Mass Spectrometric Detection and Differentiation of Enzymatically Active Abrin and Ricin Combined with a Novel Affinity Enrichment Technique.

Abrin and ricin are toxic proteins produced by plants. Both proteins are composed of two subunits, an A-chain and a B-chain. The A-chain is responsible for the enzymatic activity, which causes toxicity. The B-chain binds to glycoproteins on the cell surface to direct the A-chain to its target. Both toxins depurinate 28S rRNA, making it impossible to differentiate these toxins based on only their enzymatic activity. We developed an analytical workflow for both ricin and abrin using a single method and sample. We have developed a novel affinity enrichment technique based on the ability of the B-chain to bind a glycoprotein, asialofetuin. After the toxin is extracted with asialofetuin-coated magnetic beads, an RNA substrate is added. Then, depurination is detected by a benchtop matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometer to determine the presence or absence of an active toxin. Next, the beads are subjected to tryptic digest. Toxin fingerprinting is done on a benchtop MALDI-TOF MS. We validated the assay through sensitivity and specificity studies and determined the limit of detection for each toxin as nanogram level for enzymatic activity and µg level for toxin fingerprinting. We examined potential cross-reactivity from proteins that are near neighbors of the toxins and examined potential false results in the presence of white powders.

Bio-barcode triggered isothermal amplification in a fluorometric competitive immunoassay for the phytotoxin abrin.

Abrin is one of the most toxic phytotoxins to date, and is a potential biological warfare agent. A bio-barcode triggered isothermal amplification for fluorometric determination of abrin is described. Free abrin competes with abrin-coated magnetic microparticles (MMP) probes to bind to gold nanoparticle (AuNP) probes modified with abrin antibody and bio-barcoded DNA. Abundant barcodes are released from the MMP-AuNP complex via dithiothreitol treatment. This triggers an exponential amplification reaction (EXPAR) that is monitored by real-time fluorometry, at typical excitation/emission wavelengths of 495/520 nm. The EXPAR assay is easily operated, highly sensitive and specific. It was used to quantify abrin in spiked commercial samples. The detection limit (at S/N = 3; for n = 6) is 5.6 pg·mL-1 which is considerably lower than previous reports. This assay provides a universal sensing platform and has great potential for determination of various analytes, including small molecules, proteins, DNA, and cells. Graphical abstract Schematic representation of the bio-barcode triggered exponential amplification reaction (EXPAR) for a fluorometric competitive immunoassay for abrin. The limit of detection is 5.6 pg mL-1 with a large dynamic range from 10 pg mL-1 to 1 µg mL-1.

Rapid Detection of Abrin Toxin and Its Isoforms in Complex Matrices by Immuno-Extraction and Quantitative High Resolution Targeted Mass Spectrometry.

Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1-1 µg/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, we have developed an innovative and rapid multiepitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin-containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Using an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 h. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation, and simultaneous quantification of abrin and its isoforms by mass spectrometry.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Sequestration of the abrin A chain to the nucleus by BASP1 increases the resistance of cells to abrin toxicity.

Abrin, a type II ribosome-inactivating protein, comprises A and B subunits wherein the A subunit harbours toxin activity and the B subunit has a galactose-specific lectin activity. The entry of the protein inside the cell is through the binding of the B chain to cell surface glycoproteins followed by receptor-mediated endocytosis and retrograde transport. A previous study from our laboratory showed that different cell lines exhibited differences of as great as ~200-fold in abrin toxicity, prompting the present study to compare the trafficking of the toxin within cells. Observations made in this regard revealed that the abrin A chain, after being released into the cytosol, is sequestered into the nucleus through interaction with a cellular protein of ~25 kDa, BASP1 (brain acid-soluble protein 1). The nuclear localization of the A chain is seen predominantly in cells that are less sensitive to abrin toxicity and dependent on the levels of BASP1 in cells. The sequestration by BASP1 renders cells increasingly resistant to the inhibition of protein synthesis by abrin and the nucleus act as a sink to overcome cellular stress induced by the toxin.

A functional quantitative polymerase chain reaction assay for ricin, Shiga toxin, and related ribosome-inactivating proteins.

The potent toxins ricin, abrin, and other ribosome-inactivating proteins deadenylate a specific base in 28S ribosomal RNA that destroys ribosomes and leads to cell death. We have taken advantage of the fact that reverse transcriptase preferentially inserts an adenine opposite to an abasic site in RNA to create a quantitative polymerase chain reaction (PCR) assay to detect the damage. This assay detects as little as 30pg of ricin. We used the assay to study enzymatic properties of ricin such as pH and temperature optima (pH 4.5-5.0 and 60 degrees C).

Diphtheria toxin entry into cells is facilitated by low pH.

At neutral pH, NH4Cl and chloroquine protected cells against diphtheria toxin. A brief exposure of the cells to low pH (4.5-5.5) at 37 degrees completely abolished this protection. When, to cells preincubated with diphtheria toxin and NH4Cl, neutralizing amounts of anti-diphtheria toxin were added before the pH was lowered, the toxic effect was considerably reduced, but it was not completely abolished. A much stronger toxic effect was seen when antibodies were added immediately after incubation at low pH. Upon a short incubation with diphtheria toxin at low pH, the rate of protein synthesis in the cells decreased much faster than when the normal pH was maintained. The data suggest that, at low pH, diphtheria toxin (or its A fragment) penetrates directly through the surface membrane of the cell. The possibility is discussed that, when the medium has a neutral pH, the entry of diphtheria toxin involves adsorptive endocytosis and reduction of the pH in the vesicles possibly by fusion with lysosomes. Low pH did not facilitate the entry of the closely related toxins abrin, ricin, and modeccin.

Structural basis for neutralization of cytotoxic abrin by monoclonal antibody D6F10.

Abrin, an extremely cytotoxic Type II ribosome-inactivating protein (RIP), is a potential bio-warfare agent. Abrin A-chain (ABA) depurinates an adenosine of sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, thereby arresting protein synthesis and leading to cell death. Monoclonal antibody (mAb) D6F10 is the only known antibody that neutralizes ABA's activity in cell-free systems as well as abrin's toxicity in vitro and in vivo. However, how binding of mAb D6F10 to abrin interferes with abrin's catalytic activity at ribosome is still poorly understood. To provide structural basis for mAb D6F10-mediated rescue of ribosome inactivation by abrin, we determined crystal structures of ABA with and without substrate analogs. The structures of ABA-substrate analogs and ribosome were used in an experiment-guided computational protocol, to construct the ABA-Ribosome complex. A homology model of the variable region (Fv ) of mAb D6F10 was generated and docked with the apo-ABA structure to construct the ABA-D6F10 Fv complex. Structural superposition of ABA common to ABA-D6F10 Fv and ABA-Ribosome complexes reveals steric hindrance as the primary mechanism by which mAb D6F10 neutralizes abrin. In contrast to ABA alone, ABA bound to mAb D6F10 is unable to access the SRL on the ribosome owing to steric clashes of mAb D6F10 with the ribosome. Crystal structures of ABA also reveal a catalytic water molecule implicated in hydrolyzing N-glycosidic bond of the susceptible adenosine by RIPs. Furthermore, our strategy provides structural details of steric hindrance important for neutralization of ricin, another RIP, by mAb 6C2 and hence is of wide applicability. ENZYME: EC3.2.2.22. DATABASE: Structural data have been deposited in the Protein Data Bank (PDB) under the accession numbers 5Z37, 5Z3I, and 5Z3J.

[Ribosome-inactivating lectins from plants].

There is a heterogeneous group of plant proteins which are able to enzymatically inactivate ribosomes by depurination of an invariant adenine from the 28 S ribosomal RNA. Some of these proteins are heterodimers having a lectin subunit which is joined by disulfide bond to the enzymatic subunit. Ricin and abrin which are among the most toxic substances known belong to the last group. This review focuses on the structure of the heterodimeric plant ribosome-inactivating proteins, the way of their action on ribosome, biosynthesis, intracellular trafficking, and their possible usage in medicine.

Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals.

Receptors for a cytotoxic lectin, abrin and their role in cell intoxication.

The nature of binding of abrin to Chinese hamster ovary cells was examined in relation to the ensuing intoxication of the treated cells. Approx. 20% of [125I] abrin bound to CHO cells at 37 degree C was found to be resistant to the addition or presence of 0.1 M lactose. The extent of lactose-resistant binding depended inversely upon the temperature of incubation. Among various proteins, lectins and sugars, only non-labeled abrin could strongly inhibit the lactose-resistant binding of [125I] abrin. Lactose-resistant binding could lead to an inhibition of cellular protein synthesis and to a loss of cell viability. Abrin molecules bound at the lactose-sensitive and lactose-resistant binding sites apparently have an equal probability of being internalized by CHO cells. Binding of approx. 3.10(3) abrin molecules per CHO cell was required to elicit 50% loss of cell viability regardless of whether the binding occurs in the presence or absence of lactose. The result of a cross-linking experiment suggested that a membrane protein with an Mr of about 45 000 may be responsible for the lactose-resistant binding of abrin.

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.

Suppression of DTT-induced aggregation of abrin by alphaA- and alphaB-crystallins: a model aggregation assay for alpha-crystallin chaperone activity in vitro.

The eye lens small heat shock proteins (sHSP), alphaA- and alphaB-crystallins, have been shown to function like molecular chaperones, both in vitro and in vivo. It is essential to assess the protective effect of alphaA- and alphaB-crystallins under native conditions to extrapolate the results to in vivo conditions. Insulin and alpha-lactalbumin have widely been used to investigate the chaperone mechanism of alpha-crystallin under native conditions. Due to its smaller size, insulin B-chain may not represent the binding of putative physiological substrate proteins. As it stands, the aggregation of alpha-lactalbumin and binding of alpha-crystallin to it varies under different experimental conditions. Abrin, a ribosome inactivating protein isolated from the seeds of Abrus precatorius, consists of a 30 kDa A-chain and a lectin-like B-chain of 33 kDa joined by a single disulfide bond. Reduction of the disulfide link between the two chains of abrin leads to the aggregation of the B-chain. In this study, we demonstrate that dithiothreitol (DTT)-induced aggregation of abrin B-chain could be monitored by light scattering similar to that of insulin. Moreso, this process could be suppressed by recombinant human alphaA- and alphaB-crystallins in a concentration dependent manner, notably by binding to aggregation prone abrin B-chain. SDS-PAGE and HPLC gel filtration analysis indicate that there is a soluble complex formation between alpha-crystallin and abrin B-chain. Interestingly, in contrast to insulin, there is no significant difference between alphaA- and alphaB-crystallin in suppressing the aggregation of abrin B-chain at two different temperatures (25 and 37 degrees C). HSP26, an another small heat shock/alpha-crystallin family protein, was also able to prevent the DTT-induced aggregation of abrin. These results suggest that due to relatively larger size of its B-chain (33 kDa), compared to insulin B-chain (about 3 kDa), abrin may serve as a better model substrate for in vitro chaperone studies of alpha-crystallin and as well as other sHSP.

Native and/or asialo-Tamm-Horsfall glycoproteins Sd(a+) are important receptors for Triticum vulgaris (wheat germ) agglutinin and for three toxic lectins (abrin-a, ricin and mistletoe toxic lectin-I).

The binding properties of human Tamm-Horsfall Sd(a+) urinary glycoprotein (THGP) and asialo-THGP with Triticum vulgaris agglutinin(WGA) and three toxic lectins (abrin-a, ricin, and Mistletoe toxic lectin-I) were investigated by quantitative precipitin and precipitin inhibition assays. Both glycoproteins reacted strongly with abrin-a, precipitating over 80% of the lectin nitrogen tested. THGP also bound well to mistletoe toxic lectin-I and precipitated 86% of this lectin added, while the precipitability of its asialo product decreased by 28%. The native glycoprotein completely precipitated the WGA added, but its reactivity was reduced dramatically after desialylation. On the contrary, the poor reactivity of THGP with ricin increased substantially after removal of sialic acid and completely precipitated the lectin added. The glycoprotein-lectin interactions were inhibited by one or several of the following haptens, p-NO2-phenyl alpha GalNAc, p-NO2-phenyl beta GalNAc, Gal beta 1-->4GlcNAc, Gal beta 1-->4Glc, GlcNac beta 1-->4GlcNAc and/or GlcNAc. From the above results, it is concluded that native and/or Tamm-Horsfall glycoproteins serve as important receptors for these three toxic lectins and for WGA.

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.

The history of ricin, abrin and related toxins.

Ricin, abrin and related plant toxins have played interesting and important roles in the history of clinical medicine and biomedical research. The use of these proteins in medical treatment since ancient times is reviewed. Later the proteins played important roles in the early days of immunological research and some of the fundamental principles of immunology were discovered with toxic proteins of this group. During the last three decades the mechanism of action of the toxins was elucidated. This led to a major effort to target the toxins to malignant cells. Ricin has been used in bioterrorism. Recently, the toxins have played important roles as experimental models to elucidate the intracellular trafficking of endocytosed proteins.

Carbohydrate specificity of a toxic lectin, abrin A, from the seeds of Abrus precatorius (jequirity bean).

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Gal beta1-->4GlcNAc (II), Gal alpha1-->4Gal (E), and Gal beta1-->3GalNAc (T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the (D)Fuc pyranose ring to penta-saccharides and probably internal Gal alpha,beta-->; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Gal alpha1-->3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I >> Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.

Abrin poisoning.

Abrin is a toxic protein obtained from the seeds of Abrus precatorius (jequirity bean), which is similar in structure and properties to ricin. Abrin is highly toxic, with an estimated human fatal dose of 0.1-1 microgram/kg, and has caused death after accidental and intentional poisoning. Abrin can be extracted from jequirity beans using a relatively simple and cheap procedure. This satisfies one criterion of a potential chemical warfare agent, although the lack of large scale production of jequirity seeds means that quantity is unavailable for ready mass production of abrin for weapons. This contrasts with the huge cultivation of Ricinus seeds for castor oil production. At the cellular level, abrin inhibits protein synthesis, thereby causing cell death. Many of the features observed in abrin poisoning can be explained by abrin-induced endothelial cell damage, which causes an increase in capillary permeability with consequent fluid and protein leakage and tissue oedema (the so-called vascular leak syndrome). Most reported cases of human poisoning involve the ingestion of jequirity beans, which predominantly cause gastrointestinal toxicity. Management is symptomatic and supportive. Experimental studies have shown that vaccination with abrin toxoid may offer some protection against a subsequent abrin challenge, although such an approach is unlikely to be of benefit in a civilian population that in all probability would be unprotected.

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.