RNA N-glycosidase Research Articles

Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 A resolution and refined to R factors of 20.9% (Rfree = 23.6%) and 20.9 (Rfree = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains alpha1 and gamma2 of the ML-I B-chain separated by approximately 62 A from each other. The favoured binding of galactose in subdomain alpha1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar-lectin complex. In the galactose-binding site II of subdomain gamma2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar-protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit beta1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound.

The 1.4 Å structure of dianthin 30 indicates a role of surface potential at the active site of type 1 ribosome inactivating proteins

Ribosome inactivating proteins (RIPs) are plant proteins with enzymatic activity identified as rRNA N-glycosidase (EC 3.2.2.22), which cleaves the N-glycosidic bond of a specific adenine on the ricin/sarcin region of rRNA, thus causing inhibition of protein synthesis. They also depurinate extensively DNA and other polynucleotides. The three-dimensional structure of dianthin 30, a type 1 (single-chain) RIP of Dianthus caryophyllus (leaves), is now described at 1.4 Å, a resolution never achieved before for any RIP. The fold typical of RIPs is conserved, despite some differences in the loop regions. The general structure comparison by superimposed α-carbon (249 atoms) and the sequence alignment by structure for dianthin 30 and saporin-S6 give a root mean square deviation of 0.625 Å. Despite the differences reported for the biological activities of the two RIPs, their structures fit quite well and both show a protein segment containing strands β7, β8, and β9 shorter than other RIPs. However, the surface electrostatic potential in the active site region neatly distinguishes dianthin 30 from saporin-S6. The possible relationship between the charge distribution and the behavior of the proteins toward different substrates is discussed.

Effect of N- and C-terminal deletions on the RNA N-glycosidase activity and the antigenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

Karasurin-A, from root tubers of Trichosanthes kirilowii var. japonica, is a type I ribosome-inactivating protein (RIP) that displays activity of RNA N-glycosidase to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in the ribosome. We expressed recombinant proteins of karasurin-A and its various mutants with N- or C-terminal deletions in Escherichia coli as fusion proteins with maltose-binding protein (MBP), and compared their enzymatic activities and antigenicities. Muteins of karasurin-A generated by deleting either the first 100 N-terminal or the last 30 C-terminal amino acid residues lost activity of RNA N-glycosidase. The mutant proteins whose 80 N-terminal or 20 C-terminal amino acids were deleted could depurinate rRNA although the activities were decreased drastically. The antigenicities of the recombinant proteins were considerably reduced by deleting 20 amino acid residues from either N- or C-terminal regions.

Trichomislin, a novel ribosome-inactivating protein, induces apoptosis that involves mitochondria and caspase-3

Trichomislin, a novel ribosome-inactivating protein, was cloned from the genome of Trichosanthes kirilowii Maxim. The gene was recombined to prokaryotic expression vector and the protein was purified by cation-exchange chromatography. The secondary structure of trichomislin was measured by circular-dichroism analysis and the ratios of α-helices and β-sheets were calculated. Trichomislin could inhibit the synthesis of protein in rabbit reticulocyte lysate systems and its reaction mechanism was to inactivate ribosome as an rRNA N-glycosidase. Antitumor analyses indicated trichomislin induced the apoptosis and inhibited the growth of choriocarcinoma cells. Further investigation showed that trichomislin could bind to and enter choriocarcinoma cells, and then increase the caspase-3 activity in a time-dependent manner. At the same time, the concentration of cytochrome c in cytosol increased while that in mitochondria decreased. These results suggested that trichomislin induced apoptosis by releasing cytochrome c from mitochondria which then triggered the caspase family member activation.

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.

The N-Glycosidase Activity of the Ribosome-inactivating Protein ME1 Targets Single-stranded Regions of Nucleic Acids Independent of Sequence or Structural Motifs

ME(1), a type I ribosome-inactivating protein (RIP), belongs to a family of enzymes long believed to possess rRNA N-glycosidase activity directed solely at the universally conserved residue A4324 in the sarcin/ricin loop of large eukaryotic and prokaryotic rRNAs. We have investigated the effect of modifying the structure of nonribosomal RNA substrates on their interaction with ME(1) and other RIPs. ME(1) was shown to depurinate a variety of partially denatured nucleic acids, randomly removing adenine residues from single-stranded regions and, to a lesser extent, guanine residues from wobble base-pairs in hairpin stems. A defined sequence motif was not required for recognition of non-paired adenosines and cleavage of the N-glycosidic bond. Substrate recognition and ME(1) activity appeared to depend on the physical availability of nucleotides, and denaturation of nucleic acid substrates increased their interaction with ME(1). Pretreatment of mRNA at 75 degrees C rather than 60 degrees C, for example, lowered the apparent K(D) from 87.1 to 73.9 nm, making it more vulnerable to depurination by RIPs. Exposure to ME(1) in vitro completely abolished the infectivity of partially denatured RNA transcripts of the potato spindle tuber viroid, suggesting that RIPs may target invading nucleic acids before they reach host ribosomes in vivo. Our data suggest that the extensive folding of many potential substrates interferes with their ability to interact with RIPs, thereby blocking their inactivation by ME(1) (or other RIPs).

Bacterial expression of biologically active recombinant musarmin 1 from bulbs of Muscari armeniacum L. and Miller

Musarmins are type 1 ribosome-inactivating proteins with N-glycosidase activity on the 28 S rRNA that are present in bulbs of Muscari armeniacum L. and Miller at rather low concentrations. In the present work, a cDNA fragment coding for musarmin 1 was sub-cloned and expressed in Escherichia coli. The recombinant protein (rMU1) was synthesised as a polypeptide of 295 amino acids that was delivered to the periplasm and processed. Recombinant musarmin 1 present in the periplam has two forms: insoluble with a molecular mass of 29,423 and soluble with a molecular mass of 29,117 because of a small proteolytic shortening with respect to the insoluble one, presumably in the C-terminal. The yield of protein homogeneous by polyacrylamide gel electrophoresis was 23 mg l −1 of bacterial culture. The recombinant musarmin 1 forms isolated from both the soluble and the insoluble (upon refolding) fractions retained full translational inhibitory and 28 S rRNA N-glycosidase activities as compared with the native protein. The recombinant protein displayed great stability towards trypsin, collagenase, rat plasma and rat liver protein extract, but was sensitive to the action of papain and proteinase K. The easy availability and full activity of the recombinant musarmin 1 makes it a good candidate for the preparation of immunotoxins for targeted therapy and for the construction of transgenic plants expressing it as antipathogenic agent.

Ricin Triggers Apoptotic Morphological Changes through Caspase-3 Cleavage of BAT3

Ricin, one of type II ribosomal inactivating proteins, inhibits protein biosynthesis by its RNA N-glycosidase activity. By yeast two-hybrid screening, the human BAT3 (HLA-B-associated transcript 3) was isolated as a ricin A-chain interacting protein. A canonical caspase-3 cleavage site, DEQD(1001) was found at the C-terminal region of BAT3. Ricin induced the apoptosis by activating caspase-3 and leading to the cleavage of BAT3 at 4 h after treatment while DNA laddering at 24 h. The cleavage is completely inhibited by zDEVD-fmk, a caspase-3 specific inhibitor. In addition, cleavage of BAT3 is blocked in caspase-3-deficient MCF-7 cells, indicating that BAT3 is a novel caspase-3 substrate. Evidence indicates that caspase-3 activated by ricin acts on BAT3 at the caspase cleavage site, DEQD(1001) to release a C-terminal fragment designated CTF-131. The CTF-131 induces phosphatidylserine exposure, cell rounding, and chromatin condensation as ricin does. Moreover, silencing expression of endogenous BAT3 concomitantly suppresses ricin-induced apoptosis. Together, our results suggest a model that ricin triggers morphological changes of apoptosis by caspase-3-mediated proteolytic activation of BAT3.

Mistletoe lectin I is a sialic acid-specific lectin with strict preference to gangliosides and glycoproteins with terminal Neu5Ac alpha 2-6Gal beta 1-4GlcNAc residues.

Mistletoe lectin I (ML-I) is a type II ribosome-inactivating protein, which inhibits the protein biosynthesis at the ribosomal level. ML-I is composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding specificities. Using comparative solid-phase binding assays along with electrospray ionization tandem mass spectrometry, ML-I was shown to preferentially bind to terminally alpha2-6-sialylated neolacto series gangliosides from human granulocytes. IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer were identified as ML-I receptors, whereas the isomeric alpha2-3-sialylated neolacto series gangliosides were not recognized. Only marginal binding of ML-I to terminal galactose residues of neutral glycosphingolipids with a Galbeta1-4Glc or Galbeta1-4GlcNAc sequence was determined, whereas a distal Galalpha1-4Gal, GalNAcbeta1-3Gal, or GalNAcbeta1-4Gal disaccharide did not bind at all. Among the glycoproteins investigated in Western blot and microwell adsorption assays, only those carrying Neu5Acalpha2-6Galbeta1-4GlcNAc residues, exclusively, predominantly, or even as less abundant constituents in an assembly with Neu5Acalpha2-3Galbeta1-4GlcNAc-terminated glycans, displayed high ML-I binding capacity. From our data we conclude that (i) ML-I has to be considered as a sialic acid- and not a galactose-specific lectin and (ii) neolacto series gangliosides and sialoglycoproteins with type II glycans, which share the Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, are true ML-I receptors. This strict preference might help to explain the immunostimulatory potential of ML-I toward certain leukocyte subpopulations and its therapeutic success as a cytotoxic anticancer drug.

Induction of cytokines by toxins that have an identical RNA N-glycosidase activity: Shiga toxin, ricin, and modeccin.

Shiga toxin (Stx) has an A1-B5 subunit structure, and the A subunit is an RNA N-glycosidase that inhibits cellular protein synthesis. We previously reported that in Caco-2 cells Stx induced cytokines and that the RNA N-glycosidase activity was essential for the cytokine induction. It is known that the binding of the Stx-B subunit to its receptor glycolipid, Gb3, mediates an A subunit-independent signal in some types of cells, but the involvement of this signal in the cytokine induction is unclear. In this study, we investigated whether RNA N-glycosidase itself induces cytokines. IL-8 production was enhanced by Stx, ricin, and modeccin, three toxins that inhibit protein synthesis through an identical RNA N-glycosidase activity, but not by two other types of protein synthesis inhibitors, diphtheria toxin and cycloheximide. The RNA N-glycosidase-type toxins showed a similar induction pattern of cytokine mRNAs. Brefeldin A, a Golgi apparatus inhibitor, completely suppressed the cytokine induction by the toxins. Analysis by using inhibitors of toxin binding and also Stx-B subunit showed that the cytokine-inducing activity was independent of Gb3-mediated signaling. These results indicate that RNA N-glycosidase itself induces the cytokine production and that intracellular transport of toxins through the Golgi apparatus is essential for the activity.

Both N- and C-terminal regions are essential for cinnamomin A-chain to deadenylate ribosomal RNA and supercoiled double-stranded DNA.

Cinnamomin is a type II ribosome-inactivating protein and its A-chain exhibits RNA N-glycosidase activity to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in ribosome, arresting protein synthesis at the elongation step. In this report, deadenylation of both rRNA and supercoiled DNA by native and recombinant cinnamomin A-chain expressed in Escherichia coli was demonstrated. However, the mutants of cinnamomin A-chain devoid of N-terminal 52 or/and C-terminal 51 amino acid residues lost both the activity of RNA N-glycosidase and the ability to release adenines from supercoiled DNA. Additionally, supercoiled DNA could not be cleaved into nicked and linear forms by these mutants. These results indicate that both N- and C-terminal regions are essential for the cinnamomin A-chain to deadenylate rRNA and supercoiled DNA. It was suggested that phosphodiester bonds in the extensively deadenylated region of supercoiled DNA would become fragile and liable to be broken spontaneously owing to the existence of tension in the supercoiled DNA.

An apoptotic inducer, aralin, is a novel type II ribosome-inactivating protein from Aralia elata

We recently found that aralin, a novel cytotoxic protein consisting of two subunits, from Aralia elata selectively induces apoptosis in transformed cells as compared to normal cells. Here we report that aralin is a lectin specific for galactose (Gal) and its derivatives, and possesses RNA N-glycosidase activity as a new type II ribosome-inactivating protein (RIP). The RNA N-glycosidase activity of aralin was detected in cell-free and whole cell systems by the generation of an R-fragment from 28S rRNA. Coinciding with appearance of the R-fragment in aralin-treated cells, significant inhibition of protein synthesis was observed prior to the onset of apoptosis. Aralin-evoked cell death was efficiently repressed by the addition of Gal and its derivatives. Interestingly, melibiose preferentially protected normal cells from apoptosis as compared with transformed cells. Using rhodamine-coupled aralin, the aralin receptor could be clearly detected around the cell surface of transformed cells, but to a lesser extent on normal cells. Receptor binding was suppressed by Gal. These results indicate that aralin is incorporated into cells via its Gal-containing cell surface receptor and induces apoptosis through its RIP activity. Moreover, the expression level and/or structural changes of the aralin receptor may affect the sensitivity toward aralin.

Purification and characterization of Moschatin, a novel type I ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells.

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of approximately 29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

The role of tyrosine residues in the RNA N-glycosidase activity of cinnamomin A-chain.

Cinnamomin is a type II ribosome-inactivating protein (RIP) and its A-chain (CTA) is a RNA N-glycosidase. It is observed that modification of tyrosine residues by N-acetylimidazole (N-AI) causes almost complete loss of CTA activity. Adenine partially protects tyrosine residues from modification by N-AI. It is proposed that tyrosine residues are involved in the active site of CTA and they are crucial in recognition and binding of ribosomal RNA. Tryptophan residues of CTA are also studied by NBS modification.

Induction of apoptosis by protein fraction isolated from the leaves of Mirabilis jalapa L on HeLa and Raji cell-line

SUMMARY The leaves of Mirabilis jalapa L contains protein fraction presumed ribosome-inactivating protein (RIP). RIP is a group of protein that has RNA N-glycosidase activity that is capable to inhibit protein synthesis. Protein fraction of the plant was shown to be cytotoxic on HeLa cell-line, however, the mechanism by which the protein kill the cells is not identified yet, whether trough apoptosis, necrosis, or other mechanism. This research aim to study the mechanism of cell death caused by the protein fraction isolated from the leaves of this plant on HeLa and Raji cell-line, as representative of different kind of cancer cells. Results showed that protein fraction isolated from the leaves of Mirabilis jalapa L was more cytotoxic to HeLa cell-line (LC50: 0.65 mg/ml) than to Raji cell-line (1.815 mg/ml) on 48 hours incubation time. Moreover, it was demonstrated that the death of HeLa cells caused by the protein fraction was due to induction of apoptosis, while on Raji cell-line was due to non-apoptosis way, presumably via necrosis.

High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system

Cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we review our recent advances in a cell-free protein synthesis system prepared from wheat embryos. We first addressed and resolved the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins. We found that conventional wheat germ extracts contained the RNA N-glycosidase tritin and other inhibitors such as thionin, ribonucleases, deoxyribonucleases, and proteases that originate from the endosperm and inhibit translation. Extensive washing of wheat embryos to eliminate endosperm contaminants has resulted in extracts with a high degree of stability and activity. To maximize the translation yield and throughput of the system, we then focused on developing the following issues: optimization of the ORF flanking regions, a new strategy to construct PCR-generated DNAs for screening, and design of an expression vector for large-scale protein production. The resulting system achieves high-throughput expression, with a PCR-directed system at least 50 genes that can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Under the dialysis mode of reaction, the system with the expression vector can maintain productive translation for 14 days. The cell-free system described here bypasses most of the biological processes and lends itself to robotic automation for high-throughput expression of genetic information, thus opening up many possibilities in the post-genome era.

Purification and characterization of Luffin P1, a ribosome-inactivating peptide from the seeds of Luffa cylindrica

A peptide designated Luffin P1 was purified from the seeds of Luffa cylindrica. Its molecular mass was determined to be 5226.1 Da by MALDI-TOF MS analysis. The purified Luffin P1 shows a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate with IC 50 of 0.88 nM. Its reaction mechanism is the same as that of the ribosome-inactivating protein trichosanthin, which is an rRNA N-glycosidase. Besides, the results of gel filtration chromatography suggested the existence of polymers of Luffin P1 and polymerization of Luffin P1 enhanced its rRNA N-glycosidase activity. Luffin P1 was the smallest peptide yet reported that has translational inhibitory activity. The cDNA and deduced amino acid sequence of Luffin P1 has also been determined.

Substrate binding and catalysis in trichosanthin occur in different sites as revealed by the complex structures of several E85 mutants.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have been revealed. Here we report the crystal structures of several E85 mutant TCS complexes with adenosine-5'-monophosphate (AMP) and adenine. In E85Q TCS/AMP and E85A TCS/AMP, near the active site of the molecule and parallel to the aromatic ring of Tyr70, an AMP molecule is bound to the mutant without being hydrolyzed. In the E85R TCS/adenine complex, the hydrolyzed product adenine is located in the active pocket where it occupies a position similar to that in the TCS/NADPH complex. Significantly, AMP is bound in a position different to that of adenine. In comparison with these structures, we suggest that there are at least two subsites in the active site of TCS, one for initial substrate recognition as revealed by the AMP site and another for catalysis as represented by the NADPH site. Based on these complex structures, the function of residue 85 and the mechanism of catalysis are proposed.

Refolding of partially thermo-unfolded cinnamomin A-chain mediated by B-chain

The pure cinnamomin A-chain is unstable compared to that in the mixture of A- and B-chain or in intact cinnamomin molecule either being stored at 4 °C or being heated. When being heated at 45 °C for 20 min, the A-chain generates partially unfolded intermediate and loses its tertiary structure as monitored by circular dichroism (CD) and tryptophan fluorescence, thus resulting in the inactivity of its RNA N-glycosidase albeit it retains most of its secondary structures. This partially unfolded intermediate is sensitive to protease, exhibiting property of a molten globule. The changes in conformation and activity are irreversible upon cooling. The partially unfolded intermediate can fully restore its RNA N-glycosidase activity in the presence of cinnamomin B-chain. The phenomenon, that the cinnamomin B-chain mediates the refolding of partially unfolded A-chain, probably plays an important role in the intracellular transport of the cytotoxic protein, i.e., keeping the structural stability of A-chain and refolding partially unfolded A-chain that occasionally appeared in the process of intracellular transport, to avoid the destiny of proteolysis that occurs in most denatured proteins in cell.

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.