Single-chain Ribosome-inactivating Protein Research Articles

Pokeweed (Phytolacca americana L.) antiviral protein inhibits Zucchini yellow mosaic virus infection in a dose-dependent manner in squash plants

Pokeweed antiviral protein (PAP) of Phytolacca americana L. (pokeweed) is a single-chain ribosome-inactivating protein (RIP) characterized by its ability to depurinate plant ribosomes. Here, we isolated, cloned, and expressed the ribosome inactivating protein (RIP) gene, designated as pokeweed antiviral protein type 1 (PAP I), from the summer leaves of pokeweed collected from the Black Sea region (Turkey). Our findings presented here provide direct evidence that exogenous application of PAP I causes concentration-dependent inhibition of Zucchini yellow mosaic virus (ZYMV) infection on squash plants. Squash plants were exposed to PAP I protein with and without DMSO for four consecutive days. Regular spraying of approximately 30 kDa recombinant PAP I at 2 µg mL?1 concentration prevented treated plants from mechanical virus infection. PAP I showed antiviral activity in 9 plants out of 15 inoculated plants. Remarkably, simultaneous application of PAP, DMSO, and ZYMV did not prevent virus infection, suggesting that PAP did not have any effect on viral RNA. In the absence of ZYMV the purified peptide was not cytotoxic for squash plants, although a reduction of plant size, possibly caused by host ribosome depurination, was observed.

Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

Ebulin from dwarf elder (Sambucus ebulus L.): a mini-review.

Sambucus ebulus L. (dwarf elder) is a medicinal plant, the usefulness of which also as food is restricted due to its toxicity. In the last few years, both the chemistry and pharmacology of Sambucus ebulus L. have been investigated. Among the structural and functional proteins present in the plant, sugar-binding proteins (lectins) with or without anti-ribosomal activity and single chain ribosome-inactivating proteins (RIPs) have been isolated. RIPs are enzymes (E.C. 3.2.2.22) that display N-glycosidase activity on the 28S rRNA subunit, leading to the inhibition of protein synthesis by arresting the step of polypeptide chain elongation. The biological role of all these proteins is as yet unknown. The evidence suggests that they could be involved in the defense of the plant against predators and viruses or/and a nitrogen store, with an impact on the nutritional characteristics and food safety. In this mini-review we describe all the isoforms of ebulin that have to date been isolated from dwarf elder, as well as their functional characteristics and potential uses, whilst highlighting concern regarding ebulin toxicity.

Arabidopsis Bax Inhibitor-1 inhibits cell death induced by pokeweed antiviral protein in Saccharomyces cerevisiae.

Apoptosis is an active form of programmed cell death (PCD) that plays critical roles in the development, differentiation and resistance to pathogens in multicellular organisms. Ribosome inactivating proteins (RIPs) are able to induce apoptotic cell death in mammalian cells. In this study, using yeast as a model system, we showed that yeast cells expressing pokeweed antiviral protein (PAP), a single-chain ribosome-inactivating protein, exhibit apoptotic-like features, such as nuclear fragmentation and ROS production. We studied the interaction between PAP and AtBI-1 (Arabidopsis thaliana Bax Inhibitor-1), a plant anti-apoptotic protein, which inhibits Bax induced cell death. Cells expressing PAP and AtBI-1 were able to survive on galactose media compared to PAP alone, indicating a reduction in the cytotoxicity of PAP in yeast. However, PAP was able to depurinate the ribosomes and to inhibit total translation in the presence of AtBI-1. A C-terminally deleted AtBI-1 was able to reduce the cytotoxicity of PAP. Since anti-apoptotic proteins form heterodimers to inhibit the biological activity of their partners, we used a co-immunoprecipitation assay to examine the binding of AtBI-1 to PAP. Both full length and C-terminal deleted AtBI-1 were capable of binding to PAP. These findings indicate that PAP induces cell death in yeast and AtBI-1 inhibits cell death induced by PAP without affecting ribosome depurination and translation inhibition.

Shiga toxin 1 and ricin A chain bind to human polymorphonuclear leucocytes through a common receptor

The main cause of acute renal failure in children is HUS (haemolytic uraemic syndrome), a consequence of intestinal infections with Escherichia coli strains producing Stx (Shiga toxins). Stx released in the gut by the non-invasive bacteria reach the bloodstream and are targeted to cerebral and renal endothelium triggering HUS. PMN (polymorphonuclear leucocytes) seem to be involved in Stx delivery through an unidentified membrane receptor (Kd=10⁻⁸ M; 2×10⁵ binding sites) which does not allow internalization. Some experts in the field have defined the Stx-PMN interaction as non-specific and of little biological significance. In the present study, we show that the A chain of ricin, the well-known plant RIP (ribosome-inactivating protein), interacts with PMN (Kd=10⁻⁹ M; 2×10⁵ binding sites) competing for the same receptor that recognizes Stx, whereas diphtheria toxin and several agonists of TLRs (Toll-like receptors) or the mannose receptor were ineffective. No toxic effects of ricin A chain on PMN were observed, as assessed by measuring protein synthesis and the rate of spontaneous apoptosis of leucocytes. Moreover, two single-chain RIPs (gelonin and saporin S6) had the same competing effect. Thus RIPs and Stx1 share structural similarities, the same enzymatic activity and a common receptor on PMN. These observations reveal that the Stx-PMN interaction is specific, confirming that PMN recognize molecular patterns common to different foreign molecules.

An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo

BackgroundFew treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC), shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs) such as Shiga-like Toxin 1 (SLT-1) represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP) derived from the cytotoxic A subunit of SLT-1 (SLT-1A), harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1AIYSNKLM) allowing the toxin variant to selectively target and kill human melanoma cells.ResultsSLT-1AIYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1AIYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AIYSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1AIYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1AIYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values < 0.001). SLT-1AIYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice.ConclusionsThese results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1AIYSNKLM can specifically kill human melanoma cells in vitro and in vivo.

Ribosome Inactivating Proteins (RIPs) from Momordica charantia for Anti Viral Therapy

This review describes the nature and applications of ribosome inactivating proteins (RIPs) from Momordica charantia (bitter melon). RIPs from the plant kingdom have received much attention in biomedical research because they target conserved host protein synthesis machinery and show specificity towards human and animal cell targets. Recent studies aimed at unravelling the enzymatic activities of the M charantia RIPs provide a structural basis for their activities. It has been reported that RIPs are member of the single chain ribosome inactivating protein (SCRIP) family which act irreversibly on ribosome by removing adenine residue from eukaryotic ribosomal RNA. Various activities of RIPs include anti-tumor, broad anti-viral, ribonuclease and deoxyribonuclease. MAP30 (Momordica Anti-HIV Protein), alpha- and beta-momorcharins inhibit HIV replication in acutely and chronically infected cells and thus are considered potential therapeutic agent in HIV infection and AIDS. Further, MAP30 improved the efficacy of anti-HIV therapy when used in combination with other anti-viral drugs. MAP30 holds therapeutic promise over other RIPs because not only it is active against infection and replication of both HSV and HIV but is non toxic to normal cells. Here we review the nature, action, structure function relationship and applications of RIPs from Momordica charantia and evaluate their potential for anti-cancer and anti-viral therapy.

Different neuronal toxicity of single-chain ribosome-inactivating proteins on the rat retina

Objective To investigate the neurotoxicity of two structurally similar single chains of ribosome-inactivating proteins (RIPs): trichosanthin (TCS) and ricin A chain (RTA). Methods TCS, RTA and Ricinus communis agglutinin (RCA, a multi-chain RIP for comparison) were separately injected into rat eyes. Saline was used as control. The data on cell counts, retinal thickness and histopathological scores were collected, and the TUNEL method (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) was used to study the mode of cell death. Results TCS caused distinct retinal changes at 1 nmol. Its toxic effects were most pronounced on the cells of the outer nuclear layer, less so on those of the inner nuclear layer, and little on the ganglion cells. Apoptosis was the predominant type of cell death induced by TCS. In contrast, RTA and RCA, both at 0.01 nmol, brought about acute retinal inflammation and necrosis. Conclusion TCS can eliminate specific retinal cells by apoptosis, while RTA and RCA cause retinitis. The B chain of type II RIPs is not obligatory for their neurotoxicity. The RIPs may be useful for creating retinal models and TCS may be useful for the chemical treatment of retinoblastoma.

Expression of a ribosome-inactivating protein gene in bitter melon is induced by Sphaerotheca fuliginea and abiotic stimuli

The gene encoding a single-chain, ribosome-inactivating protein (SCRIP) was cloned from bitter melon (Momordica charantia L.) leaves infected with the fungus, Sphaerotheca fuliginea, by RT-PCR. The ORF was 861 bp. The ribosome-inactivating protein was expressed in E. coli and, when purified, it inhibited the growth of Fusarium solani [corrected] Northern blot analysis revealed that RIP transcripts rapidly accumulated in leaves 1-day post inoculation with Sphaerotheca fuliginea and reached a peak at 3 d. The expression pattern of RIP induced by methyl jasmonate and salicylic acid were different from that of pathogen-induced expression. Mechanical wounding, silver nitrate and osmotic stress stimulated only a slight accumulation of RIP transcripts. Abscisic acid also induced transcription of RIPs. The signal compounds, ethylene and okadaic acid, induced a moderate accumulation of RIP transcripts.

The C‐terminus of pokeweed antiviral protein has distinct roles in transport to the cytosol, ribosome depurination and cytotoxicity

Pokeweed antiviral protein (PAP) produced by pokeweed plants is a single-chain (type I) ribosome-inactivating protein (RIP) that depurinates ribosomes at the alpha-sarcin/ricin loop of the large rRNA, resulting in inhibition of translation. Unlike the type II RIPs, which have an active and a binding moiety, PAP has only the active moiety. The mechanism by which toxins without a binding moiety gain access to cytosolic ribosomes is not known. We set up yeast as a simple and genetically tractable system to investigate how PAP accesses ribosomes and showed that the mature form of PAP is targeted to the cytosol from the endomembrane system in yeast. In the present study, we performed a systematic deletion analysis to identify the signal required for transport of PAP to the cytosol. We demonstrate here that processing of the C-terminal extension and sequences at the C-terminus of the mature protein are critical for its accumulation in the cytosol. Using a series of PAP mutants, we identified the C-terminal signal and demonstrated that it is distinct from the sequences required for ribosome depurination and cytotoxicity. The C-terminal motif showed sequence similarity to type II RIPs that retrotranslocate from the endoplasmic reticulum to the cytosol. These results demonstrate that a conserved sequence at the C-terminus of a type I RIP mediates its transport to the cytosol and suggest that type I and II RIPs may use a common signal to enter the cytosol.

Truncations of gelonin lead to a reduction in its cytotoxicity

Gelonin is a single chain ribosome-inactivating protein (RIP) with potential applications as a bullet of immunoconjugate for the treatment of cancer and AIDS. Using truncated forms of gelonin, we now report the relationship between its conformation and function. Circular dichroism (CD) and fluorescence spectra show that the N-terminus forms beta-sheets whereas the C-terminus contains alpha-helices of secondary structures. Biological experiments indicate that all gelonin truncation mutants lose partial toxicity compared to intact gelonin, an effect most strongly seen with C-terminally truncated gelonin. Similar evidence is also provided using a DNase-like activity assay. In addition, the intact gelonin exhibits the highest cytotoxicity to cancer cells. These results suggest that truncations of the terminal region of gelonin negatively regulate its function dominantly and that, due to its toxicity, intact gelonin is an important potential immunoconjugate.

N‐terminal deletion affects catalytic activity of saporin toxin

Single-chain ribosome inactivating proteins (RIPs) are cytotoxic components of macromolecular pharmaceutics for immunotherapy of cancer and other human diseases. Saporin belongs to a family of single-chain RIPs sharing sequence and structure homology. In a preliminary attempt to define an active saporin polypeptide of minimum size we have generated proteins with deletions at the N-terminus and at the C-terminus. An N-terminal (sapDelta1-20) deletion mutant of saporin displayed defective catalytic activity, drastically reduced cytotoxicity but increased ability to interact with liposomes inducing their permeabilization at low pH. A C-terminal (sapDelta239-253) deletion mutant showed instead a moderate reduction in cytotoxic activity. A substantial alteration of secondary structure was evidenced by Fourier transformed infrared spectroscopy (FTIR) in the sapDelta1-20 mutant. It can be hypothesized that the defective functions of sapDelta1-20 are due to alterations of its spatial configuration.

Molecular characterization and systemic induction of single-chain ribosome-inactivating proteins (RIPs) in sugar beet (Beta vulgaris) leaves

Sugar beet (Beta vulgaris L.) leaves contain virus-inducible type 1 (single chain) ribosome-inactivating proteins that have been named beetins. The structural and functional characterization, the cellular location, and the potential role of beetins as antiviral agents are reported here. Beetins are formed of a single polypeptide chain with a varying degree of glycosylation and strongly inhibited in vitro protein synthesis in rabbit reticulocyte lysates (IC50=1.15 ng ml(-1)) and a Vicia sativa L. cell-free system (IC50=68 ng ml(-1)) through the single depurination of the large rRNA. Beetins trigger the multidepurination of tobacco mosaic virus (TMV) genomic RNA which underwent extensive degradation upon treatment with acid aniline. Beetins are extracellular proteins that were recovered from the apoplastic fluid. Induction of sugar beet RIPs with either H2O2 or artichoke mottled crinkle virus (AMCV) was observed in leaves distant from the site of application of such elicitors. The external application of purified beetin to sugar leaves prevented infection by AMCV which supports the preliminary hypothesis that beetins could be involved in plant systemic acquired resistance subjected to induction by phytopathogens.

Mitochondrial DNA D-loop as a new target of Saporin 6 nuclease activity

The single-chain ribosome-inactivating proteins (RIPs) from plant origin, including Saporin 6 from the seeds of Saponaria officinalis, are ribotoxins known to act as N-glycosidases which depurinate the conserved alpha sarcin loop of large rRNAs. As a consequence, the eukaryotic ribosomes become inactivated, thereby arresting the protein synthesis at the elongation step. RIPs are currently under study as antiviral and antiproliferative agents. Additional in vitro activities of RIPs against either RNA or DNA have been recently described. A specific nuclease activity on plasmidic DNA was demonstrated by either purified or bacterial-recombinant molecules. We report here that human mitochondrial DNA (mtDNA) is a new specific target of Saporin 6 nuclease activity. A unique site of cleavage has been identified and mapped within the most variable part of the D-loop region of the covalently closed circular mtDNA molecule.

Cytotoxicity of ribosome-inactivating protein saporin is not mediated through α 2-macroglobulin receptor

Saporin is a single chain ribosome-inactivating protein produced by the plant Saponaria officinalis. Several isoforms of saporin have been isolated from various parts of the plant. In the present study recombinant saporin isoforms 5 and 6 were produced in Escherichia coli. Saporin-6 was found to be more active than saporin-5 in its N-glycosidase, cytotoxic, and genomic DNA fragmentation activities. Earlier, saporin has been shown to bind low-density lipoprotein receptor-related protein (LRP), however, in this study the sensitivities of LRP-negative and LRP-positive cell lines were found to be similar towards saporin-6 toxicity suggesting the internalization of saporin not to be solely dependent on the expression of LRP on eukaryotic cells.

The Cytotoxic Activity of Ribosome-inactivating Protein Saporin-6 Is Attributed to Its rRNA N-Glycosidase and Internucleosomal DNA Fragmentation Activities

Saporin-6 produced by the plant Saponaria officinalis belongs to the family of single chain ribosome-inactivating proteins. It potently inhibits protein synthesis in eukaryotic cells, by cleaving the N-glycosidic bond of a specific adenine in 28 S rRNA, which results in the cell death. Saporin-6 has also been shown to be active on DNA and induces apoptosis. In the current study, we have investigated the roles of rRNA depurination and the activity of saporin-6 on genomic DNA in its cytotoxic activity. The role of putative active site residues, Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208), and two invariant residues, Tyr(16) and Arg(24), proposed to be important for structural stability of saporin-6, has been investigated in its catalytic and cytotoxic activity. These residues were mutated to alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A. We show that for the RNA N-glycosidase activity of saporin-6, residues Tyr(16), Tyr(72), and Arg(179) are absolutely critical; Tyr(120) and Glu(176) can be partially dispensed with, whereas Trp(208) and Arg(24) do not appear to be involved in this activity. The residues Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208) were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr(16) and Arg(24) do not appear to be required for the DNA fragmentation. The study shows that saporin-6 possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required.

Pokeweed Antiviral Protein Regulates the Stability of Its Own mRNA by a Mechanism That Requires Depurination but Can Be Separated from Depurination of the α-Sarcin/Ricin Loop of rRNA

Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, alpha-sarcin/ricin loop in the large rRNA, resulting in inhibition of protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeast Saccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to the N-glycosidase activity of the protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs.

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.

Musarmins: three single-chain ribosome-inactivating protein isoforms from bulbs of Muscari armeniacum L. and Miller

Three new ribosome-inactivating protein (RIP; EC 3.2.2.22) isoforms that we have named musarmins (MUs) 1, 2 and 3 have been isolated from the bulbs of Muscari armeniacum L. and Miller by ion-exchange chromatography and gel filtration. Analysis by electrophoresis revealed that they are single-chain proteins and mass spectrometry analysis afforded M r values of 28,708, 30,003 and 27,626 for MUs 1, 2 and 3, respectively. Musarmins strongly inhibited protein synthesis carried out by mammalian ribosomes, with IC 50 values in the 0.14–0.24 nM range but not that carried out by plant cell-free systems or HeLa cells. MUs promote the single depurination of rabbit reticulocyte 28 S rRNA. cDNA cloning of genes coding for musarmins revealed that they contain open reading frames of 298, 294 and 295 aminoacids for MU1, MU2 and MU3, respectively. Mature MU1, MU2 and MU3 contain 277, 273 and 273 aminoacids, respectively suggesting post-translational C-terminal processing. An untranslated mRNA coding for an ORF very similar to that of MU3 was detected in leaves. Each of the four MU genes contains an intron. In contrast to other RIPs, MUs are present only in bulbs and are not induced in leaves either by senescence, or by treatment of leaves with H 2O 2 or salicylic acid, or by growth in darkness. Therefore, these proteins could play a non-vital role in plants; for instance, as anti-pathogens and protective agents only in some stages of the plant life cycle (237).

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.