Characterization of a cDNA encoding ricin E,a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.     

Homology between ricin and Ricinus communie agglutinin: Amino terminal sequence analysis and protein synthesis inhibition studies

The existence of three forms of ricin and two forms of the Ricinus communis agglutinin (RCA) was established using cation exchange chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis. The preparation of the RCA we obtained was 60–75 times more potent than ricin in the agglutination of erythrocytes, but was about 4% as effective as an inhibitor of cell-free protein synthesis. When reduced with 2-mercaptoethanol, the RCA was activated 3000-fold as an inhibitor of cell-free protein synthesis, whereas ricin was activated about 600-fold by the same treatment. A mixture of the RCA A chains was about one-fifth as effective as the ricin A chain in the inhibition of cell-free protein synthesis. The purified polypeptide subunits of the castor bean lectins were subjected to automated Edman degradation. The sequence for 17 of the first 19 residues of the agglutinin A chain was determined. The first seven residues of the ricin A chain were determined and they are identical with those of the RCA A chain. Nineteen turns of Edman degradation on the RCA B chain resulted in the identification of 18 amino acids. The sequence determined for the first 17 residues of the ricin B chain was identical with that of the RCA B chain. It is likely that the identity of the ricin/RCA A and B chain sequences extends further along the polypeptide chains than the sequences we have determined. The similar structural and catalytic potentials of the RCA and ricin suggest that they bear a precursor-product relationship.     

Real-time cytotoxicity assay for rapid and sensitive detection of ricin from complex matrices

Background

In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits.

Methodology/Findings

This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index–time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material.

Conclusions/Significance

The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices.     

The primary sequence of Ricinus communis agglutinin. Comparison with ricin

A mixture of synthetic oligonucleotides representing all possible sequences of a peptide present in the ricin B chain has been used to screen a cDNA library constructed using ripening castor bean seed poly(A+) RNA. The eight largest recombinant plasmids selected, by hybridization, a single mRNA species whose translational product was identified as preprolectin by immunoprecipitation. Restriction enzyme analysis of these clones demonstrated that two classes were present representing sequences complementary to two distinct but closely related preprolectin mRNA species. The nucleotide sequence of the cloned cDNA from one of these classes encodes preproricin and has been presented elsewhere (Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265-270). The nucleotide sequence of the second class is presented here and shown to represent prepro-Ricinus communis agglutinin. The entire coding sequence was deduced from two overlapping cDNA clones having inserts of 1668 and 1151 base pairs. The coding region defines a preproprotein with a 24-amino acid N-terminal signal sequence preceding the A chain (266 amino acids) which is joined to the B chain (262 amino acids) by a 12-amino acid linking peptide. The protein was confirmed as R. communis agglutinin since the deduced B chain N-terminal sequence corresponds exactly with that determined for purified R. communis agglutinin B chain over a region where several residue differences occur in the ricin B chain. The nucleotide and deduced amino acid sequences of the R. communis agglutinin precursor are compared with those of the ricin precursor.     

Formation of γ-BTC in Flooded Rice Field Soils Treated with γ-BHC

The amino acids of the B-chains of two abrins (designated as abrin-a and abrin-b) from the seeds of Abrus precatorius have been sequenced. The sequence of the B-chain of abrin-a was solved by analysis of peptides derived by enzymatic digestions with trypsin, Iysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The sequence of the B-chain of abrin-b was analyzed by sequence analysis of tryptic peptides and comparing these sequences with those of corresponding peptides of the B-chain of abrin-a. The B-chains of abrin-a and abrin-b consist of 268 amino acid residues and share 256 identical residues. Comparison of their sequences with that of the ricin B-chain shows that 60% of the residues of both abrin B-chains are identical to those of the ricin B-chain and that two saccharide-binding sites in ricin B-chain identified by a crystallographic study are highly conserved in both abrin B-chains.     

Effects of Iodination on Cytoagglutination by and Toxicity of Ricinus communis Lectins

Iodinations of two Ricinus communis lectins, ricin D and hemagglutinin (CBH), with potassium iodide at pH 7.0 and 0°C led to inactivation of the cytoagglutinating activity on sarcoma 180 ascites tumor cells as well as the toxicity to HeLa cells of ricin D, whereas the cytoagglutinating activity of CBH was affected slightly. In the presence of lactose, which binds to ricin D, one tyrosyl residue in the B-chain of ricin D was protected from iodination and 40% of the cytoagglutinating activity was retained. This protection against iodination was not observed in the presence of glucose, which does not bind to ricin D. This suggested that the protected tyrosyl residue in the B-chain of ricin D may be situated at or near the saccharide binding site and directly involved in the binding to the saccharide moieties of the cellular receptors.

Adsorption of the iodinated ricin D to Sepharose 4B indicated that one of the two saccharide binding sites in ricin D is still intact and participates in the binding to saccharide: ricin D was altered from divalent to monovalent by the iodination.

We found from binding experiments with ¹²⁵I-labeled iodinated ricin D to HeLa cells, that the low toxicity of the iodinated ricin D may be attributed mainly to its decreased internalization into the cells and that the divalent binding of ricin D to the cellular receptors is important for this internalization.     

Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D

The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.     

The removal of carbohydrates from ricin with endoglycosidases H,F and D and α-mannosidase

Recently, several investigators have explored the possibility of targetting ricin to designated cell types in animals by its linkage to specific antibodies. There is evidence, however, that the mannose-containing oligosaccharide chains on ricin are recognised by reticuloendothelial cells in the liver and spleen and so cause the immunotoxins to be removed rapidly from the blood stream. In the present study we analysed the carbohydrate composition of ricin and examined enzymic methods for removing the carbohydrate. The carbohydrate analysis ricin A-chain revealed the presence of one residue of xylose and one of fucose in addition to mannose and N-acetylglucosamine which had been detected previously. The B-chain contained only mannose and N-acetylglycosamine. Ricin A-chain is heterogeneous containing two components of molecular weight 30 000 and 32 000. Strong evidence was found that the heavier form of the A-chain contains an extra carbohydrate unit which is heterogeneous with respect to concanavalin A binding and sensitivity to endoglycosidase H. The lower molecular weight form of A-chain did not bind concanavalin A and was insusceptible to endoglycosidases. Only one of the two high mannose oligosaccharide units on the isolated B-chain could be removed by endoglycosidases H or F, whereas both were removable after denaturation of the polypeptide by SDS. Both the isolated A- and B-chains were sensitive to α-mannosidase. Intact ricin was resistant to endoglycosidase treatment and was only slightly sensitive to α-mannosidase. The addition of SDS allowed endoglycosidase H to remove both of the B-chain oligosaccharides from intact ricin and increased the toxin's sensitivity to α-mannosidase. In conclusion, extensive enzymic deglycosylation of ricin may only be possible if the A- and B-chains are first separated, treated with enzymes and then recombined to form the toxin.     

The expression of functional ricin B-chain in Saccharomyces cerevisiae

Yeast cells transformed with plasmids containing ricin B-chain coding sequences expressed this heterologous protein. When ricin B-chain was expressed in a form which resulted in its deposition in the yeast cytosol it formed insoluble aggregates which were devoid of galactose-binding activity. In contrast, when DNA fusions were constructed, in which the B-chain coding sequence was preceded by either the preproalpha-factor leader sequence or the native preproricin signal sequence, the recombinant B-chain products were soluble and biologically active. Both the homologous yeast signal peptide and the heterologous plant signal peptide directed the expressed product into the lumen of the yeast endoplasmic reticulum. As a result, the recombinant B-chain products were processed at the N-terminus, glycosylated and folded into an active conformation, presumably stabilized by correct intrachain disulphide bond formation.     

Interaction of pneumococcal S-14 polysaccharide with lectins from Ricinus communis,Triticum vulgaris, and bandeiraea simplicifolia

Two purified lectins, namely, wheat-germ agglutinin (from Triticum vulgaris) and the hemagglutinin from Ricinus communis seeds, readily form a precipitate with pneumococcal S-14 polysaccharide, whereas the Bandeiraea simplicifolia lectin (BS 1) does not. Exhaustive periodate oxidation and borohydride reduction of S 14 modifies terminal β-D-galactopyranosyl residues, as well as chain D-glucopyranosyl residues, and abolishes reactivity with both the R. communis lectin and wheat-germ agglutinin. Controlled periodate oxidation followed by Smith degradation cleaves only terminal β-D-galactopyranosyl residues, giving a linear polymer, the structure of which was determined by methylation analysis. This derived polymer, containing (1→6)-linked 2-acetamido-2-deoxy-β-D-glucosyl residues, readily precipitated wheat-germ agglutinin, but not the R. communis lectin.     

Volkensin from Adenia volkensii Harms (kilyambiti plant), a type 2 ribosome-inactivating protein.

Volkensin, a type 2 ribosome-inactivating protein from the roots of Adenia volkensii Harms (kilyambiti plant) was characterized both at the protein and nucleotide level by direct amino acid sequencing and cloning of the gene encoding the protein. Gene sequence analysis revealed that volkensin is encoded by a 1569-bp ORF (523 amino acid residues) without introns, with an internal linker sequence of 45 bp. Differences in residues present at several sequence positions (reproduced after repeated protein sequence analyses), with respect to the gene sequence, suggest several isoforms for the volkensin A-chain. Based on the crystallographic coordinates of ricin, which shares a high sequence identity with volkensin, a molecular model of volkensin was obtained. The 3D model suggests that the amino acid residues of the active site of the ricin A-chain are conserved at identical spatial positions, including Ser203, a novel amino acid residue found to be conserved in all known ribosome-inactivating proteins. The sugar binding site 1 of the ricin B-chain is also conserved in the volkensin B-chain, whilst in binding site 2, His246 replaces Tyr248. Native volkensin contains two free cysteinyl residues out of 14 derived from the gene sequence, thus suggesting a further disulphide bridge in the B chain, in addition to the inter- and intrachain disulphide bond pattern common to other type 2 ribosome-inactivating proteins.     

Membrane destabilization by ricin

Ricin is a promising candidate for the treatment of cancer because it can be selectively targeted to tumor cells via linkage to monoclonal antibodies. Biochemical evidence suggests that escape of ricin or its ribosome-inactivating subunit from an intracellular compartment is mediated by retrograde transport to the endoplasmic reticulum and subsequent direction into the ER-associated degradation pathway. Alternatively, lipase activity of ricin may facilitate leakage from endocytic vesicles. We have observed ricin-mediated release of macromolecular dyes from lipid vesicles that mimic the composition of endosomal membranes. Release of small molecules occurs to the same extent, suggesting an all-or-none mechanism due to bilayer destabilization. The level of accompanying membrane fusion depends on vesicle composition. Since it takes 24 h of incubation before the first traces of lysolipids are detectable by matrix-assisted laser desorption/ionization mass spectrometry, membrane destabilization is not due to the lipase activity of ricin.Abbreviations CF Carboxyfluorescein - DPhPC Diphytanoyl-phosphatidylcholine - DPA Dipicolinic acid - EDTA Ethylendiamine-tetracetate - ER Endoplasmic reticulum - ERAD ER-associated degradation - FRET Fluorescence-resonance energy transfer - GM₁ Monosialoganglioside - MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry - MES 2-Morpholino-ethanesulfonic acid - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine) - PC Phosphatidylcholine - PE Phosphatidylethanolamine - PG Phosphatidylglycerol - Rh-PE N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine - RIP Ribosome-inactivating protein - RTA A-chain of ricin - RTB B-chain of ricin - TES N-[Tris-(hydroxymethyl)-methyl]-2-aminoethansulfonic acid - TOF Time-of-flight     

The specific binding of ricin and its polypeptide chains to rat liver ribosomes and ribosomal subunits

Ricin from Ricinus communis was isolated and the binding of ³H-reductively alkylated or ¹²⁵I-iodinated ricin was studied by incubating the toxic protein with ribosomes and isolating the ricin-ribosome complex by centrifugation. Neither of the labeled ricin derivatives nor ³H-labeled A chain bound Escherichia coli ribosomes, but both bound rat liver ribosomes in a reproducible manner. ³H-labeled ricin bound in a ratio of 1 mol/mol of ribosomes with a dissociation constant of 3 μm as calculated from a Scatchard plot. Similarly, ³H-labeled B chain isolated from ricin also bound in a one-to-one complex with a dissociation constant of 1 μm. The binding of ricin and ricin B chain was sensitive to lactose, while the binding of reduced ricin or ricin A chain was not prevented by lactose. Reduced ¹²⁵I-labeled ricin in the presence of lactose and ³H-labeled A chain bound with a ratio of 2 mol/mol of ribosomes. It was further demonstrated that ³H-labeled ricin A chain bound only to the 60S ribosomal subunit and not to the 40S ribosomal subunit. The dissociation constant for the binding was 2 μm both in the presence and absence of lactose and 2 mol of A chain were bound per mole of 60S ribosomal subunit.     

Binding of saccharides to ricin E isolated from small castor beans

The binding of saccharides to ricin E isolated from small castor beans was studied by equilibrium dialysis and spectroscopy. Equilibrium dialysis data indicate that ricin E has two galactose-binding sites, a high affinity site (HA-site) and a low affinity site (LA-site). The binding of specific saccharides to ricin E induces a shift of the fluorescence spectrum to shorter wavelength by 3 nm and UV-difference spectra with a maximum at 290 nm and a negative intensity around 300 nm. The interaction of ricin E with its specific saccharides was analyzed in terms of the variation of the intensity at 320 nm in the fluorescence spectrum and the magnitude of the negative intensity at 300 nm in the UV-difference spectra as functions of saccharide concentration. The results indicate that these spectroscopic changes are representative of the binding of saccharides to the LA-site, which contains a tryptophan residue. By comparing the association constants of saccharides for ricin E with those for ricin D, isolated from the large castor beans, it was found that the HA of ricin E binds saccharides with an affinity of less than one-half that of ricin D, while the saccharide-binding abilities of the LA-site of the two ricins were about the same.     

The specific cytotoxicity of immunoconjugates containing blocked ricin is dependent on the residual binding capacity of blocked ricin: evidence that the membrane binding and A-chain translocation activities of ricin cannot be separated.

Recently we have developed blocked ricin, a derivative of native ricin in which the galactose-binding sites of the B-chain are blocked by covalent modification with affinity ligands. This modification impedes the binding function of the B-chain, while sparing its ability to facilitate the entry of the toxic subunit of ricin, the A-chain, into the cytoplasm. Immunotoxins prepared with blocked ricin approach the cytotoxic potency of native ricin with antibody-dependent specificity. Here we report that the high cytotoxic potency of these immunoconjugates, which is attributed to the preserved translocation function of the ricin B-chain, is dependent on the minimal residual lectin activity of blocked ricin. Our findings support the notion that two functions of ricin, membrane binding and translocation, cannot be separated.     

Preparation and properties of a hybrid toxin of modeccin A-chain and ricin B-chain

Hybrid molecules were prepared from the A- and B-chains of the two toxic lectins ricin and modeccin by dialyzing mixtures of isolated chains to allow a disulfide bridge to be formed between them. Whereas the hybrid consisting of ricin A-chain and modeccin B-chain was non-toxic, the converse hybrid, modeccin A-chain/ricin B-chain, was even more toxic to Vero cells than were the parent toxins, native ricin and modeccin. A number of drugs (NH₄Cl, monensin, trifluoperazine, verapamil, ionophore A23187) which protect cells against modeccin, but not against ricin, protected to some extent against the toxic hybrid, but less so than against native modeccin. The possibility is discussed that the modeccin A-chain of the hybrid may enter the cytosol by two routes, one which is highly efficient and identical to that used by native modeccin and another less efficient one which cannot be used by native modeccin.     

Structures of sugar chains of ricin D

The toxic lectin, ricin D, contains mannose, fucose, xylose, and N-acetylglucosamine as sugar components. Sugar chains are linked to Asn-10 of the A-chain, and to Asn-95 and Asn-135 of the B-chain (Funatsu, G. et al. (1978) Agric. Biol. Chem. 42, 501-503; Araki, T. & Funatsu, G. (1985) FEBS Lett. 191, 121-124). Asparagine-linked sugar chains of each glycopeptide from ricin D were liberated by hydrazinolysis followed by N-acetylation. The reducing end residues of the sugar chains were coupled with 2-aminopyridine and the pyridylamino (PA-) derivatives obtained were purified by gel-filtration and reversed-phase HPLC. Eight main PA-sugar chains were obtained from three glycopeptides and the structures of these sugar chains were determined by component analysis, stepwise exoglycosidase digestions, partial acetolysis, and 500 MHz 1H-NMR spectroscopy. The results show that oligomannose type sugar chains (Man6-7GlcNAc2) are linked to Asn-95; Man5-7 GlcNAc2 and M4X (structure, see below) to Asn-135 of the B-chain, and M3FX and M3X to Asn-10 of the A-chain. (Formula: see text).     

Interaction of ricin and its constituent polypeptides with dipalmitoylphosphatidylcholine vesicles

The interaction of ricin and of its constituent polypeptides, the A- and B-chain, with dipalmitoylphosphatidylcholine (DPPC) vesicles was investigated. The A- and B-chain were individually associated with DPPC vesicles, although the intact ricin was not associated. The maximum binding and association constants were evaluated to be 154 micrograms per mg of DPPC and Ka = 2.30 X 10(5) M-1 for the A-chain, and 87 micrograms per mg of DPPC and Ka = 14.5 X 10(5) M-1 for the B-chain, respectively. The A-chain could induce the phase transition release of carboxyfluorescein from DPPC vesicles to a greater extent than the B-chain, whereas the release induced by the intact ricin was negligible. The evidence indicated that the hydrophobic regions on the A-chain and on the B-chain were buried inside when the two chains constituted the intact ricin molecule through one interchain disulfide bond, and that the A-chain caused perturbation of the DPPC bilayer at the phase transition temperature with consequent leakage of carboxyfluorescein.     

The preparation of deglycosylated ricin by recombination of glycosidase-treated A- and B-chains: effects of deglycosylation on toxicity and in vivo distribution

Deglycosylation of ricin may be necessary to prevent the entrapment of antibody-ricin conjugates in vivo by cells of the reticuloendothelial system which have receptors that recognise the oligosaccharide side chains on the A- and B-chains of the toxin. Carbohydrate-deficient ricin was therefore prepared by recombining the A-chain, which had been treated with alpha-mannosidase, with the B-chain, which had been treated with endoglycosidase H or alpha-mannosidase or both. By recombining treated and untreated chains, a series of ricin preparations was made having different carbohydrate moieties. The removal of carbohydrate from the B-chain did not affect the ability of the toxin to agglutinate erythrocytes, and alpha-mannosidase treatment of the A-chain did not affect its ability to inactivate ribosomes. The toxicity of ricin to cells in culture was only reduced in those preparations containing B-chain that had been treated with alpha-mannosidase, when a 75% decrease in toxicity was observed. The toxicity of the combined ricin preparation to mice varied from double to half that of native ricin, depending on the chain(s) treated and the enzymes used. Removal of carbohydrate greatly reduced the hepatic clearance of the toxin and the levels of toxin in the blood were correspondingly higher. These results suggest that antibody-ricin conjugates prepared from deglycosylated ricin would be cleared more slowly by the liver, inflict less liver damage, and have greater opportunity to reach their target.     

Effect of pH on the conformation and stability of the plant toxin ricin

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.