mediated by their interaction with galactose rather than mannose receptors. Presumably, the affinity of ribosome-inactivating proteins to galactose receptors increases with increasing amount of saccharides bound to the protein molecule. The modulating effect of ribosome-inactivating proteins of type II on the respiratory burst of neutrophils induced the chemotactic peptide is due to the structural peculiarities of these proteins.     

Nonspecific deadenylation on sarcin/ricin domain RNA catalyzed by gelonin under acidic conditions

Gelonin is a single-chain ribosome-inactivating protein that can hydrolyze the glycosidic bond of a highly conserved adenosine residue in the sarcin/ricin domain (SRD) of the largest RNA in ribosome and thus irreversibly inhibit protein synthesis. Recently, the specificity in substrate recognition was challenged by the fact that gelonin could remove adenines from some other oligoribonucleotide substrates. However, the site specificity of gelonin to deadenylate various substrates were unknown. Hereby, the effect of pH values upon site specificity of the deadenylation activity of gelonin was studied using the synthetic oligoribonucleotide (named SRD RNA) that mimicked the ribosomal SRD. Interestingly, gelonin gradually acquired the ability to nonspecifically remove adenines from SRD RNA when pH values changed from neutral to acidic conditions. Another two SRD RNA mutants, either with the conserved adenosine deleted or with the tetraloop converted, showed very similar cleavage style to wild-type SRD RNA, underscoring the important role of pH value in site specificity of recognition by gelonin. Furthermore, the RNA N-glycosidase activity of gelonin was also enhanced with the decreasing of pH values. In addition, no obvious change was observed in the molecular conformation of gelonin at various pH values. Taken together, our data implied that the protonation of adenosines in SRD RNA was potentially an important factor for the nonspecific deadenlyation by gelonin.     

Primary amino acid sequence of alpha-trichosanthin and molecular models for abrin A-chain and alpha-trichosanthin

Ricin A-chain, abrin A-chain, and alpha-trichosanthin are members of a larger group of proteins called ribosome-inactivating proteins. These proteins all function to catalytically inactivate eukaryotic 60 S ribosomal subunits leading to rapid shutdown of protein synthesis. They are homologous in sequence and are probably evolutionarily related. We have determined the complete primary amino acid sequence of alpha-trichosanthin and have found it to be homologous, as expected, to that of abrin A-chain and ricin A-chain. A crystal structure for ricin, which includes ricin A-chain and ricin B-chain, has been determined from x-ray diffraction data. Based on the sequence homologies of these proteins, we fit the primary sequences of abrin A-chain and alpha-trichosanthin to the backbone structure for ricin A-chain and have generated energy-minimized molecular models for them. These models should prove useful in studying the structural-functional relationships of these proteins in particular and of the class in general.