Studies on the structure and properties of the lectins from Abrus precatorius and Ricinus communis.

The amino acid composition of the isolated A- and B-chains of the toxic lectins abrin and ricin was determined and compared. Even though the two toxins originate from widely different plants, statistical analysis of the amino acid content indicates extensive homologies in the amino acid sequence of the 4 chains. The intact lectins contain no free SH-groups whereas the isolated A- and B-chains contain close to one free SH-group each. The results indicate that in both toxins the A- and B-chains are connected by a single S-S bond. The B-chains of abrin and ricin contain similar amounts of mannose and glucosamine. The A-chain of ricin also contains some carbohydrate, whereas the A-chain of abrin appears not to be a glycoprotein. The non-toxic abrus and ricinus agglutinins contain more carbohydrate than abrin and ricin. The isoelectric points of the different lectin preparations were measured by isoelectrofocusing. The intact lectins are much more resistant to heat, freezing and chemical treatments than the isolated A- and B-chains. The intact lectins are also very resistant to treatment with proteolytic enzymes, whereas the isolated chains are easily digested. Evidence indicating that the toxins and their chains undergo extensive conformational changes upon reduction of the S-S bond is discussed.     

Identification of the ricin lipase site and implication in cytotoxicity

Ricin is a heterodimeric plant toxin and the prototype of type II ribosome-inactivating proteins. Its B-chain is a lectin that enables cell binding. After endocytosis, the A-chain translocates through the membrane of intracellular compartments to reach the cytosol where its N-glycosidase activity inactivates ribosomes, thereby arresting protein synthesis. We here show that ricin possesses a functional lipase active site at the interface between the two subunits. It involves residues from both chains. Mutation to alanine of catalytic serine 221 on the A-chain abolished ricin lipase activity. Moreover, this mutation slowed down the A-chain translocation rate and inhibited toxicity by 35%. Lipase activity is therefore required for efficient ricin A-chain translocation and cytotoxicity. This conclusion was further supported by structural examination of type II ribosome-inactivating proteins that showed that this lipase site is present in toxic (ricin and abrin) but is altered in nontoxic (ebulin 1 and mistletoe lectin I) members of this family.     

The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins

Ricin is a potent cytotoxic protein derived from the higher plant Ricinus communis that inactivates eukaryotic ribosomes. In this paper we have studied the mechanism of action of ricin A-chain on rat liver ribosomes in vitro. Our findings indicate that the toxin inactivates the ribosomes by modifying both or either of two nucleoside residues, G4323 and A4324, in 28 S rRNA. These nucleotides are located close to the alpha-sarcin cleavage site and become resistant to all ribonucleases tested. The examination of the lability of phosphodiester bonds of these nucleotides to both mild alkaline digestion and aniline treatment at acidic pH suggests that the base of A4324 is removed by the toxin. This unique activity of ricin A-chain was also observed when naked 28 S rRNA is used as a substrate, indicating that the toxin directly acts on the RNA. Similar activity on 28 S rRNA is also exhibited by abrin and modeccin, ricin-related toxins, suggesting a general mechanistic pathway for ribosome inactivation by lectin toxins.     

Brefeldin-A enhancement of ricin A-chain immunotoxins and blockade of intact ricin, modeccin, and abrin

After binding, the protein toxins ricin, abrin, and modeccin are endocytosed and processed through the cell's vesicular system in a poorly understood fashion, prior to translocation to the cytosol. The role of the Golgi apparatus in toxin processing was studied using brefeldin-A (BFA), a fungal metabolite which blocks Golgi function. At concentrations that inhibit secretion of interleukin-2 (IL-2), BFA blocks ricin, modeccin, and abrin intoxication of a lymphocyte derived cell line (Jurkat). Paradoxically, BFA enhances the toxicity of two ricin A-chain immunotoxins targeted against distinct cell surface determinants. BFA concentrations which are optimal for immunotoxin enhancement are below those needed to affect ricin intoxication or IL-2 secretion. BFA blockade of ricin does not involve effects on ricin endocytosis, toxin translocation to the cytosol, or the enzymatic activity of toxin A-chain. In contrast, BFA has no effect on immunotoxin processing but does enhance the immunotoxin translocation step. It is concluded that: 1) intact Golgi function is required for holotoxin processing. 2) Intact Golgi function is not required for holotoxin translocation. 3) Golgi function is tightly linked to immunotoxin translocation. 4) BFA has effects on vesicular routing in addition to the block of Golgi function in secretion which has been reported.     

Conformation similarities of ricin A-chain and trichosanthin

The conformation of ricin A-chain from castor bean was studied by circular dichroism at pH 4.7, 7 and 9 and compared with that of trichosanthin from the Chinese herb Tianhuafen. The CD spectra of ricin A-chain and trichosanthin were nearly identical at each of the three pHs. Analysis of the data indicated that, like trichosanthin, ricin A-chain had about 29% alpha-helix and 42% beta-sheet but no beta-turn. However, there was a subtle difference in the CD spectra in 20 mM sodium dodecyl sulfate, the addition of which at pH 7 slightly increased the helicity and decreased the content of beta-sheet of ricin A-chain in contrast to a larger increase in helicity at the expense of beta-sheet for trichosanthin, thus indicating a different stability against the surfactant. Native ricin A-chain and trichosanthin had about the same amount of secondary structure, which supports the belief that a high degree of sequence homology of the two proteins [Zhang & Wang (1986) Nature 321, 477-478] may lead to a conformational similarity between them, even though the two proteins are not taxonomically related.     

Ribosome inactivation by the toxic lectins abrin and ricin. Kinetics of the enzymic activity of the toxin A-chains.

A sensitive test system for toxin-treated ribosomes was worked out by treating rabbit reticulocyte ribosomes with abrin A-chain, ricin A-chain or ricinus agglutinin A-chain, adding neutralizing amounts of specific antitoxins and testing for polyphenylalanine-synthesizing activity in a system where the concentration of elongation factors and ribosomes were varied. The strongest inhibition was obtained in the presence of low concentrations of elongation factor (EF-2). The activity of the ribosomes decreased with time of incubation with the toxin A-chains. Addition of anti-toxins stopped further inactivation. In systems containing untreated and toxin-treated ribosomes the ability to polymerize phenylalanine was proportional to the concentration of untreated ribosomes. There was a linear relationship between toxin A-chain concentration and the number of ribosomes inactivated per minute. The inactivation rate increased with temperature, and the estimated activation energy was 10.6 kcal (44.3 kJ). Linewaver-Burk plots of the data obtained by incubating various ribosome concentrations with toxins indicated a molecular activity of about 1500 ribosomes/minute for abrin and ricin A-chains and 100 ribosomes/minute for ricinus agglutinin A-chain. The apparent Michaelis constant was 0.1-0.2 muM for all three A-chains. The activity of the A-chains in the intact cell is discussed.     

Ribosomal RNA identity elements for ricin A-chain recognition and catalysis

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose at position A4324 in eukaryotic 28 S rRNA. The requirements for the recognition by ricin A-chain of this nucleotide and for the catalysis of cleavage were examined using a synthetic oligoribonucleotide that reproduces the sequence and the secondary structure of the RNA domain (a helical stem, a bulged nucleotide, and a 17-member single-stranded loop). The wild-type RNA (35mer) and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type oligoribonucleotide the ricin A-chain catalyzed reaction has a Km of 13.55 microM and a Kcat of 0.023 min-1. Recognition and catalysis by ricin A-chain has an absolute requirement for A at the position that corresponds to 4324. The helical stem is also essential; however, the number of base-pairs can be reduced from the seven found in 28 S rRNA to three without loss of identity. The nature of these base-pairs can affect catalysis. A change of the second set from one canonical (G.C) to another (U.A) reduces sensitivity to ricin A-chain; whereas, a change of the third pair (U.A----G.C) produces supersensitivity. The bulged nucleotide does not contribute to identification. Hydrolysis is affected by altering the nucleotides in the universal sequence surrounding A4324 or by changing the position in the loop of the tetranucleotide GA(ricin)GA: all of these mutants have a null phenotype. If ribosomes are treated first with alpha-sarcin to cleave the phosphodiester bond at G4325 ricin can still catalyze depurination at A4324. This implies that cleavage by alpha-sarcin at the center of what has been presumed to be a 17 nucleotide single-stranded loop in 28 S rRNA produces ends that are constrained in some way. On the other hand, hydrolysis by alpha-sarcin of the corresponding position in the synthetic oligoribonucleotide prevents recognition by ricin A-chain. The results suggest that the loop has a complex structure, affected by ribosomal proteins, and this bears on the function in protein synthesis of the alpha-sarcin/ricin rRNA domain.     

Structural and functional studies of cinnamomin, a new type II ribosome-inactivating protein isolated from the seeds of the camphor tree.

Cinnamomin is a new type II ribosome-inactivating protein (RIP). Its A-chain exhibits RNA N-glycosidase activity to inactivate the ribosome and thus inhibit protein synthesis, whereas the glycosylated B-chain is a lectin. The primary structure of cinnamomin, which exhibits approximately 55% identity with those of ricin and abrin, was deduced from the nucleotide sequences of cDNAs of cinnamomin A- and B-chains. It is composed of a total of 549 amino-acid residues: 271 residues in the A-chain, a 14-residue linker and 264 residues in the B-chain. To explore its biological function, the cinnamomin A-chain was expressed in Escherichia coli with a yield of 100 mg per L of culture, and purified through two-step column chromatography. After renaturation, the recovery of the enzyme activity of the expressed A-chain was 80% of that of native A-chain. Based on the modeling of the three-dimensional structure of the A-chain, the functional roles of five amino acids and the only cysteine residues were investigated by site-directed mutagenesis or chemical modification. The conserved single mutation of the five amino-acid residues led to 8-50-fold losses of enzymatic activity, suggesting that these residues were crucial for maintaining the RNA N-glycosidase activity of the A-chain. Most interestingly, the strong electric charge introduced at the position of the single cysteine in A-chain seemed to play a role in enzyme/substrate binding.     

Purification of immunotoxins containing ricin A-chain and abrin A-chain using Blue Sepharose CL-6B

We describe a method for separating antibody from immunotoxins by affinity chromatography on Cibacron blue F3GA coupled to Sepharose (Blue Sepharose). The antibody did not bind to the gel. The immunotoxins were bound by their ricin A-chain or abrin A-chain moiety and could be recovered in high yield and purity using mild elution conditions. The method is suitable for the large-scale purification of immunotoxins.     

The RNA N-glycosidase activity of ricin A-chain

The RNA N-glycosidase activity of ricin A-chain has been characterized. When rat liver ribosomes were used as substrates, the A-chain cleaved the N-glycosidic bond at A-4324 in 28S rRNA. An apparent Michaelis constant (Km) for the reaction was determined to be 2.6 microM and the turnover number (Kcat) was 1777 min-1. When naked rRNA was the substrate, the A-chain cleaved the same bond in 28S rRNA but at a greatly reduced rate. The Km value was 5.8 microM. The results suggest that the A-chain has a similar affinity for 28S rRNA in both ribosomes and the naked states. When the deproteinized Escherichia coli rRNA was the substrates, ricin A-chain cleaved a N-glycosidic bond at A-2600 in 23S rRNA which corresponds to the ricin-site in 28S rRNA of rat liver ribosomes, while the A-chain has little activity on 23S rRNA in the ribosomes. The results suggest that ricin A-chain acts directly on RNA by recognizing a certain structure in the molecules. Using the secondary structure models for each species of rRNA, we have deduced a loop and stem structure having GAGA in the loop to be a minimum requirement for the substrate of ricin A-chain.     

Carbohydrate-specifically polyethylene glycol-modified ricin A-chain with improved therapeutic potential

Ricin A-chain, which exhibits excellent cytotoxicity to tumor cells, has been widely used as an immunotoxin source. However, it has the fatal shortcoming of poor pharmacokinetics due to the tremendous liver uptake via carbohydrate-mediated recognition. Modification of proteins with polyethylene glycol, PEGylation, has the advantages of shielding the specific sites and prolonging the biological half-life. In this study, the carbohydrate-specific PEGylation of ricin A-chain was considered to be a novel approach to overcome this limitation. The carbohydrate group of ricin A-chain was oxidized by sodium m-periodate and further specifically conjugated with hydrazide-derivatized PEG. For a comparative study, the PEGylated ricin A-chain at amino groups was prepared using the hydroxysuccinimide ester-derivatized PEG. The carbohydrate-specifically PEGylated ricin A-chain showed a markedly lower liver uptake and systemic clearance compared with the amine-directly PEGylated ricin A-chain as well as the unmodified ricin A-chain. Furthermore, carbohydrate-specifically PEGylated ricin A-chain showed a significantly higher in vitro ribosome-inactivating activity than the amine-directly PEGylated ricin A-chain. These findings clearly demonstrate that the carbohydrate-specificity as well as PEGylation plays an important role in improving the in vivo pharmacokinetic properties and in vitro bioactivity. Therefore, these results suggest that the therapeutic use of immunotoxins constructed using this carbohydrate-specifically PEGylated ricin A-chain has potential as a cancer therapy.     

Features of the structure of catalytic subunits of toxins, inhibiting protein synthesis. I. The effect of pH and interaction with the B-chain of ricin

A comparative study of gelonin and A-chains of ricin, mistletoe lectin I and diphtheria toxin was undertaken. The effect of pH was studied on: a) the conformation of the proteins under study using intrinsic fluorescence; b) interaction of these proteins with ricin B-chain using gel-filtration. Structural stability of the proteins was assessed according to denaturing action of guanidine hydrochloride and temperature, and localization of tryptophan residues was determined using fluorescence quenching by I-, Cs+ and acrylamide. All investigated proteins were shown to undergo the conformational changes when a environment became acidic. In comparison with an intact protein--gelonin, the A-chains of ricin, a mistletoe lectin and a diphtheria toxin are less stable. At pH less than 5.0 tryptophan residues became more accessible to quencher and a positive charge of the surrounding area increases (in the case of gelonin it is negatively charged). No reliable interaction of a ricin B-chain with both gelonin and A-chain of diphtheria toxin was observed. The interaction of a ricin B-chain with a A-chain of mistletoe lectin I is weaker than that with ricin A-chain and is practically pH-independent.     

Prediction of a conserved, neutralizing epitope in ribosome-inactivating proteins

The secondary structures, side-chain solvent accessibilities, and superpositioned crystal structures of the A-chain of ricin and four other plant rRNA N-glycosidases (ribosome-inactivating proteins, RIPs) were examined. Previously, a 26-residue fragment from the A-chain of ricin was determined to bind to a neutralizing monoclonal antibody. The region in the native ricin A-chain, to which this peptide corresponds, is solvent-exposed and contains a negatively charged residue that has been hypothesized to participate in the toxin's function, namely, rRNA binding and/or enzymatic activity. This region appears to be conserved in all of the structurally defined plant RIPs examined. Moreover, other plant RIPs, whose tertiary structures are, as yet, unknown, were predicted to have an analogous, solvent-exposed region containing a conserved, negatively charged residue. By analogy, these conserved structural and functional features lead to the suggestion that this exposed region represents a logical starting point for experiments designed to locate neutralizing epitopes in these RIPs. In contrast, the tertiary structure of the analogous region in a bacteria-derived RIP (Shiga toxin) is a less solvent-exposed, truncated loop and is a structure that is not as likely to be a neutralizing epitope. Because most of the amino acid residues are not conserved within this exposed region, these RIPs are predicted to be antigenically distinct.     

Studies on the variants of the protein toxins ricin and abrin.

This study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101-109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.     

Preproabrin: genomic cloning, characterisation and the expression of the A-chain in Escherichia coli

Synthetic oligonucleotides representing all possible sequences of an N-terminal and an internal region of the A-chain of abrin C were used to generate a probe specific for abrin-related sequences using the polymerase chain reaction on Abrus precatorius genomic DNA. A lambda phage library constructed from genomic DNA isolated from leaf tissue of A. precatorius was screened and positive hybridising clones were characterised by restriction enzyme analysis. The coding regions of unique clones were characterised by DNA sequencing. One clone encodes a preproprotein closely related to abrin C with 83% similarity between the A-chain sequences. Based on similarity with the ricin toxins and Ricinus communis agglutinin, the preproabrin consists of an A-chain of 251 amino acids preceded by 34 amino acids containing an N-terminal signal peptide, followed by a 14-amino-acid linker and a B-chain of 263 amino acids. The mature A-chain of the preproabrin has been expressed cytoplasmically in Escherichia coli and the soluble recombinant protein was produced at levels exceeding 6% of total cell protein. The recombinant A-chain has been purified to homogeneity and its ability to depurinate 28S rRNA in rat liver ribosomes has been demonstrated in vitro.     

Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.     

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.     

Comparative study of interaction of two type II ribosome-inactivating proteins and their A-chains with model membrane

Both cinnamomin and ricin are type II ribosome-inactivating proteins. Cinnamomin is less cytotoxic compared with ricin. In order to clarify the mechanism of their different cytotoxicities, the interaction of cinnamomin and its A-chain with model membrane was investigated and compared with that of ricin and its A-chain. It was revealed that cinnamomin is less effective than ricin in interacting with model membrane. Cinnamomin A-chain interacts with model membrane much less violently than ricin A-chain. The differences in the interaction of cinnamomin, ricin or their A-chains with model membrane might at least in part indicate the different cytotoxicity between cinnamomin and ricin.     

Cholera toxin B-subunit protects mammalian cells from ricin and abrin toxicity

The glycoproteins ricin and abrin intoxicate cells by inhibiting protein synthesis. Pretreatment of HeLa cells with cholera toxin partially protects them from ricin and abrin activity. The involvement in this phenomenon of the various effects of cholera toxin, namely, redistribution of membrane receptors elicited from protomer B and increasing cyclic AMP concentrations induced by protomer A, were studied. Substances able to enhance cyclic AMP concentrations do not affect ricin and abrin activity, while protomer B alone protects cells. In addition, the effects of several lectins on ricin or abrin toxicity were examined. Almost complete prevention of ricin or abrin activity was obtained using concanavalin A (Con A) and wheat germ agglutinin (WGA). Conversely, neither succinyl Con A nor Ulex europeus agglutinin (UEA) affected the cellular response. Both protomer B of cholera toxin and Con A did not alter the binding of ricin or abrin; they seem to protect cells by altering membrane structure.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.