Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.     

Depurination of plant ribosomes by pokeweed antiviral protein

Mammalian ribosomes have been shown to be enzymatically modified by ribosomal inactivating protein (RIPs) via specific depurination of rRNA. Here we report that ribosomes isolated from wheat germ contain intact and undepurinated rRNA and are depurinated by pokeweed antiviral protein (PAP). Pokeweed ribosomes isolated under the same conditions are depurinated. Total RNA isolated from pokeweed in the presence of strong denaturants was found to pbe partially depurinated. We conclude that wheat germ ribosomes are resistant to the endogenous RIP, tritin, but are sensitive to PAP and that pokeweed ribosomes can be depurinated by the N-glycosidase activity of endogenous PAP during isolation.     

A switch-on mechanism to activate maize ribosome-inactivating protein for targeting HIV-infected cells

Maize ribosome-inactivating protein (RIP) is a plant toxin that inactivates eukaryotic ribosomes by depurinating a specific adenine residue at the α-sarcin/ricin loop of 28S rRNA. Maize RIP is first produced as a proenzyme with a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP with full N-glycosidase activity. This naturally occurring switch-on mechanism provides an opportunity for targeting the cytotoxin to pathogen-infected cells. Here, we report the addition of HIV-1 protease recognition sequences to the internal inactivation region and the activation of the maize RIP variants by HIV-1 protease in vitro and in HIV-infected cells. Among the variants generated, two were cleaved efficiently by HIV-1 protease. The HIV-1 protease-activated variants showed enhanced N-glycosidase activity in vivo as compared to their un-activated counterparts. They also possessed potent inhibitory effect on p24 antigen production in human T cells infected by two HIV-1 strains. This switch-on strategy for activating the enzymatic activity of maize RIP in target cells provides a platform for combating pathogens with a specific protease.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Isolation, Crystallization, and Investigation of Ribosomal Protein S8 Complexed with Specific Fragments of rRNA of Bacterial or Archaeal Origin

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8–RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.     

Wheat ribosome-inactivating proteins: Seed and leaf forms with different specificities and cofactor requirements

Distinct forms of ribosome-inactivating proteins were purified from wheat (Triticum aestivum L.) germ and leaves and termed tritin-S and tritin-L, respectively. These differ in size and charge and are antigenically unrelated. They are both RNA N-glycosidases which act on 26S rRNA in native yeast (Saccharomyces cerevisiae) ribosomes by the removal of A3024 located in a universally conserved sequence in domain VII which has previously been identified as the site of action of ricin A-chain. Tritin-S and tritin-L differ in both their ribosome substrate specificities and cofactor requirements. Tritin-S shows only barely detectable activity on ribosomes from the endosperm, its tissue of synthesis, whereas tritin-L is highly active on leaf ribosomes. Additionally, tritin-S is inactive on wheat germ, tobacco leaf and Escherichia coli ribosomes but active on rabbit reticulocyte and yeast ribosomes. Tritin-L is active on ribosomes from all of the above sources. Tritin-S, unlike tritin-L shows a marked requirement for ATP in its action.Abbreviations CM carboxymethyl - FPLC fast protein liquid chromatography - NEPHGE non-equilibrium pH gradient gel electrophoresis - PAP pokeweed antiviral protein - RIP ribosome-inactivating protein A.J.M. was the recipient of a U.K. Science and Engineering Research Council CASE studentship sponsored by Agricultural Genetics Company Ltd., Cambridge CB4 4GG, UK.     

The barley 60 kDa jasmonate-induced protein (JIP60) is a novel ribosome-inactivating protein

The N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosome-inactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticulocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.     

紫茉莉悬浮细胞培养体系的建立

为建立紫茉莉(Mirabilis jalapa L.)悬浮细胞培养体系,以紫茉莉无菌苗叶片诱导的愈伤组织为材料,筛选紫茉莉悬浮细胞的适宜培养体系。结果表明,紫茉莉愈伤组织在MS+2,4-D 1 mg L-1+KT 0.5 mg L-1的液体培养基中悬浮继代培养3~4次,能得到稳定的悬浮细胞系。培养基的pH值为5.5~5.9,蔗糖浓度为30 g L-1更适合悬浮细胞的生长。紫茉莉悬浮细胞的生长曲线大致呈S型。最佳继代培养时间是10 d,培养液的体积为40 mL时,接种量为7.5 mL,可以较好地保持悬浮细胞系。1 L培养液中可提取分泌蛋白(0.42±0.15) g。这些有助于对悬浮细胞提取分泌蛋白的研究。     

Investigation of ribosome binding by the Shiga toxin A1 subunit, using competition and site-directed mutagenesis.

The enzymatic subunit of Shiga toxin (StxA1) is a member of the ribosome-inactivating protein (RIP) family, which includes the ricin A chain as well as other examples of plant toxins. StxA1 catalytically depurinates a well-conserved GAGA tetra-loop of 28S rRNA which lies in the acceptor site of eukaryotic ribosomes. The specific activities of native StxA1, as well as mutated forms of the enzyme with substitutions in catalytic site residues, were measured by an in vitro translation assay. Electroporation was developed as an alternative method for the delivery of purified A1 polypeptides into Vero cells. Site-directed mutagenesis coupled with N-bromosuccinimide modification indicated that the sole tryptophan residue of StxA1 is required for binding it to the 28S rRNA backbone. Northern analysis established that the catalytic site substitutions reduced enzymatic activity by specifically interfering with the capacity of StxA1 to depurinate 28S rRNA. Ribosomes were protected from StxA1 by molar excesses of tRNA and free adenine, indicating that RIPs have the capacity to enter the acceptor site groove prior to binding and depurinating the GAGA tetra-loop.     

Ribosome-inactivating and adenine polynucleotide glycosylase activities in Mirabilis jalapa L. tissues

Several tissues of Mirabilis jalapa L. (Nyctaginaceae) were assayed for inhibition of translation by a rabbit reticulocyte lysate (as a signal of ribosome-inactivating activity) and for adenine DNA glycosylase activity, activities that are both due to the presence of a class of enzymes called ribosome-inactivating proteins (RIPs), currently classified as rRNA N-glycosylases (EC ). These activities were highest in seed; intermediate in flower bud, immature seed, sepal + gynoecium, leaf, and root; and very low in all other tissues. By cation-exchange chromatography, four protein peaks with inhibitory activity on cell-free translation were identified in extracts from seeds, and two proteins were isolated from peaks 1 and 4, all of which have the properties of single-chain type 1 RIP. One is Mirabilis antiviral protein (MAP), so far purified only from roots. The second is a new protein that we propose to call MAP-4. The distribution of MAP and MAP-4 in several tissues was determined with a novel experimental approach based on liquid chromatography/mass spectrometry. The direct enzymatic activity of MAP on several substrates is described here for the first time. MAP depurinated not only rRNA in intact ribosomes, thus inhibiting protein synthesis, but also other polynucleotides such as poly(A), DNA, and tobacco mosaic virus RNA. Autologous DNA was depurinated more extensively than other polynucleotides. Therefore, the enzymatic activity of this protein may be better described as adenine polynucleotide glycosylase activity rather than rRNA N-glycosylase activity. Finally, MAP does not cross-react immunologically with other commonly utilized RIPs.     

Engineering a switch-on peptide to ricin A chain for increasing its specificity towards HIV-infected cells

Background

Ricin is a type II ribosome-inactivating protein (RIP) that potently inactivates eukaryotic ribosomes by removing a specific adenine residue at the conserved α-sarcin/ricin loop of 28S ribosomal RNA (rRNA). Here, we try to increase the specificity of the enzymatically active ricin A chain (RTA) towards human immunodeficiency virus type 1 (HIV-1) by adding a loop with HIV protease recognition site to RTA.

Methods

HIV-specific RTA variants were constructed by inserting a peptide with HIV-protease recognition site either internally or at the C-terminal region of wild type RTA. Cleavability of variants by viral protease was tested in vitro and in HIV-infected cells. The production of viral p24 antigen and syncytium in the presence of C-terminal variants was measured to examine the anti-HIV activities of the variants.

Results

C-terminal RTA variants were specifically cleaved by HIV-1 protease both in vitro and in HIV-infected cells. Upon proteolysis, the processed variants showed enhanced antiviral effect with low cytotoxicity towards uninfected cells.

Conclusions

RTA variants with HIV protease recognition sequence engineered at the C-terminus were cleaved and the products mediated specific inhibitory effect towards HIV replication.

General significance

Current cocktail treatment of HIV infection fails to eradicate the virus from patients. Here we illustrate the feasibility of targeting an RIP towards HIV-infected cells by incorporation of HIV protease cleavage sequence. This approach may be generalized to other RIPs and is promising in drug design for combating HIV.     

Isolation and characterization of a cDNA clone encoding the anti-viral protein from Phytolacca americana

Phytolacca anti-viral protein (PAP) was purified from Phytolacca leaves and the N-terminal was sequenced. A cDNA library was made from mRNAs isolated from Phytolacca leaves and cDNA clones for PAP were identified using oligonucleotide probes derived from the N-terminal amino acid sequence. The PAP-cDNA clone was sequenced from both directions. The predicted amino acid sequence of PAP was compared with the amino acid sequences of other ribosome-inactivating proteins. The identities of these proteins to PAP ranged from 29 to 38%, and a region was found in each with a sequence similar to the PAP sequence (AIQMVSEAARFKYI). Southern blot analysis indicates that PAP is encoded by a multi-gene family.Abbreviations MAP Mirabilis jalapa anti-viral protein - PAP Phytolacca anti-viral protein - SO6 30 kDa ribosome-inactivating protein from the seeds of Saponaria officinalis     

Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with uracil and uridine

Ribosome inactivating protein (RIP) catalyzes the cleavage of glycosidic bond formed between adenine and ribose sugar of ribosomal RNA to inactivate ribosomes. Previous structural studies have shown that RNA bases, adenine, guanine, and cytosine tend to bind to RIP in the substrate binding site. However, the mode of binding of uracil with RIP was not yet known. Here, we report crystal structures of two complexes of type 1 RIP from Momordica balsamina (MbRIP1) with base, uracil and nucleoside, uridine. The binding studies of MbRIP1 with uracil and uridine as estimated using fluorescence spectroscopy showed that the equilibrium dissociation constants (K_D) were 1.2 × 10⁻⁶ M and 1.4 × 10⁻⁷ M respectively. The corresponding values obtained using surface plasmon resonance (SPR) were found to be 1.4 × 10⁻⁶ M and 1.1 × 10⁻⁷ M, respectively. Structures of the complexes of MbRIP1 with uracil (Structure-1) and uridine (Structure-2) were determined at 1.70 and 1.98 Å resolutions respectively. Structure-1 showed that uracil bound to MbRIP1 at the substrate binding site but its mode of binding was significantly different from those of adenine, guanine and cytosine. However, the mode of binding of uridine was found to be similar to those of cytidine. As a result of binding of uracil to MbRIP1 at the substrate binding site, three water molecules were expelled while eight water molecules were expelled when uridine bound to MbRIP1.     

Molecular Cloning of a cDNA Encoding Ribosome Inactivating Protein from Amaranthus viridis and Its Expression in E. coli

In order to isolate a cDNA clone of ribosome inactivating protein (RIP), a cDNA library was constructed in Uni-ZAP XL vector with poly(A) RNA purified from leaves of Amaranthus viridis. To get the probe for screening the library, PCR of phage DNA was conducted using the vector primer and degenerate primer designed from a conserved putative active site of the RIPs. Twenty-six cDNA clones from about 600,000 plaques were isolated, and one of these clones was fully sequenced. It was 1,047 bp and contained an open reading frame encoding 270 amino acids. The deduced amino acid sequence had a putative signal sequence of 17 amino acids and a putative active site (AIQMVAEAARFFKYIE) conserved in other RIPs. E. coli cells expressing A. viridis RIP cDNA did not grow well as compared to control cells, indicating that recombinant A. viridis RIP presumably inactivated E. coli ribosomes. In addition, recombinant A. viridis RIP cDNA produced by E. coli had translation inhibition activity in vitro.     

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.Revisions requested 30 September 2004; Revisions received 22 October 2004     

Type 1 ribosome-inactivating proteins depurinate plant 25S rRNA without species specificity.

Four different type 1 ribosome-inactivating proteins (RIPs) with RNA N-glycosidase activity were tested for their ability to attack the large rRNA of plant ribosomes derived from tobacco plants, as well as from the plant species from which the particular RIP had been isolated. Incubation of tobacco ribosomes with RIPs isolated from either Phytolacca americana L. (pokeweed), Dianthus barbatus L. (carnation), Spinacia oleracea L. (spinach) or Chenopodium amaranthicolor Coste and Reyn. (chenopodium) rendered the 25S rRNA sensitive to aniline-catalyzed hydrolysis, generating a single rRNA-fragment of about 350 nucleotides. The same fragment was generated when rRNAs from pokeweed, carnation, spinach or chenopodium ribosomes were aniline-treated without any deliberate treatment of the ribosomes with the respective RIP. This indicated that ribosomes from all RIP-producing plants were already inactivated by their own RIPs during preparation. These results demonstrate that plant ribosomes are generally susceptible to RIP attack, including modification by their own RIPs. Direct sequencing of the newly generated fragments revealed that a single N-glycosidic bond at an adenosine residue within the highly conserved sequence 5'-AGUACGAGAGGA-3' was cleaved by all of the RIPs investigated, a situation also found in animal, yeast and Escherichia coli ribosomes.     

A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from Phytolacca americana, is characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. In this study, we present evidence that PAP is associated with ribosomes and depurinates tobacco ribosomes in vivo by removing more than one adenine and a guanine. A mutant of pokeweed antiviral protein, PAPn, which has a single amino acid substitution (G75D), did not bind ribosomes efficiently, indicating that Gly-75 in the N-terminal domain is critical for the binding of PAP to ribosomes. PAPn did not depurinate ribosomes and was non-toxic when expressed in transgenic tobacco plants. Unlike wild-type PAP and a C-terminal deletion mutant, transgenic plants expressing PAPn did not have elevated levels of acidic pathogenesis-related (PR) proteins. PAPn, like other forms of PAP, did not trigger production of salicylic acid (SA) in transgenic plants. Expression of the basic PR proteins, the wound-inducible protein kinase and protease inhibitor II, was induced in PAPn-expressing transgenic plants and these plants were resistant to viral and fungal infection. These results demonstrate that PAPn activates a particular SA-independent, stress-associated signal transduction pathway and confers pathogen resistance in the absence of ribosome binding, rRNA depurination and acidic PR protein production.     

A ribosomal protein is specifically recognized by saporin, a plant toxin which inhibits protein synthesis.

Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of the eukaryotic ribosome (ribosome inactivating proteins, RIPs). The site of action of RIPs (A4324 in the rRNA from rat liver) is in a loop structure whose nucleotide sequence all around the target adenine is also conserved in those species which are completely or partially insensitive to RIPs. In this paper we identify a covalent complex between saporin (the RIP extracted from Saponaria officinalis) and ribosomal proteins from yeast (Saccharomyces cerevisiae), by means of chemical crosslinking and immunological or avidin-biotin detection. The main complex (mol. wt. congruent to 60 kDa) is formed only with a protein from the 60 S subunit of yeast ribosomes, and is not detected with ribosomes from E. coli, a resistant species. This observation supports the hypothesis for a molecular recognition mechanism involving one or more ribosomal proteins, which could provide a 'receptor' site for the toxin and favour optimal binding of the target adenine A4324 to the active site of the RIP.     

New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L. and Bougainvillea spectabilis Willd.

New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC₅₀ (concentration causing 50% inhibition) in the 10⁻¹⁰ M range, and by various cell lines, with IC₅₀s in the 10⁻⁸–10⁻⁶ M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237–282) with an LD₅₀ (concentration that is 50% lethal) ≤ 8 mg · kg⁻¹ body weight, whilst the RIP from Bougainvillea spectabilis had an LD₅₀ >32 mg · kg⁻¹. The N-terminal sequence of the two RIPs from Basella rubra had 80–93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV. Received: 5 March 1997 / Accepted: 27 May 1997