Mirabilis Antiviral Protein Research Articles

Identification of Peptides Compounds from Mirabilis jalapa L. (Caryophyllales: Nyctaginaceae) Potentially as a Biopesticide

Mirabilis Antiviral Protein (MAP) is a specific protein found in the Mirabilis jalapa. The MAP has many promising benefits, one of them is that it can be used as biopesticides. However, the structure and visualization of protein from MAP have not been identified. This study investigated the modelling structures of MAP compounds which potentially become bioinsecticide for insect and the interaction between bonding ligand-receptor in the surface cell. This research used In silico models that refers to the BIOPEP database with the bioinformatics tools methods. The screening of MAP compounds was conducted through hydrolysis steps by 30 enzymes to become new peptides. The new Antimicrobial Peptides (AMPs) were analyzed by a multidimensional statistical analysis using four predicted algorithms, Support Vector Machines (SVM), Random Forest (RF), a Neural Network Artificial (ANN) and Discriminant Analysis (DA) that were available in the Anti-Microbial Peptides Collection (CAMP Database). The results were identified six new types of AMPs from 449 AMPs during the in-silico proteolysis process toward M. jalapa. This peptide predicted the function become antimicrobials by inactivating the ribosomes. There were obtained three of the best peptide structures from M. jalapa that have the potential as toxin compounds (biopesticides). Through the visualization of the three AMPs, models of biopesticide structures are WIFKTVEEIKLVMGLLKSS, IKLVMGLLKSS, and ITNIRTKVA with the residual category 6-22, and the characterization of molecular weight was 561,7 to 2971,4 g/mol. The AMPs fulfill the Boman Index which the measurement of protein-peptide affinity to build the biological interactions, ranging from -0,51 to 2,98. Further, these renewable peptides can be used to determine the pathways that occur in targeted insect pests as biopesticides.

Co-silencing of the Mirabilis antiviral protein (MAP) permits virus-induced gene silencing (VIGS) of other genes in Four O’Clock plants (Mirabilis jalapa)

SummaryVirus-induced gene silencing (VIGS) is a powerful and rapid technique for the analysis of gene function during plant growth and development. We attempted to use VIGS for the functional analysis of genes associated with floral senescence and abscission in Four O’Clock plants (Mirabilis jalapa). Plants infected with a Tobacco Rattle Virus (TRV) vector containing a fragment of the phytoene desaturase (PDS) gene did not show the expected photo-bleaching phenotype. We hypothesised that the failure of VIGS in this species might be due to the presence of the Mirabilis antiviral protein (MAP) which is known to have potent activity in preventing viral infection. Plants infected with a TRV vector containing fragments of both the PDS and MAP genes showed the typical photo-bleaching phenotype in photosynthetic tissues, and this was accompanied by reductions in the abundance of PDS and MAP transcripts. We also demonstrated reduced transcript abundance (and the expected phenotypes) when VIGS with a TRV-MAP vector was used to silence the chlorata42 (Ch42) and the proliferating cell nuclear antigen (PCNA) genes. Silencing of the 3,4-dihydroxy-L-phenylalanine 5-glucosyl transferase (DOPA5GT) gene reduced pigmentation in stems, leaves, and flowers. This is the first direct demonstration of the role of an enzyme that has been proposed to be a key step in the synthesis of betalain pigments. Co-silencing of endogenous anti-viral proteins may increase the range of taxa that are amenable to the use of VIGS for functional gene analysis.

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.

Isolation and characterization of cDNAs encoding ribosome inactivating protein from Dianthus sinensis L.

To isolate a ribosome inactivating protein (RIP) gene, six plant species were surveyed for antiviral activity. Crude proteins extracted from these plants were tested for the antiviral activity against tobacco mosaic virus (TMV) in Nicotiana glutinosa. All the plants, Spinacia oleracea, Amaranthus lividus, Dianthus superbus, Dianthus sinensis and Celosia cristata, with an exception of Oenanthe stolonifera, presented 70-90% inhibition of viral infectivity. In an attempt to search for the RIP gene from D. sinensis, partial cDNA was obtained by polymerase chain reaction (PCR) of the poly(A)+ RNA from D. sinensis leaves. DNA gel blot analysis showed that D. sinensis has multi-copy RIP genes. The expression of RIP gene was investigated in the flower, leaf, root and stem of D. sinensis, and was found to be most abundant in the leaf. Using the partial cDNA as a probe, seven full-length cDNAs were isolated from a library prepared from D. sinensis leaves. They were divided into three groups on the basis of their nucleotide sequence homology. The three representative clones, cDsRIP1, cDsRIP2 and cDsRIP3 were completely sequenced. They all had an open reading frame of 882 bp. The cDsRIP2 showed 79% homology with dianthin 30 and saporin genes; 59% with PAP and Mirabilis antiviral protein MAP genes. From the analysis of deduced amino acid sequences, it was predicted that D. sinensis RIP cDNAs might have a putative signal peptide of 23 amino acid residues at their N-terminus. When the cDNA was expressed in E. coli, the bacteria was unable to grow upon IPTG induction, suggesting that expression of the gene renders toxicity to E. coli cells.

Antiviral and Antiviroid Activity of MAP-Containing Extracts from Mirabilis jalapa Roots.

Extracts of Mirabilis jalapa (Nyctaginaceae), containing a ribosome inactivating protein (RIP) called Mirabilis antiviral protein (MAP), were tested against infection by potato virus X, potato virus Y, potato leaf roll virus, and potato spindle tuber viroid. Root extracts of M. jalapa sprayed on test plants 24 h before virus or viroid inoculation inhibited infection by almost 100%, as corroborated by infectivity assays and the nucleic acid spot hybridization test. Antiviral activity of MAP extracts was observed against mechanically transmitted viruses but not against aphid-transmitted viruses. Purified MAP showed the same antiviral effect as the crude extracts.

Improved crystals of the toxic protein MAP by protein engineering towards the host specificity.

Mirabilis anti-viral protein (MAP) is a ribosome-inactivating protein from Mirabilis jalapa L. Since MAP is effective over a broad spectrum of species, the protein is difficult to express in heterologous hosts such as Escherichia coli. Recently, we obtained a MAP mutant, Y72F which exhibits a lower (1/100) activity against E. coli ribosomes while retaining almost full activity against mammalian cells [Habuka, Miyano, Kataoka, Tsuge & Noma (1992). J. Biol. Chem. 267, 7758-7760]. For the crystallographic studies, the Y72F MAP expression vector with an OmpA leading sequence was constructed and expressed in E. coli. The Y72F MAP mutant was then isolated and purified from the cell culture medium. Crystals were grown using the crystallization conditions for the native MAP crystals [Miyano et al. (1992). J. Mol. Biol. 226, 281-283]: 50% ammonium sulfate containing 50 mM ammonium citrate and 2 mM adenine sulfate, pH 5.4. The crystals belong to space group P3(1)21 (or P3(2)21) with a = b = 104.1 and c = 134.3 A. The crystals are isomorphous with the wild-type crystals but diffract to higher resolution. Imaging-plate photographs of the Y72F mutant showed sharp intense spots without the streaking observed in the native crystals.

Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP)

Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A4324 in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes. Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.

Crystallization and preliminary X-ray crystallographic analysis of Mirabilis antiviral protein

Mirabilis antiviral protein is a single-chain ribosome-inactivating protein purified from the tuberous root of Mirabilis jalapa L. We obtained several forms of crystals of the protein by the hanging drop vapor diffusion method, but most of these crystals were not suitable for X-ray crystallography. After refining the growth conditions, crystals of Crystallographic quality were grown in 20-μl droplets of an equi-volume mixture of 1.5% ( w v ) protein solution and a reservoir solution containing 49 to 50% ( w v ) ammonium sulfate and 50 m m-ammonium citrate (pH 5.4) at room temperature. Addition of 2 m m-adenine sulfate reduced twinning and “crystal shower”. The resulting trigonal crystals diffract beyond 2.5 Å resolution using a rotating anode X-ray generator. The space group was determined to be P3 121 or P3 221 ( a = b = 103.9 A ̊ , c = 134.6 A ̊ , α = β = 90 °, γ = 120 °) based on their precession photography of h0 l and hk0 zones. There seems to be three monomers in an asymmetric unit for V M = 2.51 A ̊ 3/Da .

Specificities of RNA N-glycosidase activity of Mirabilis antiviral protein variants.

Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, inactivates both eukaryotic and prokaryotic ribosomes by means of site-specific RNA N-glycosidase activity. In order to identify the site of this activity, some amino acid residues of MAP, conserved in homologous ribosome-inactivating proteins, were altered to other amino acids by replacing DNA fragments of the total synthetic gene of MAP. When the in vitro proteins synthesis of rabbit reticulocyte was treated with MAP variants secreted into culture media of Escherichia coli transformants, the inhibitory effect of R26L and R48L (R26L designates MAP variant with Arg-26 changed to Leu) was found to be similar to that of native MAP. Both purified Y72F and Y118F had the same effect as native MAP, and E168D had a slightly weaker effect. In contrast, on the protein synthesis of E. coli, Y118F had one-tenth the effect of native MAP, and Y72F and E168D approximately one-hundredth the effect. These three variant proteins also exhibited reduced RNA N-glycosidase activity on substrate E. coli ribosomes. These results suggest that Tyr-72 and Glu-168 are involved in RNA N-glycosidase activity. When the R171K gene was expressed in E. coli, an N-glycosidic bond of the 23 S rRNA of the host ribosome was found to be cleaved, although no product of the gene could be detected. This suggests that MAP variants can maintain their N-glycosidase activity when the conserved Glu-168 and Arg-171 are changed to similarly charged residues.

Substantial increase of the inhibitory activity of Mirabilis antiviral protein by an elimination of the disulfide bond with genetic engineering.

Mirabilis antiviral protein (MAP) is a rigid, heat-stable protein composed of 250 amino acids with an intramolecular disulfide bond. MAP inhibits the in vitro protein synthesis of rabbit reticulocyte with approximately one-thirtieth the activity of the ricin A chain, a homologous protein with no such bond (Habuka, N., Murakami, Y., Noma, M., Kudo, T., and Horikoshi, K. (1989) J. Biol. Chem. 264, 6629-6637; Habuka, N., Akiyama, K., Tsuge, H., Miyano, M., Matsumoto, T., and Noma, M. (1990) J. Biol. Chem. 265, 10988-10992). The bond is presumed to induce some structural perturbation that alters the mode of interaction with the substrate ribosome and thus lowers the activity. To confirm this hypothesis, a mutant MAP gene in which the codons of both cysteines were replaced by those of serines was constructed and expressed in Escherichia coli, and its product (C36/22OS) was purified. In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, C36/220S showed the same mobility as that of MAP reduced by 2-mercaptoethanol, whereas nonreduced MAP showed faster migration. The inhibitory activity of C36/220S was approximately 22 times higher than that of native MAP, that is the mutant had an IC50 of 0.16 nM for the protein synthesis of the rabbit reticulocyte system, whereas the native MAP had an IC50 of 3.5 nM. The results indicate that the activity of MAP is increased by the elimination of the disulfide bond, and this supports the hypothesis.

Escherichia coli ribosome is inactivated by Mirabilis antiviral protein which cleaves the N-glycosidic bond at A2660 of 23 S ribosomal RNA

Ribosome-inactivating proteins (RIPs) are known to inactivate eukaryotic ribosomes, which results in the inhibition of protein synthesis, but there has been no evidence that they inactivate the ribosomes of Escherichia coli. Recently, Mirabilis antiviral protein (MAP), a RIP, has been shown to inhibit the protein synthesis of E. coli as well as eukaryotes. To elucidate its mechanism, E. coli ribosomes treated with MAP were analyzed by polyacrylamide/agarose composite gel electrophoresis and RNA sequencing using reverse transcriptase with DNA primer. The 23 S rRNAs, with an A 260 value for ribosomes of 15, were completely cleaved in vitro by a 30 minute treatment with MAP at a concentration of 100 n m at 37°C and a subsequent treatment with aniline. However, they were not affected by ricin A-chain under the same conditions. The primer extension of DNA polymerization stopped before A2660 of 23 S rRNA in RNA sequencing. Furthermore, both 16 S and 23 S rRNAs were cleaved by the MAP and aniline treatments when naked E. coli rRNAs were used as substrates, and the primer extension stopped before bases A2660 and A1014, respectively, in RNA sequencing. As the A2660 region has been shown to interact with the elongation factors EF-Tu and EF-G these results indicate that MAP cleaves the N-glycosidic bond at A2660 in E. coli 23 S RNA resulting in the inactivation of the ribosome.

DNA sequence of Mirabilis antiviral protein (MAP), a ribosome-inactivating protein with an antiviral property, from mirabilis jalapa L. and its expression in Escherichia coli

We cloned a cDNA for Mirabilis antiviral protein (MAP), a ribosome-inactivating protein (RIP), which inhibits the mechanical transmission of plant virus and the in vitro protein synthesis of both prokaryotes and eukaryotes. The cDNA consisted of 1066 nucleotides and could encode 278 amino acids. The major part of the amino acid sequence (from Ala29 to Ser278) was identical with the sequence of native MAP as determined by protein sequencing. An NH2-terminal extrapeptide (28 amino acid residues) of MAP was comparable with the signal peptides of plant proteins accumulating in the vacuole. A stable hairpin structure was predicted in the 3'-noncoding region of the cDNA. Tandem repeated sequences were found downstream from the hairpin structure. They were composed of triple complete repeats of a heptanucleotide with preceding and following hexa-nucleotide repeats. The cDNA was expressed in Escherichia coli based on the T7 expression system. The product encoded by the cDNA was confirmed to be MAP precursor by Western blotting followed by immunological analysis. The growth of the transformants was inhibited by the expression of the gene. MAP precursor also seemed to inhibit the protein synthesis of E. coli just as native MAP has been observed to do.

Expression and secretion of Mirabilis antiviral protein in Escherichia coli and its inhibition of in vitro eukaryotic and prokaryotic protein synthesis.

Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, exhibits inhibitory effects on both plant virus infection and protein synthesis. To study these functions by site-specific mutagenesis, the total synthetic gene of MAP was constructed and expressed in Escherichia coli. However, the growth of the host was inhibited by the products, and the yield of MAP was very low. To improve the system for expressing MAP, an expression vector, pSH7, was constructed. This vector is based on the high copy number plasmid pUC19 and includes PL promoter and temperature-sensitive cI857 repressor. The plasmid also contains the ompA signal sequence and the total synthetic MAP gene. The MAP gene was expressed and its product was secreted into the culture medium after E. coli transformants were cultivated at 30 degrees C and the temperature was raised to 42 degrees C. The secreted MAP was then purified and characterized. This protein was identical to native MAP as determined by its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the amino acid sequence at the NH2 terminus, and its inhibitory effect on in vitro protein synthesis. MAP was found to inhibit the in vitro protein synthesis of rabbit reticulocyte and wheat germ. It further showed an IC50 concentration of approximately 200 nM in an E. coli in vitro translation system in contrast to ricin A-chain, a well known ribosome-inactivating protein.

A potent plant virus inhibitor found in Mirabilis jalapa L.

An inhibitor of plant virus infection was found in the extracts of Mirabilis jalapa L. The inhibitor, a protein designated as MAP (Mirabilis antiviral protein), showed highly potent activity against mechanical transmission of viruses, tobacco mosaic (TMV), cucumber green mottle mosaic, potato Y, turnip mosaic and cucumber mosaic. Almost complete inhibition was achieved when MAP at 0.8μg/ml was applied on the upper surface of leaves of Xanthi nc tobacco 24hr before TMV inoculation, and 50% inhibition when MAP at 10μg/ml was applied on the under surface of leaves. MAP induced systemic resistance to TMV infection in tobacco plants when it was applied on their basal leaves 24hr before TMV inoculation onto the upper ones. MAP showed no inhibition when it was applied 1hr after TMV inoculation. Antiserum against MAP gave positive reaction in the agar gel with leaf extracts of M. jalapa and did not with those of the other Chenopodiales plants such as Basella rubra, Boerhaavia diffusa, Bougainvillea sp., Chenopodium amaranticolor and Phytolacca americana. Concentration of MAP was successfully quantified using enzyme-linked immunosorbent assay. MAP content in the root tissue was exceedingly higher than in the other parts of the plant and that of an yellow-flower trait of M. jalapa was as high as 1.1mg/g fresh weight. M. jalapa seems to be a promising source of antiviral substances for the practical use.

Amino acid sequence of Mirabilis Antiviral Protein, Total Synthesis of Its Gene and Expression in Escherichia coli

We have determined the complete amino acid sequence of Mirabilis antiviral protein (MAP). MAP is composed of 250 amino acids having a combined molecular weight of 27,833 and contains 23 lysine residues and 7 arginine residues. The amino acid sequence of MAP has 24% homology with the Ricin D-A chain. To carry out systematic structure-function studies of MAP, we have accomplished the total synthesis of its gene. We designed a synthetic MAP gene containing 12 unique restriction sites that were on the average 65 base pairs apart. Thirty synthetic oligonucleotides were enzymatically joined to form DNA duplexes. These were strategically synthesized to have EcoRI and HindIII cohesive ends and were cloned in pUC19. Nine blocks of the synthetic fragments were assembled in pUC19 to form the MAP gene consisting of 759 base pairs. The correctness of the connecting reactions was confirmed by step-wise sequencing of each assembled fragment as well as the total gene. When expressed under control of the tac promoter in Escherichia coli, the synthetic gene gave a protein similar to the native MAP. This was confirmed by an enzyme-linked immunosorbent assay and Western blotting analysis.