Ricin

Biosynthesis

Ricin is synthesized in the endosperm of castor oil plant seeds.^[3] The ricin precursor protein is 576 amino acid residues in length and contains a signal peptide (residues 1–35), the ricin A chain (36–302), a linker peptide (303–314), and the ricin B chain (315–576).^[4] The N-terminal signal sequence delivers the prepropolypeptide to the endoplasmic reticulum (ER) and then the signal peptide is cleaved off. Within the lumen of the ER the propolypeptide is glycosylated and a protein disulfide isomerase catalyzes disulfide bond formation between cysteines 294 and 318. The propolypeptide is further glycosylated within the Golgi apparatus and transported to protein storage bodies. The propolypeptide is cleaved within protein bodies by an endopeptidase to produce the mature ricin protein that is composed of a 267 residue A chain and a 262 residue B chain that are covalently linked by a single disulfide bond.^[3]

Structure

In terms of structure, ricin closely resembles abrin-a, an isomer of abrin. The quaternary structure of ricin is a globular, glycosylated heterodimer of approximately 60–65 kDa.^[5] Ricin toxin A chain and ricin toxin B chain are of similar molecular weights, approximately 32 kDa and 34 kDa, respectively.

• Ricin toxin A chain (RTA) is an N-glycoside hydrolase composed of 267 amino acids.^[6] It has three structural domains with approximately 50% of the polypeptide arranged into alpha-helices and beta-sheets.^[7] The three domains form a pronounced cleft that is the active site of RTA.
• Ricin toxin B chain (RTB) is a lectin composed of 262 amino acids that is able to bind terminal galactose residues on cell surfaces.^[8] RTB forms a bilobal, barbell-like structure lacking alpha-helices or beta-sheets where individual lobes contain three subdomains. At least one of these three subdomains in each homologous lobe possesses a sugar-binding pocket that gives RTB its functional character.

While other plants contain the protein chains found in ricin, both protein chains must be present to produce toxic effects. For example, plants that contain only protein chain A, such as barley, are not toxic because without the link to protein chain B, protein chain A cannot enter the cell and do damage to ribosomes.^[9]

Entry into the cytoplasm

Ricin B chain binds complex carbohydrates on the surface of eukaryotic cells containing either terminal N-acetylgalactosamine or beta-1,4-linked galactose residues. In addition, the mannose-type glycans of ricin are able to bind to cells that express mannose receptors.^[10] RTB has been shown to bind to the cell surface on the order of 10⁶–10⁸ ricin molecules per cell surface.^[11]

The profuse binding of ricin to surface membranes allows internalization with all types of membrane invaginations. The holotoxin can be taken up by clathrin-coated pits, as well as by clathrin-independent pathways including caveolae and macropinocytosis.^[12][13] Intracellular vesicles shuttle ricin to endosomes that are delivered to the Golgi apparatus. The active acidification of endosomes is thought to have little effect on the functional properties of ricin. Because ricin is stable over a wide pH range, degradation in endosomes or lysosomes offers little or no protection against ricin.^[14] Ricin molecules are thought to follow retrograde transport via early endosomes, the trans-Golgi network, and the Golgi to enter the lumen of the endoplasmic reticulum (ER).^[15]

For ricin to function cytotoxically, RTA must be reductively cleaved from RTB to release a steric block of the RTA active site. This process is catalysed by the protein PDI (protein disulphide isomerase) that resides in the lumen of the ER.^[16][17] Free RTA in the ER lumen then partially unfolds and partially buries into the ER membrane, where it is thought to mimic a misfolded membrane-associated protein.^[18] Roles for the ER chaperones GRP94,^[19] EDEM^[20] and BiP^[21] have been proposed prior to the 'dislocation' of RTA from the ER lumen to the cytosol in a manner that uses components of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. ERAD normally removes misfolded ER proteins to the cytosol for their destruction by cytosolic proteasomes. Dislocation of RTA requires ER membrane-integral E3 ubiquitin ligase complexes,^[22] but RTA avoids the ubiquitination that usually occurs with ERAD substrates because of its low content of lysine residues, which are the usual attachment sites for ubiquitin.^[23] Thus, RTA avoids the usual fate of dislocated proteins (destruction that is mediated by targeting ubiquitinylated proteins to the cytosolic proteasomes). In the mammalian cell cytosol, RTA then undergoes triage by the cytosolic molecular chaperones Hsc70 and Hsp90 and their co-chaperones, as well as by one subunit (RPT5) of the proteasome itself, that results in its folding to a catalytic conformation,^[19][24] which de-purinates ribosomes, thus halting protein synthesis.

Ribosome inactivation

RTA has rRNA N-glycosylase activity that is responsible for the cleavage of a glycosidic bond within the large rRNA of the 60S subunit of eukaryotic ribosomes.^[25] RTA specifically and irreversibly hydrolyses the N-glycosidic bond of the adenine residue at position 4324 (A4324) within the 28S rRNA, but leaves the phosphodiester backbone of the RNA intact.^[26] The ricin targets A4324 that is contained in a highly conserved sequence of 12 nucleotides universally found in eukaryotic ribosomes. The sequence, 5'-AGUACGAGAGGA-3', termed the sarcin-ricin loop, is important in binding elongation factors during protein synthesis.^[27] The depurination event rapidly and completely inactivates the ribosome, resulting in toxicity from inhibited protein synthesis. A single RTA molecule in the cytosol is capable of depurinating approximately 1500 ribosomes per minute.

Depurination reaction

Within the active site of RTA, there exist several invariant amino acid residues involved in the depurination of ribosomal RNA.^[14] Although the exact mechanism of the event is unknown, key amino acid residues identified include tyrosine at positions 80 and 123, glutamic acid at position 177, and arginine at position 180. In particular, Arg180 and Glu177 have been shown to be involved in the catalytic mechanism, and not substrate binding, with enzyme kinetic studies involving RTA mutants. The model proposed by Mozingo and Robertus,^[7] based on X-ray structures, is as follows:

1. Sarcin-ricin loop substrate binds RTA active site with target adenine stacking against tyr80 and tyr123.
2. Arg180 is positioned such that it can protonate N-3 of adenine and break the bond between N-9 of the adenine ring and C-1' of the ribose.
3. Bond cleavage results in an oxycarbonium ion on the ribose, stabilized by Glu177.
4. N-3 protonation of adenine by Arg180 allows deprotonation of a nearby water molecule.
5. Resulting hydroxyl attacks ribose carbonium ion.
6. Depurination of adenine results in a neutral ribose on an intact phosphodiester RNA backbone.

Symptoms

Because the symptoms are caused by failure to make protein, they may take anywhere from hours to days to appear, depending on the route of exposure and the dose. When ingested, gastrointestinal symptoms can manifest within six hours; these symptoms do not always become apparent. Within two to five days of exposure to ricin, its effects on the central nervous system, adrenal glands, kidneys, and liver appear.^[29]

Ingestion of ricin causes pain, inflammation, and hemorrhage in the mucosal membranes of the gastrointestinal system. Gastrointestinal symptoms quickly progress to severe nausea, vomiting, diarrhea, and difficulty swallowing (dysphagia). Haemorrhage causes bloody feces (melena) and vomiting blood (hematemesis). The low blood volume (hypovolemia) caused by gastrointestinal fluid loss can lead to organ failure in the pancreas, kidney, liver, and GI tract and progress to shock. Shock and organ failure are indicated by disorientation, stupor, weakness, drowsiness, excessive thirst (polydipsia), low urine production (oliguria), and bloody urine (hematuria).^[29]

Symptoms of ricin inhalation are different from those caused by ingestion. Early symptoms include a cough and fever.^[29]

When skin or inhalation exposure occur, ricin can cause an allergic reaction to develop. This is indicated by swelling (edema) of the eyes and lips; asthma; bronchial irritation; dry, sore throat; congestion; skin redness (erythema); skin blisters (vesication); wheezing; itchy, watery eyes; chest tightness; and skin irritation.^[29]

Treatment

An antidote has been developed by the UK military, although as of 2006 it has not yet been tested on humans.^[32][33] As of 2005 another antidote developed by the US military has been shown to be safe and effective in lab mice injected with antibody-rich blood mixed with ricin, and has had some human testing.^[34] Monoclonal antibodies are under scientific investigation as a possible treatment for ricin poisoning.^[35]

Symptomatic and supportive treatments are available for ricin poisoning. Existing treatments emphasize minimizing the effects of the poison. Possible treatments include intravenous fluids or electrolytes, airway management, assisted ventilation, or giving medications to remedy seizures and low blood pressure. If the ricin has been ingested recently, the stomach can be flushed by ingesting activated charcoal or by performing gastric lavage. Survivors often develop long-term organ damage. Ricin causes severe diarrhea and vomiting, and victims can die of circulatory shock or organ failure; inhaled ricin can cause fatal pulmonary edema or respiratory failure. Death typically occurs within 3–5 days after oral ingestion.^[29]

Prevention

Vaccination is possible by injecting an inactive form of protein chain A.^[9] This vaccination is effective for several months due to the body's production of antibodies to the foreign protein. In 1978 Bulgarian defector Vladimir Kostov survived a ricin attack similar to the one on Georgi Markov, probably due to his body's production of antibodies. When a ricin-laced pellet was removed from the small of his back it was found that some of the original wax coating was still attached. For this reason only small amounts of ricin had leaked out of the pellet, producing some symptoms but allowing his body to develop immunity to further poisoning.^[9]

Sources

The seeds of Ricinus communis are commonly crushed to extract castor oil. As ricin is not oil-soluble, little is found in the extracted castor oil.^[9] The extracted oil is also heated to more than 80 °C (176 °F) to denature any ricin that may be present.^[9] The remaining spent crushed seeds, called variously the "cake", "oil cake", and "press cake", can contain up to 5% ricin.^[36] While the oil cake from coconut, peanuts, and sometimes cotton seeds can be used as cattle feed or fertilizer, the toxic nature of castor beans precludes their oil cake from being used as feed unless the ricin is first deactivated by autoclaving.^[37] Accidental ingestion of Ricinus communis cake intended for fertilizer has been reported to be responsible for fatal ricin poisoning in animals.^[28][38]

Deaths from ingesting castor plant seeds are rare, partly because of their indigestible seed coat, and because some of the ricin is deactivated in the stomach.^[5] The pulp from eight beans is considered dangerous to an adult.^[39] Rauber and Heard have written that close examination of early 20th century case reports indicates that public and professional perceptions of ricin toxicity "do not accurately reflect the capabilities of modern medical management".^[40]

Most acute poisoning episodes in humans are the result of oral ingestion of castor beans, 5–20 of which could prove fatal to an adult. Swallowing castor beans rarely proves to be fatal unless the bean is thoroughly chewed. The survival rate of castor bean ingestion is 98%.^[9] In 2013 a 37-year-old woman in the United States survived after ingesting 30 beans.^[41] In another case, a man ingested 200 castor beans mixed with juice in a blender and survived.^[42] Victims often manifest nausea, diarrhea, fast heart rate, low blood pressure, and seizures persisting for up to a week.^[28] Blood, plasma, or urine ricin or ricinine concentrations may be measured to confirm diagnosis. The laboratory testing usually involves immunoassay or liquid chromatography-mass spectrometry.^[43]

History

The United States investigated ricin for its military potential during World War I.^[48] At that time it was being considered for use either as a toxic dust or as a coating for bullets and shrapnel. The dust cloud concept could not be adequately developed, and the coated bullet/shrapnel concept would violate the Hague Convention of 1899 (adopted in U.S. law at 32 Stat. 1903), specifically Annex §2, Ch.1, Article 23, stating "... it is especially prohibited ... [t]o employ poison or poisoned arms".^[49]

During World War II the United States and Canada studied ricin in cluster bombs.^[50] Though there were plans for mass production and several field trials with different bomblet concepts, the end conclusion was that it was no more economical than using phosgene. This conclusion was based on comparison of the final weapons, rather than ricin's toxicity (LCt₅₀ ~10 mg/min·m³).^{[citation needed]} Ricin was given the military symbol W or later WA.^{[citation needed]} Interest in it continued for a short period after World War II, but soon subsided when the US Army Chemical Corps began a program to weaponize sarin.^[51]

The Soviet Union possessed weaponized ricin. The KGB developed weapons using ricin which were used outside the Soviet bloc, most famously in the Markov assassination.^[52][53]

Control

In spite of ricin's extreme toxicity and utility as an agent of chemical/biological warfare, production of the toxin is difficult to limit. The castor bean plant from which ricin is derived is a common ornamental and can be grown at home without any special care.

Under both the 1972 Biological Weapons Convention and the 1997 Chemical Weapons Convention, ricin is listed as a schedule 1 controlled substance. Despite this, more than 1 million metric tons (1,100,000 short tons) of castor beans are processed each year, and approximately 5% of the total is rendered into a waste containing negligible concentrations of undenatured ricin toxin.^[54]

Ricin is several orders of magnitude less toxic than botulinum or tetanus toxin, but the latter are harder to come by. Compared to botulinum or anthrax as biological weapons or chemical weapons, the quantity of ricin required to achieve LD₅₀ over a large geographic area is significantly more than an agent such as anthrax (tons of ricin vs. only kilogram quantities of anthrax).^[55] Ricin is easy to produce, but is not as practical or likely to cause as many casualties as other agents.^[30] Ricin is easily denatured by temperatures over 80 °C (176 °F) meaning many methods of deploying ricin would generate enough heat to denature it.^[36] Once deployed, an area contaminated with ricin remains dangerous until the bonds between chain A or B have been broken, a process that takes two or three days.^[9] In contrast, anthrax spores may remain lethal for decades. Jan van Aken, a German expert on biological weapons, explained in a report for The Sunshine Project that Al Qaeda's experiments with ricin suggest their inability to produce botulinum or anthrax.^[56]