Detection of Abrin-Like and Prepropulchellin-Like Toxin Genes and Transcripts Using Whole Genome Sequencing and Full-Length Transcript Sequencing of Abrus precatorius.

The sequenced genome and the leaf transcriptome of a near relative of Abrus pulchellus and Abrus precatorius was analyzed to characterize the genetic basis of toxin gene expression. From the high-quality genome assembly, a total of 26 potential coding regions were identified that contain genes with abrin-like, pulchellin-like, and agglutinin-like homology, with full-length transcripts detected in leaf tissue for 9 of the 26 coding regions. All of the toxin-like genes were identified within only five isolated regions of the genome, with each region containing 1 to 16 gene variants within each genomic region (<1 Mbp). The Abrusprecatorius cultivar sequenced here contains genes which encode for proteins that are homologous to certain abrin and prepropulchellin genes previously identified, and we observed substantial diversity of genes and predicted gene products in Abrus precatorius and previously characterized toxins. This suggests diverse toxin repertoires within Abrus, potentially the results of rapid toxin evolution.

A chimeric protein of abrin and Abrus precatorius agglutinin that neutralizes abrin mediated lethality in mice.

Abrin, a type II ribosome inactivating protein from the Abrus precatorius plant, is extremely toxic. It has been shown to be 75 times more potent than its infamous sister toxin, ricin and their potential use in bio-warfare is a cause of major concern. Although several vaccine candidates are under clinical trials for ricin, none are available against abrin. The present study proposes a chimeric protein, comprising of 1-123 amino acids taken from the A chain of abrin and 124-175 amino acids from Abrus precatorius agglutinin A chain, as a vaccine candidate against abrin intoxication. The design was based on the inclusion of the immunogenic region of the full length protein and the minimal essential folding domains required for inducing neutralizing antibody response. The chimera also contains the epitope for the only two neutralizing antibodies; D6F10 and A7C4, reported against abrin till now. Active immunization with the chimera protected all the mice challenged with 45 X LD50 of abrin. Also, passive transfer of antibodies raised against the chimera rescued all mice challenged with 50 X LD50 of toxin. Hence the chimeric protein appears to be a promising vaccine candidate against abrin induced lethality.

Truncated abrin A chain expressed in Escherichia coli: a promising vaccine candidate.

Abrin toxin (AT) is a highly potent toxin, and is classified as one of the most important biological warfare and bioterrorism agents. There is currently no approved vaccine for AT. Therefore, the development of an effective vaccine is important in the prevention of intoxication by abrin. In this study, five vectors containing different gene of truncated abrin toxin A chain (tATA) fragments were constructed, and two of them (tATA1(1-126), tATA4(1-188)) were successfully expressed as a soluble form in E.coli strain. Both of the two tATA retained most of their immunogenicity with either low or no toxic effects as determined by both in vitro and in vivo assays. They were used to immunize BALB/c mice three times at an interval of three weeks apart. As a result, the tATA1 can elicite 80% protective efficacy against i.p. challenge of 5×LD50 of abrin, and the tATA4 provides a better protection, which can elicite 100% protective efficacy against intraperitoneal challenge of 40×LD50 of abrin. The superior fragment (tATA4(1-188)) should be considered as a promising vaccine candidate for further investigations.

A recombinant chimeric protein containing B chains of ricin and abrin is an effective vaccine candidate.

Both ricin toxin (RT) and abrin toxin (AT) are 2 important toxin agents as potantial bioweapons. A dual subunit vaccine against RT and AT exposure is a promising option for developing prophylactic vaccination. In this study, we constructed a dual vaccine with RT B chain and AT B chain named RTB-ATB. The RTB-ATB chimeric protein was expressed in Escherichia coli (E. coli), and the purified protein was used to evaluate the immune response by a 2 × 2 × 2 × 2 factorial design. The main effects included dose of RTB-ATB, route of immunization injection, immunization time interval, and dose of native toxins challenge. For 2 × LD(50) challenge of RT or AT, 100% of the RTB-ATB immunized mice survived and regained or exceeded their initial weights within 10 days. For 4 × LD(50) challenge, different routes of immunization injection caused significant difference (P < 0.05), intraperitoneal (i.p.) administration of immunogen protected mice better than the subcutaneous (s.c.) administration. In conclusion, when administered i.p. to mice with 25 µg per mouse and immunization time interval Π in the absence of adjuvant, the chimeric protein elicited a stronger immune response and protected the animals from a dose of native toxins which was 4 times higher than their LD(50) in unvaccinated mice. Besides, the RTB-ATB chimeric protein could induce specific neutralizing antibodies against these 2 toxins. We anticipate that this study will open new possibilities in the preparation of RTB-ATB dual subunit vaccine against the exposure to deadly RT and AT.

Antitumor effects of recombinant antivascular protein ABRaA-VEGF121 combined with IL-12 gene therapy.

Development and neoplastic progression strongly rely on tumor microenvironment cells. Various kinds of cells that form such tumor milieu play substantial roles in angiogenesis and immunosuppression. Attempts to inhibit tumor vascularization alter tumor milieu and enhance immune response against the tumor. Anticancer therapeutic strategy bringing together antiangiogenic and immunostimulating agents has emerged as a promising approach. We here investigated whether therapy directed against preexisting vessels, combined with an immunomodulatory factor would be equally effective in arresting tumor growth. To this goal, we investigated the effectiveness of ABRaA-vascular endothelial growth factor isoform 121 (VEGF121), an antivascular drug constructed by us. It is a fusion protein composed of VEGF121, and abrin A chain (translation-inhibiting toxin). We used it in combination with interleukin (IL-12) gene therapy and tried to inhibit B16-F10 melanoma tumor growth. ABRaA-VEGF121 is a chimeric recombinant protein capable of destroying tumor vasculature and triggering necrosis in the vicinity of damaged vessels. IL-12 cytokine, in turn, activates both specific and non-specific immune responses. Our results demonstrate that combination of ABRaA-VEGF121 antivascular agent with immunostimulatory cytokine IL-12 indeed inhibits tumor growth more effectively than either agent alone, leading to complete cure of ca. 20 % mice. Post-therapeutic analysis of tumors excised from mice treated with combination therapy showed decreased numbers of blood microvessels in the tumor microenvironment, lowered numbers of regulatory T lymphocytes, as well as showed higher levels of CD4(+) and CD8(+) as compared to control mice. It seems that bringing together antivascular strategy and the action of immunostimulating agents indeed inhibits growth of tumors.

Selection and characterization of human anti-MAGE-A1 scFv and immunotoxin.

Melanoma-associated antigen (MAGE) is expressed on the surface of multiple tumor cell types and is a promising target of biotherapeutic drug delivery via the anti-MAGE-A1 antibody. In this study, a human single-chain variable fragment (scFv) antibody phage library was generated and applied to recombinant MAGE-A1-coated immunotubes by phage display technology. The soluble anti- MAGE-A1 scFv was expressed and purified by immobilized metal-chelated affinity chromatography (IMAC). The anti-MAGE-A1 scFv could bind native MAGE-A1 confirmed by enzyme-linked immunosorbent assay (ELISA), dot blot, and immunoprecipitation (IP) analysis. The immunotoxin was expressed and purified by IMAC successfully. The results indicated that the human anti-MAGE-A1 immunotoxin could provide a valuable drug for clinic cancer therapy.

Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

A recombinant mutant abrin A chain expressed in Escherichia coli can be used as an effective vaccine candidate.

We used site-directed mutagenesis to mutate two key amino acid residues, Glu164 and Arg167, of abrin A chain (ABRA), creating a mutant ABRA(E164AR167L). The mutant ABRA(mABRA) encoded by mABRA(E164AR167L) was expressed in the cytoplasm of Escherichia coli, and used to develop an effective vaccine to protect mice against native abrin intoxication. The cytotoxicity of mABRA was dramatically reduced as compared to that of recombinant ABRA(rABRA) and native abrin, but the antigenicity and immunogenicity remained the same. Balb/c mice were vaccinated with purified mABRA, and survival was evaluated after challenge with native abrin. Mice that were given three vaccinations developed a protective immune response that was 100% protective against an intraperitoneal (i.p.) administration of 10×LD50 of native abrin. Furthermore, the sera from immunized mice provided complete passive protection for naive mice. This study describes the generation of a substantial amount of mABRA from E. coli and the potential application of mABRA as an effective vaccine candidate for humans, to protect against a high-dose of native abrin.

A biophysical elucidation for less toxicity of agglutinin than abrin-a from the seeds of Abrus precatorius in consequence of crystal structure.

X-ray crystal structure determination of agglutinin from Abrus precatorius in Taiwan is presented. The crystal structure of agglutinin, a type II ribosome-inactivating protein (RIP) from the seeds of Abrus precatorius in Taiwan, has been determined from a novel crystalline form by the molecular replacement method using the coordinates of abrin-a as the template. The structure has space group P4(1)2(1)2 with Z = 8, and been refined at 2.6 A to R-factor of 20.4%. The root-mean-square deviations of bond lengths and angles from the standard values are 0.009 A and 1.3 degrees. Primary, secondary, tertiary and quaternary structures of agglutinin have been described and compared with those of abrin-a to a certain extent. In subsequent docking research, we found that Asn200 of abrin-a may form a critical hydrogen bond with G4323 of 28SRNA, while corresponding Pro199 of agglutinin is a kink hydrophobic residue bound with the cleft in a more compact complementary relationship. This may explain the lower toxicity of agglutinin than abrin-a, despite of similarity in secondary structure and the activity cleft of two RIPs.

Chimeric protein ABRaA-VEGF121 is cytotoxic towards VEGFR-2-expressing PAE cells and inhibits B16-F10 melanoma growth.

It has been known that VEGF(121) isoform can serve as a carrier of therapeutic agents targeting tumor endothelial cells. We designed and constructed synthetic cDNA that encodes a chimeric protein comprising abrin-a (ABRaA) toxin A-chain and human VEGF(121). Expression of the ABRaA-VEGF(121) chimeric protein was carried out in E. coli strain BL21(DE3). ABRaA-VEGF(121) preparations were isolated from inclusion bodies, solubilized and purified by affinity and ion-exchanged chromatography (Ni-agarose and Q-Sepharose). Finaly, bacterial endotoxin was removed from the recombinant protein. Under non-reducing conditions, the recombinant protein migrates in polyacrylamide gel as two bands (about 84 kDa homodimer and about 42 kDa monomer). ABRaA-VEGF(121) is strongly cytotoxic towards PAE cells expressing VEGFR-2, as opposed to VEGFR-1 expressing or parental PAE cells. The latter are about 400 times less sensitive to the action of this fusion protein. The biological activity of the ABRaA domain forming part of the chimeric protein was assessed in vitro: ABRaA-VEGF(121) inhibited protein biosynthesis in a cell-free translation system. Preincubation of ABRaA-VEGF(121) with antibody neutralizing the biological activity of human VEGF abolished the cytotoxic effect of the chimeric protein in PAE/KDR cells. Experiments in vivo demonstrated that ABRaA-VEGF(121) inhibits growth of B16-F10 murine melanoma tumors.

Abrin-a A chain expressed as soluble form in Escherichia coli from a PCR-synthesized gene is catalytically and functionally active.

Abrin-a A chain (ABRaA) is a potent plant toxin, which possesses N-glycosylase activity toward eukaryotic 28S rRNA, and may have potential use in cancer therapy. To improve levels of expression in Escherichia coli, the gene encoding ABRaA was optimized by replacing rare codons with high-frequency ones, and synthesized using two-step PCR. The optimized ABRaA was cloned into the pET-His vector, and highly expressed in cytoplasm of E. coli. The yield of the purified recombinant (r) ABRaA proteins was up to 80 mg/l of induced culture. The rABRaA was one-step purified to homogeneity and its RNA-N-glycosylase ability to inhibit protein biosynthesis in a cell-free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The MTT assay showed that it also had a killing effect on human hepatoma cell line SMMC-7721 and myeloma cell line Sp2/0. For the first time, ABRaA expressed as soluble form in E. coli from a PCR-synthesized gene is catalytically and functionally active.

Abrus pulchellus type-2 RIP, pulchellin: heterologous expression and refolding of the sugar-binding B chain.

Abrus pulchellus type-2 RIP, or pulchellin, is a heterodimeric glycoprotein found in A. pulchellus seeds. These chimerolectins, like all type-2 RIPs, are characterized as highly toxic proteins with enzymatic and lectin properties performed by two separate polypeptide subunits. Intending to obtain pure and homogeneous protein for structural and biological studies, the A. pulchellus type-2 RIP lectin subunit or pulchellin binding chain encoding gene fragment (PBC) was cloned. Oligonucleotides based on the sequence homologies between other RIPs like abrin and ricin were synthesized and used to amplify the complete PBC from A. pulchellus genomic DNA. The amplification product was inserted into plasmid pET28a to express the recombinant PBC (rPBC) in Escherichia coli BL21(DE3). The rPBC was expressed as inclusion bodies that were recovered and denatured in a buffer containing urea. Repeated dialysis rounds against the oxidation buffer, which presented the redox pair cysteine-cystine, D-galactose, and decreasing urea concentrations, conducted the protein refolding. The refolding process of rPBC was successfully confirmed by biological assays and circular dichroism.

Cloning, expression of the abrin-a A-chain in Escherichia coli and measurement of the biological activities in vitro.

The coding sequence of abrin-a A-chain (ABRaA) gene was obtained by RT-PCR and cloned into the expression vector pET28b. The mature ABRaA has been highly expressed in the cytoplasm of Escherichia coli by 1 mmol/L IPTG induction, and the yield of the soluble recombinant protein was 4 mg/L of induced culture. The recombinant ABRaA was purified to be homogeneity. The biological activities of expressed ABRaA were demonstrated in vitro. It strongly inhibited the protein biosynthesis of rabbit reticulocyte lysates, with an IC(50) of 0.08 nmol/L. It also depurinated 28 S rRNA through cleaving at the A4324 site in rat liver ribosomes by its N-glycosidase activity. These data suggested that the recombinant ABRaA could be used for the preparation of immunotoxins as a potential cancer chemotherapeutic agent.

Restoration of lectin activity to an inactive abrin B chain by substitution and mutation of the 2 gamma subdomain.

Abrin is a heterodimeric plant protein that occurs in several isoforms (abrin-a, abrin-b, abrin-c and abrin-d), whose B chains are believed to either have (abrin-a and abrin-d) or lack (abrin-b and abrin-c) the ability to bind galactose. The 5' signal sequence and toxin B chain (ATB)-coding region were excised from a preproabrin cDNA [K. A. Wood, J. M. Lord, E. J. Wawrzynczak, and M. Piatak (1991) Eur. J. Biochem. 198, 723-732], tentatively identified as abrin-c, which was predicted to lack lectin activity, and fused in-frame to generate pre-ATB cDNA. Transcripts, synthesized in vitro from pre-ATB cloned into the transcription vector pSP64T, were expressed after microinjection into Xenopus oocytes. The recombinant ATB was shown, using a qualitative sugar-binding assay, to be devoid of lectin activity. Lectin activity could not be restored to this nonbinding ATB by replacing the 2 gamma subdomain with the corresponding galactose-binding 2 gamma subdomain from ricin B chain, but it was restored by replacement with the active galactose-binding 2 gamma subdomain from a different abrin isoform (abrin-a). The putative galactose-binding pocket of the nonbinding ATB 2 gamma subdomain contained a His residue at the position occupied by a residue with an aromatic side chain (Tyr or Trp) in functional 2 gamma subdomains. Mutationally converting this His to either Tyr or Trp restored lectin activity to the nonbinding ATB, emphasizing the contribution of an aromatic side chain in a functional 2 gamma subdomain galactose-binding site for members of this lectin family.

Identification of amino acid residues of abrin-a A chain is essential for catalysis and reassociation with abrin-a B chain by site-directed mutagenesis.

Abrin is a toxic protein consisting of two subunits, an enzymatic A chain (ABRaA) and a lectin-active B chain (ABRaB), linked by a disulfide bond. Site-directed mutagenesis was performed using PCR to study how the conserved amino acid residues, Tyr74, Tyr113, Glu164 and Trp198, around the active site of ABRaA are involved in enzyme catalysis, enzyme-substrate recognition and reassociation of ABRaA with ABRaB. The protein biosynthesis inhibitory activities of Y74F, Y113F and W198F were decreased moderately to that of wild type reABRaA, while that of E164Q decreased dramatically. Kinetic analysis showed that the kat of Y74F, Y113F and W198F resembled that of wild type, while the Km increased significantly. W198F did not reassociate with ABRaB to form heterodimers, while Y74F, Y113F and E164Q did. SDS-PAGE analysis of ABRaA treated with trypsin showed that reABRaA, Y74F, Y113F and E164Q survived digestion, whereas W198F was not protected from digestion. CD spectra revealed that W198F showed significant conformational changes. These observations suggest that E164 is directly involved in catalysis, and Tyr74, Tyr113 and Trp198 in substrate binding, while Trp198 also plays an important role in maintaining the conformation of ABRaA required for its reassociation with ABRaB.

Probing the domain structure of abrin-a by tryptic digestion.

Abrin-a is a potent plant toxin that consists of A and B chains linked by a disulfide bond. The abrin-a A chain (AaTA) has N-glycosidase activity while the abrin-a B chain (AaTB) has galactose-binding activity. By partial tryptic digestion, the domain structure of abrin-a was investigated. Seven tryptic fragments with molecular masses greater than 3500 Da were isolated and characterized. One fragment, designated T-21 and consisting of 153 amino acid residues, contained the major part of the second domain of AaTB and, after cross-linking of T-21 with glutaraldehyde, the reaction product had the same level of hemagglutinating activity as native abrin. When the T-21 fragment was conjugated with AaTA, the conjugate inhibited protein biosynthesis in HeLa cells. This suggests that the T-21 fragment is able to bind specifically to cells; its conjugate facilitates membrane translocation of AaTA into cells and consequently inhibits protein biosynthesis. T-21, with a molecular mass less than AaTB, is therefore a potentially useful substance for the preparation of immunotoxins.

Crystal structure of abrin-a at 2.14 A.

The crystal structure of abrin-a, a type II ribosome-inactivating protein from the seeds of Abrus precatorius, has been determined from a novel crystalline form by the molecular replacement method using the coordinates of ricin. The structure has been refined at 2.14 A to a R-factor of 18.9%. The root-mean-square deviations of bond lengths and angles from the standard values are 0.013 A and 1.82 degrees, respectively. The overall protein folding is similar to that of ricin, but there are differences in the secondary structure, mostly of the A-chain. Several parts of the molecular surface differ significantly; some of them are quite near the active site cleft, and probably influence ribosome recognition. The positions of invariant active site residues remain the same, except the position of Tyr74. Two water molecules of hydrogen-bonded active site residues have been located in the active site cleft. Both of them may be responsible for hydrolyzing the N-C glycosidic bond. The current abrin-a structure is lactose free; this is probably essential for abrin-a crystallization. The B-chain is a glycoprotein, and the positions of several sugar residues of two sugar chains linked to earlier predicted glycosylation sites were determined. One of the sugar chains is a bridge between two neighboring molecules, since one of its mannose residues is connected to the galactose binding site of the neighboring molecule. Another sugar chain covers the surface of the B-chain.

Primary structure of three distinct isoabrins determined by cDNA sequencing. Conservation and significance.

A family of toxic proteins, the isoabrins, which possess N-glycosylase activity toward eukaryotic 28 S r-RNA, may have potential use in cancer chemotherapy. By polymerase chain reaction techniques, cDNA clones of three isoabrins, carrying A and B-chain sequences, were isolated and their nucleotide sequences were determined. The isoabrins consist of an A-chain comprised of 250 of 251 amino acids, followed by a 10 amino acid linker and a B-chain of 267 amino acids. There is substantial conservation in the B-chain of the three isoabrins, with less than six amino acid substitutions, whereas as many as 46 amino acid substitutions occur in the A-chains. Based on the relationships between the biological activities and the putative amino acid sequences of the isoabrins, three isoabrins, abrin-a, -b and -d, could be identified and the potential epitope of immunological response of these isoabrins could be assigned.

The complete primary structure of abrin-a B chain.

The complete 267 amino acid sequence of abrin-a B chain was determined by analysis of peptides obtained by digestion with trypsin, chymotrypsin, lysyl endopeptidase, Staphylococcus aureus V8 protease and thermolysin. The sequence is not identical with that predicted previously by nucleotide sequencing, indicating the presence of isoforms of abrin. Comparison of the amino acid sequence of abrin-a B chain with that of ricin-D B chain reveals a high degree of sequence identity (59%). Abrin-a B chain appears to consist of two domains, each domain with subdomains (alpha, beta, gamma) of about 40 amino acid residues.

Conserved amino acid residues in ribosome-inactivating proteins from plants.

The amino acid sequences of eleven RIPs sequenced to date have been compared in the expectation that this would be useful in the location of functionally and/or structurally important sites of these molecules. In addition to several highly conserved hydrophobic amino acids, thirteen absolutely conserved residues have been found in ricin A-chain: Tyr21, Phe24, Arg29, Tyr80, Tyr123, Gly140, Ala165, Glu177, Ala178, Arg180, Glu208, Asn209 and Trp211. The role of these residues as well as of the C-terminal region have been discussed based on the results of chemical and enzymatic modifications, site-directed mutagenesis, and deletion studies.