Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes.

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.     

Dislocation of ricin toxin A chains in human cells utilizes selective cellular factors

Ricin is a potent A-B toxin that is transported from the cell surface to the cytosol, where it inactivates ribosomes, leading to cell death. Ricin enters cells via endocytosis, where only a minute number of ricin molecules reach the endoplasmic reticulum (ER) lumen. Subsequently, the ricin A chain traverses the ER bilayer by a process referred to as dislocation or retrograde translocation to gain access to the cytosol. To study the molecular processes of ricin A chain dislocation, we have established, for the first time, a human cell system in which enzymatically attenuated ricin toxin A chains (RTA(E177D) and RTA(Δ177-181)) are expressed in the cell and directed to the ER. Using this human cell-based system, we found that ricin A chains underwent a rapid dislocation event that was quite distinct from the dislocation of a canonical ER soluble misfolded protein, null Hong Kong variant of α(1)-antitrypsin. Remarkably, ricin A chain dislocation occurred via a membrane-integrated intermediate and utilized the ER protein SEL1L for transport across the ER bilayer to inhibit protein synthesis. The data support a model in which ricin A chain dislocation occurs via a novel strategy of utilizing the hydrophobic nature of the ER membrane and selective ER components to gain access to the cytosol.     

重组蓖麻毒素A链与几种天然单链核糖体失活蛋白的活性研究

采用ｐＥｘＳｅｃⅠ载体系统进行了蓖麻毒素Ａ链的原核表达,经ＣＭ－Ｓｅｐｈａｒｏｓｅ一步纯化后,获得了纯度约８０％的重组蓖麻毒素Ａ链．将其与几种天然单链核糖体失活蛋白进行了超螺旋ＤＮＡ裂解研究和无细胞体系中蛋白合成抑制试验,结果表明,重组蓖麻毒素Ａ链具有类似于天然单链核糖体失活蛋白的活性,但两种测活方法之间没有明显的相关性     

蓖麻毒素A链突变体的设计、表达与活性研究

利用蛋白质结构同源模建并结合表观静电势分析,设计了拟具有生物学活性的蓖麻毒素A链的突变体.将PCR扩增的突变体基因,导入pKK223-3载体中,于大肠杆菌(E.coli)中获得高效、可溶性表达,而且,确证了表达产物具有预期的生物学活性.     

High-level expression and simplified purification of recombinant ricin A chain.

Ricin toxin is a glycoprotein which catalytically inactivates eukaryotic ribosomes by depurination of a single adenosine residue from the 28S ribosomal RNA. The enzymatic activity is present in the A chain of the toxin molecule, whereas the B chain contains two binding sites for galactose. Since it is highly potent in inhibiting protein synthesis, the A chain is used to prepare cytotoxic conjugates effective against tumor cells. Such chimeric proteins are highly selective and have a wide range of clinical applications. Extensive preclinical studies on these conjugates require large amounts of purified A chain. Native ricin A chain is heterogeneous, since plants produce a number of isoforms of ricin toxin. Purified, native preparations often contain two types of ricin A chain which differ in the extent of glycosylation. By cloning and expressing the gene of A chain, one could obtain homogeneous toxin molecules devoid of carbohydrates. In addition, structural changes in the toxin polypeptide could be introduced by in vitro mutagenesis, which can improve the pharmacological properties and antitumor activity. Earlier methods of expression strategies using Escherichia coli have yielded only moderate levels of expression. In the present study, the coding region of ricin A chain was cloned into pET3b, a high-level expression vector under the control of the T7 promoter. Recombinant ricin A chain produced by this construct has an additional 14 amino acid residues at the NH2 terminus. Subsequently, a NdeI site was created at the 5' end of the gene by oligonucleotide-directed mutagenesis. The modified fragment was then introduced into pET3b vector to produce toxin polypeptide identical to the native sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

A method for detecting the expression of a toxic gene in cultured cells.

We have devised a rapid method for examining the expression of a toxin gene following in vitro transfection using a bacterial beta-galactosidase (lacZ) gene as a reporter gene. Ricin A chain DNA and the lacZ gene, both under the control of the immunoglobulin gene promoter and enhancer, were transfected into mouse fibroblast cells (L cells). Transient expression of the lacZ gene was detected 2 days after transfection by histochemical staining of the transfectants with 5-bromo-3-indolyl-beta-D-galactoside. Cotransfection of the ricin A chain gene resulted in a progressive reduction in the number of lacZ transfectants as the expressed toxin killed the cells. A ricin construct with the intervening sequence from the human beta-actin gene required 4 days instead of 2 days to produce the toxic effect. This is a useful method for examining the expression of toxin gene in a cell.     

Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits.

The A-subunit polypeptides of Shiga toxin, the Shiga-like toxins (SLTs), and the plant lectin ricin inactivate eucaryotic ribosomes by enzymatically depurinating 28S rRNA. Comparison of the amino acid sequences of the members of the Shiga toxin family and ricin revealed two regions of significant homology that lie within a proposed active-site cleft of the ricin A chain. In previous studies, these conserved sequences of the SLT-I and ricin A subunits have been implicated as active sites. To establish the importance of these regions of homology, we used site-directed mutagenesis to alter the A-subunit sequences of two members of the Shiga toxin family. Substitution of an aspartic acid for glutamic acid 166 of the Slt-IIA subunit decreased the capacity of the polypeptides to inhibit protein synthesis at least 100-fold in a cell-free translation system. However, this mutation did not prevent the expression of immunoreactive, full-length Slt-IIA. In addition, SLT-II holotoxin containing the mutated A subunit was 1,000-fold less toxic to Vero cells. Finally, site-directed mutagenesis was used to delete sequences encoding amino acids 202 through 213 of the Shiga toxin A subunit. Although this deletion did not prevent holotoxin assembly, it abolished cytotoxic activity.     

Modulation of toxin stability by 4-phenylbutyric acid and negatively charged phospholipids

AB toxins such as ricin and cholera toxin (CT) consist of an enzymatic A domain and a receptor-binding B domain. After endocytosis of the surface-bound toxin, both ricin and CT are transported by vesicle carriers to the endoplasmic reticulum (ER). The A subunit then dissociates from its holotoxin, unfolds, and crosses the ER membrane to reach its cytosolic target. Since protein unfolding at physiological temperature and neutral pH allows the dissociated A chain to attain a translocation-competent state for export to the cytosol, the underlying regulatory mechanisms of toxin unfolding are of paramount biological interest. Here we report a biophysical analysis of the effects of anionic phospholipid membranes and two chemical chaperones, 4-phenylbutyric acid (PBA) and glycerol, on the thermal stabilities and the toxic potencies of ricin toxin A chain (RTA) and CT A1 chain (CTA1). Phospholipid vesicles that mimic the ER membrane dramatically decreased the thermal stability of RTA but not CTA1. PBA and glycerol both inhibited the thermal disordering of RTA, but only glycerol could reverse the destabilizing effect of anionic phospholipids. In contrast, PBA was able to increase the thermal stability of CTA1 in the presence of anionic phospholipids. PBA inhibits cellular intoxication by CT but not ricin, which is explained by its ability to stabilize CTA1 and its inability to reverse the destabilizing effect of membranes on RTA. Our data highlight the toxin-specific intracellular events underlying ER-to-cytosol translocation of the toxin A chain and identify a potential means to supplement the long-term stabilization of toxin vaccines.     

Processing of preproricin in transgenic tobacco

The plant protein toxin ricin has found widespread application as a potential therapeutic agent for many human diseases and in disease-model systems such as those involving apoptosis. Genetic engineering and expression of the complete two-polypeptide chain toxin have only been possible in plants, specifically in transgenic tobacco carrying the preproricin gene under the control the cauliflower mosaic virus 35S promoter. Production of modified ricin for altered controllable activity and/or fusion therapeutics to target delivery requires knowledge of the heterologous processing that occurs when preproricin is expressed in tobacco. Here, recombinant ricin from transgenic tobacco was purified using lectin affinity chromatography and characterized using various biochemical and biophysical techniques. Coomassie blue staining of an SDS-PAGE gel of lactose-agarose purified material identified predominant proteins of 30 and 35 kDa molecular weight. Western analysis using anti-ricin a- and b-chain antibodies confirmed the expression and purification of recombinant ricin, with identical protein banding profiles to that of authentic castor-bean-derived ricin. High-resolution gel filtration chromatography characterized the lactose binding complex as a 66-kDa native molecular weight protein which could be separated into 30- and 35-kDa proteins upon incubation with the reducing agent dithiothreitol. N-terminal sequencing of the recombinant ricin a-chain revealed that an equimolar ratio of two alternately processed peptides was present, which varied by an additional amino acid derived from the signal peptide. Similar analysis of ricin b-chain again identified two forms of this polypeptide as well; however, full-length ricin b-chain and b-chain missing the first alanine residue were present at 11:1 molar ratios. Transgenic tobacco plants expressing ricin were used to develop a stable cell suspension culture system from callus induced with the growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Double sandwich enzyme-linked immunosorbent assay using anti-ricin b-chain antibodies and Western analysis identified soluble ricin in the media of the cultures, indicating that cell cultures provide a safe and simple means to produce properly processed recombinant ricin.     

The position of the proricin vacuolar targeting signal is functionally important

Ricin is synthesised as an ER-targeted precursor containing an enzymatic A chain and a galactose-binding B chain separated by a 12-amino acid linker propeptide. This internal propeptide is known to contain a sequence-specific vacuolar sorting signal whose functionality depends on the presence of an isoleucine residue. Conversion of this isoleucine to glycine completely abolished vacuolar targeting of proricin and led to its secretion. However, when this mutated signal was positioned at the C-terminus of a normally secreted reporter, vacuolar targeting of a significant fraction still occurred. Likewise, when the corrupted linker was C-terminally exposed within its natural context following the mature ricin A chain, and then co-expressed with ricin B chain, toxin heterodimers were still partially transported to tobacco cell vacuoles. By contrast, when placed at the N-terminus of the secreted reporter, or at the N-terminus of ricin B chain for co-expression with ricin A chain, the propeptide behaved most strikingly as a sequence-specific vacuolar targeting signal that, when mutated, resulted in complete secretion of the proteins. It would appear that the position of the linker peptide influences the specificity of its vacuolar targeting function.     

Studies on the galactose-binding site of ricin and the hybrid toxin man6P-ricin

N-acetylimidazole (NAI) was used to O-acetylate the plant seed toxin ricin. O-acetylation of one to two tyrosine residues per molecule of ricin inhibited ricin binding to Sepharose 4B and decreased toxicity by 90% in a protein synthesis inhibition assay in HeLa cells. Lactose, known to block the binding site on the ricin B subunit, protected ricin from NAI modification of binding or toxicity. Thus NAI, under these conditions, can be a lactose site-specific inhibitor. The lactose site-specific modification of the hybrid toxin, Man6P-ricin, performed under the same conditions, exhibited the same 90% inhibition of Man6P receptor-mediated toxicity as the galactose-containing receptor-mediated toxicity of either Man6P-ricin or ricin. Thus the ricin B chain lactose-binding site appears to be essential for the high potency of Man6P-ricin via the new cell type-specific Man6P receptor. Treatment of fibroblasts with neuraminidase exposes galactose residues, thus increasing the sensitivity to ricin eight fold. The Man6P receptor-mediated toxicity of Man6P-ricin is not affected by this treatment, although the galactose-inhibited route is potentiated eight fold. The Man6P-ricin hybrid appears to require the ricin B chain galactose-binding site to enter the cytosol after initially binding to the Man6P receptor. These data provide some insights into the proper design of hybrid toxins. We discuss a number of possible models for hybrid toxin entry.     

Structure of recombinant ricin A chain at 2.3 A.

The plant cytotoxin ricin is a heterodimer with a cell surface binding (B) chain and an enzymatically active A chain (RTA) known to act as a specific N-glycosidase. RTA must be separated from B chain to attack rRNA. The X-ray structure of ricin has been solved recently; here we report the structure of the isolated A chain expressed from a clone in Escherichia coli. This structure of wild-type rRTA has and will continue to serve as the parent compound for difference Fouriers used to assess the structure of site-directed mutants designed to analyze the mechanism of this medically and commercially important toxin. The structure of the recombinant protein, rRTA, is virtually identical to that seen previously for A chain in the heterodimeric toxin. Some minor conformational changes due to interactions with B chain and to crystal packing differences are described. Perhaps the most significant difference is the presence in rRTA of an additional active site water. This molecule is positioned to act as the ultimate nucleophile in the depurination reaction mechanism proposed by Monzingo and Robertus (1992, J. Mol. Biol. 227, 1136-1145).     

The tRNA N2,N2-dimethylguanosine-26 methyltransferase encoded by gene trm1 increases efficiency of suppression of an ochre codon in Schizosaccharomyces pombe

The plant toxin ricin has proven valuable as a membrane marker in studies of endocytosis as well as studies of different intracellular transport steps. The toxin, which consists of two polypeptide chains, binds by one chain (the B-chain) to both glycolipids and glycoproteins with terminal galactose at the cell surface. The other chain (the A-chain) enters the cytosol and inhibits protein synthesis enzymatically. After binding the toxin is endocytosed by different mechanisms, and it is transported via endosomes to the Golgi apparatus and the endoplasmic reticulum before translocation of the A-chain to the cytosol. The different transport steps have been analyzed by studying trafficking of ricin as well as modified ricin molecules.     

The primary structure of the operons coding for Shigella dysenteriae toxin and temperature phage H30 shiga-like toxin

Nucleotide(nt) sequences were determined for the toxin (SHT) operon present in the chromosome of Shigella dysenteriae 1 and for the shiga-like toxin (SLT) operon found in the lambdoid phage H30 genome. The coding sequences of the sht and slt genes differ in 4 nt with 1 nt change responsible for an amino acid replacement. The deduced amino acid sequence in the A chain of the toxins is highly homologous to that of the A chain of ricin, a plant toxin. SHT-coding mRNAs were detected by mapping the 5' termini and using blot-hybridisation; one of them was more abundant and coded only for the B subunit of SHT while the other (bi-cistronic mRNA) encoded both subunits. An IS element related to the IS3 element of Escherichia coli was found in the chromosome of S. dysenteriae near the sht operon.     

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.     

Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity.

Ricin is a potent plant toxin consisting of two disulfide-bonded subunits. The A chain of ricin is an N-glycosidase which inactivates 28 S RNA and inhibits protein synthesis. The B chain is a galactose-specific lectin with two galactose-binding sites. The genes encoding preproricin and its A and B chains have been cloned and expressed. In addition, X-ray crystallographic studies have identified the galactose-contact residues in both the high- and low-affinity galactose-binding sites of the B chain. In this study, the high-affinity galactose-contact residue of the B chain was changed from Asn-255 to Ala-255 by oligonucleotide-directed mutagenesis. The resulting mutant was sequenced to confirm the presence of a single mutation and was expressed in Cos-M6 cells. Both wild-type and mutant recombinant B chain could be immunoprecipitated with a heterologous anti-B chain antibody and both could form A-B heterodimers. However, as compared to the wild-type, the mutant B chain lacked more than 99% of its lectin activity and cytotoxicity as an A-B dimer. In conclusion, altering the contact residue of the high-affinity galactose-binding site of ricin B chain from Asn-255 to Ala-255 abrogates more than 99% of its lectin activity and the cytotoxicity of the A-B heterodimer to ricin-sensitive cells.     

Ricin B chain targeted to the endoplasmic reticulum of tobacco protoplasts is degraded by a CDC48- and vacuole-independent mechanism

The B chain of ricin was expressed and delivered to the endoplasmic reticulum of tobacco protoplasts where it disappeared with time in a manner consistent with degradation. This turnover did not occur in the vacuoles or upon secretion. Indeed, several lines of evidence indicate that, in contrast to the turnover of endoplasmic reticulum-targeted ricin A chain in the cytosol, the bulk of expressed ricin B chain was degraded in the secretory pathway.     

Modification of the carbohydrate in ricin with metaperiodate and cyanoborohydride mixtures: effect on binding, uptake and toxicity to parenchymal and non-parenchymal cells of rat liver

The carbohydrate in the toxic glycoprotein ricin was chemically modified by simultaneous treatment with sodium metaperiodate and sodium cyanoborohydride. This treatment causes oxidative cleavage of the sugar residues and reduction of the aldehyde groups which are formed to primary alcohols. The modification markedly decreased the rapid removal of ricin from the blood by hepatic non-parenchymal cells with only a relatively small increase in accumulation of the toxin by parenchymal cells. Binding, uptake and toxicity of the modified ricin in primary monolayer cultures of hepatic non-parenchymal cells were all decreased to a much greater extent than in parenchymal cells. The results indicate that native ricin binds to non-parenchymal cells by a dual recognition process which involves both interaction of cell receptors with the mannose-containing oligosaccharides of the toxin and binding of ricin to galactose-containing glycoproteins and glycolipids on the cells. However, uptake and toxicity of native ricin in non-parenchymal cells appears to result principally from entry of the toxin through the mannose recognition pathway. By contrast, uptake and toxicity of the expressed essentially through the galactose-recognition route.     

重组蓖麻毒素A链蛋白的可溶性表达、纯化与抗原性分析

用PCR方法从克隆质粒pUC19-RTA中扩增出蓖麻毒素A(RTA)链基因,序列分析正确后,亚克隆到原核表达质粒pET-His中,构建重组表达质粒pET-HisRTA,再转化到E.coliBL21(DE3)plysS中获得表达工程菌株BL21/pET-HisRTA。该工程菌在30℃经0.4mmol／LIPTG诱导4h后获得可溶性表达的目的蛋白,约占菌体总蛋白的18.45％,SDS-PAGE分析显示表达的蛋白区带与RTA相对分子量相符,约32kDa左右。表达产物经Ni-NTA亲和层析法一步纯化,蛋白纯度约达97.53％,并可得到约18mg/L重组RTA蛋白。Western印迹和间接ELISA结果证明,重组RTA蛋白与抗天然蓖麻毒素多抗可发生特异性的抗原抗体反应,具有良好的抗原性,这为制备RT特异性抗体及建立RT的检测方法奠定了基础。