Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCS(M(120-123)), TCS(E160A/E189A), with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCS(R122G), which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property.     

Fractionation of the ribosome inactivating protein preparations with triazine dyes

Aspergillins are ribosome-inactivating proteins (RIPs), isolated from several strains of Aspergillus. The interaction between Cibacron Blue F3GA and two members of this family, alpha sarcin and mitogillin, and other RIPs of type I, was studied. Alpha sarcin retention depended on pH and ionic strength. By chromatography on Affi-Gel Blue in mild experimental conditions, mitogillin and PAP-I did not interact with the dye, whereas 40% of alpha sarcin and 70-90% of briodin, RTA and gelonin were recovered in the bound fraction. In all cases, the major fraction showed a higher toxicity level in protein synthesis inhibition assays. The unbound alpha sarcin, conjugated with the anti-ovarian carcinoma monoclonal antibody MOv17, showed on OVCA 432 a cytotoxicity which was 900 times higher than that exerted by the alpha sarcin alone.     

Ribosomal protein L10a, a bridge between trichosanthin and the ribosome

Trichosanthin is a type I ribosome-inactivating protein with many pharmacological activities. The trichosanthin-coupled Sepharose affinity purification revealed a protein, which was identified by mass spectrometry as the ribosomal protein L10a. The interaction between trichosanthin and recombinant L10a was further confirmed by in vitro binding assay. Kinetic analysis by surface plasmon resonance technology revealed that L10a had a high affinity to trichosanthin with a K(D) of 7.78nM. The study with mutated forms of trichosanthin demonstrated that this specific association correlates with the ribosome-inactivating activity of trichosanthin. This finding might provide insight into the mechanisms by which trichosanthin inactivates ribosome and that underlies its pharmacological effect.     

The C-terminal fragment of the ribosomal P protein complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and the ribosome

Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by enzymatically depurinating a specific adenine residue at the sarcin-ricin loop of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation centre of the ribosome. Here, we present the 2.2 Å crystal structure of trichosanthin (TCS) complexed to the peptide SDDDMGFGLFD, which corresponds to the conserved C-terminal elongation factor binding domain of the ribosomal P protein. The N-terminal region of this peptide interacts with Lys173, Arg174 and Lys177 in TCS, while the C-terminal region is inserted into a hydrophobic pocket. The interaction with the P protein contributes to the ribosome-inactivating activity of TCS. This 11-mer C-terminal P peptide can be docked with selected important plant and bacterial RIPs, indicating that a similar interaction may also occur with other RIPs.     

Chromatographic separation of the isoforms of the ribosome inactivating protein, gelonin.

The three isoforms of gelonin were separated by affinity chromatography on concanavalin-A Sepharose into discrete components of Mr 31,500, 30,000 and 29,200. Their separation was achieved by apparent differences in interaction with the lectin due to variation in carbohydrate patterns. The Mr 30,000 component representing 67% of the total mixture was the most active in inhibiting protein synthesis in a cell free translation assay using rabbit reticulocyte lysates, although the other two were also active. An antibody prepared against the major fraction (Mr 30,000) reacted well with all three components, demonstrating immunological similarity. This purification may aid the structural elucidation of gelonin and preparation of hormonotoxins and immunotoxins.     

On the contentious sequence and glycosylation motif of the ribosome inactivating plant protein gelonin

The amino acid sequence and the glycosylation motif of the ribosome inactivating protein (RIP) gelonin are identified by Fourier transform ion cyclotron resonance mass spectrometry. Intact gelonin as isolated from the seeds of Gelonium multiflorum consists of at least three different post-translational modified forms: analysis of gelonin peptides as obtained by proteolytic digestion is consistent with the amino acid sequence published by Nolan et al. High resolution mass determination established a glycosylation pattern of GlcNAc2Man(3-5)Xyl. N189 was identified as glycosylation site. The proposed glycan structure is consistent with a standard plant N-glycosylation pattern as found in other RIP. Based on these results we suggest that gelonin is located in the vacuole of Gelonium multiflorum seeds.     

Immunoconjugate generation between the ribosome inactivating protein restrictocin and an anti-human breast carcinoma MAB

Summary Treatment of Wistar/AG rats with a single i.p. injection of 1 mg/kg of synthetic double-stranded polynucleotides, either polyadenylic-polyuridylic acid (rA_n.rU_n), or a mismatched analogue of polyinosinic-polycytidylic acid (rI_n.r(C₁₂U)_n), enhanced the cytotoxicity of natural killer (NK) cells among peripheral blood leukocytes and lung intracapillary leukocytes (LICL). The enhancement reached a peak 24 h after treatment and returned to control values after 4 days. In rats given repeated injections of double-stranded polynucleotides (2 per week), the NK cytotoxicity expressed by LICL reached more than ten times (in lytic units) the control levels between day 8, after 3 injections, and day 360, after 100 injections. No hypore-sponsiveness was observed. Moreover, NK activity was frequently and significantly higher in rats given multiple injections than in those given a single injection. In rats with experimentally induced P77 lung fibrohistiocytoma colonies, repeated injections of rI_n.r(C₁₂U)_n stimulated NK activity and reduced the number of metastatic nodules from 172 to 19. The same significant reduction (from 172 to 27) was also observed in animals given repeated injections of rA_n.rU_n. However, with two models of spontaneous metastases, significant reduction in lung metastases (M37 bronchioloalveolar carcinoma) or lack of effect (S₄T₁₉ rhabdomyosarcoma) were observed.     

Liposome-mediated delivery of ribosome inactivating proteins to cells in vitro

This study describes the liposome-mediated delivery of toxins to a variety of cells in vitro. Gelonin, a potent inhibitor of protein synthesis from Gelonium multiflorum, was delivered to the cytoplasm of TLX5 lymphoma cells most effectively by phosphatidylserine vesicles. These liposomes were also capable of inhibiting protein synthesis in XC (transformed rat fibroblasts) and phytohaemagglutinin-stimulated CBA mouse lymphocytes. Phosphatidylcholine liposomes had no capacity to deliver their contents to the cytoplasm, but the addition of cholesterol to the vesicle membrane resulted in an increased capacity. Delivery events were enhanced further by the addition of mixed bovine brain gangliosides to the membrane in the ratio 5:5:1 phosphatidylcholine/cholesterol/gangliosides. The addition of cholesterol to phosphatidylserine vesicles failed to increase the inhibitory effects of the gelonin liposomes. The A chain of diphtheria toxin encapsulated in phosphatidylserine liposomes had no inhibitory effect on the level of protein synthesis in TLX5 or Daudi cells.     

Purification, characterization and cloning of antiviral/ribosome inactivating protein from Amaranthus tricolor leaves

An antiviral protein (AVP), imparting high level of resistance against sunnhemp rosette virus (SRV) was purified from the dried leaves of Amaranthus tricolor. The purified protein (AAP-27) exhibited approximately 98% inhibition of local lesion formation at a concentration range of approximately 30 microg ml(-1). The protein was found to be highly basic glycoprotein monomer (pI approximately 9.8) of Mr 27 kDa, with neutral sugar content of 4%. The purified protein exhibited N-glycosidase and RNase activities. We have also isolated full-length cDNA clone, encoding this protein designated as A. tricolor antiviral protein-1 (AAP-1). Two primers, one designed on the basis of N-terminal sequence of the purified protein and the other from the conserved active peptides of other AVPs/RIPs were used for PCR amplification of double stranded cDNA, isolated from the leaves of A. tricolor. The amplified fragment was used as a probe for library screening. The isolated full-length cDNA consisted of 1058 nucleotides with an open reading frame encoding a polypeptide of 297 amino acids. The deduced amino acid sequence of AAP-1 has a putative active domain conserved in other AVPs/RIPs and shows varying homology to the RIPs from other plant species.     

Bifunctional plant defence enzymes with chitinase and ribosome inactivating activities from Trichosanthes kirilowii cell cultures

Three bifunctional plant enzymes (named TKC 15, TKC 28-I, and TKC 28-II) with both chitinase and RIP activity were purified from the medium of Trichosanthes kirilowii plant cell cultures. Highly purified preparations of these proteins exhibited endochitolytic activity as well as the specific 28S rRNA N-glycosidase activity characteristic of ribosome inactivating proteins (RIPs). In addition to providing a competitive advantage to the plant, these enzymes offer the possibility of improved antifungal protection when expressed in transgenic plants.     

肥皂草素类核糖核蛋白体失活蛋白的纯化及其性质研究

 利用强阳离子交换柱从Saponaria officinalis L种子中分离出一种肥皂草素(Saporin)成分。它在无细胞体系中显示了较强的抑制蛋白合成的活性,与抗体连接后能特异性杀伤靶细胞。     

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2'-deoxyadenosine triphosphate (2'-dATP) (F). These were determined at 1.67?, 1.60?, 2.20?, 1.70?, 2.07? and 1.90? resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2'-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ(1)=-66°and χ(2)=165° in structures (A) and (B); (ii) χ(1)=-95° and χ(2)=70° in structures (C), (D) and (E); and (iii) χ(1)=-163° and χ(2)=87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.     

Importance of conserved cysteine residues in the coronavirus envelope protein

Coronavirus envelope (E) proteins play an important, not fully understood role(s) in the virus life cycle. All E proteins have conserved cysteine residues located on the carboxy side of the long hydrophobic domain, suggesting functional significance. In this study, we confirmed that mouse hepatitis coronavirus A59 E protein is palmitoylated. To understand the role of the conserved residues and the necessity of palmitoylation, three cysteines at positions 40, 44, and 47 were changed singly and in various combinations to alanine. Double- and triple-mutant E proteins resulted in decreased virus-like particle output when coexpressed with the membrane (M) protein. Mutant E proteins were also studied in the context of a full-length infectious clone. Single-substitution viruses exhibited growth characteristics virtually identical to those of the wild-type virus, while the double-substitution mutations gave rise to viruses with less robust growth phenotypes indicated by smaller plaques and decreased virus yields. In contrast, replacement of all three cysteines resulted in crippled virus with significantly reduced yields. Triple-mutant viruses did not exhibit impairment in entry. Mutant E proteins localized properly in infected cells. A comparison of intracellular and extracellular virus yields suggested that release is only slightly impaired. E protein lacking all three cysteines exhibited an increased rate of degradation compared to that of the wild-type protein, suggesting that palmitoylation is important for the stability of the protein. Altogether, the results indicate that the conserved cysteines and presumably palmitoylation are functionally important for virus production.     

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.     

Site-directed polyethylene glycol modification of trichosanthin: effects on its biological activities, pharmacokinetics, and antigenicity

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modified with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antigenic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sites were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential antigenic determinants, hence masking the antigenic region and prevent binding of antibodies specific to this site. In general, mutation did not bring about significant changes in ribosome-inactivating activity, cytotoxicity, and abortifacient activity of TCS. However, the in vitro activities of PEG modified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activity 50% less than that of nTCS. Pharmacokinetics study indicated that all three PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an IgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylation) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.     

Transgenic tobacco plants with ribosome inactivating protein gene cassin from Cassia occidentalis and their resistance to tobacco mosaic virus]

Cassin, the new gene of ribosome-inactivating protein (RIP) isolated from Cassia occidentalis, was inserted into expression vector pBI121 to produce plant expression vector pBI121-cassin (Figs.1, 2). pBI121-cassin was introduced into tobacco cultivar 'K326' by the Agrobacteriurm tumefaciens transformation method and more than 100 independent transformants were obtained. Southern blot hybridization analysis showed that a single gene locus was inserted into the chromosome of the transgenic tobacco lines (Fig.5) and PCR analysis of segregation population of progeny indicated that the inheritance of transgene was dominant in transgenic lines (Fig.4, Table 1). Results of RT-PCR and Northern blot hybridization analysis showed that transgene could be transcribed correctly (Figs.5, 6) . Three self-pollination lines of transgenic T(1) and T(2) were challenged with TMV at different concentration titers by mechanical inoculation. The transgenic lines exhibited different levels of resistance to TMV with the nontransgenic plants. After both titers of TMV concentration were inoculated, transgenic lines were considered as the highly resistant type with a delay of 4-13 d in development of symptoms and 10%-25% of test plants were infected, while nontransgenic control plants were susceptible typical symptoms on the newly emerged leaves (Table 2). One T(2) line, T(2)-8-2-1, was regarded as an immune type because it did not show any symptoms during 70 d and all plants were shown to be virus free by ELISA tests.     

The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sδ. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A⁵¹ residue on 28Sδ. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T.cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.