Ricin

Ricin , from the castor oil plant Ricinus communis, is a highly toxic, naturally occurring protein. A dose as small as a few grains of salt can kill an adult. The LD50 of ricin is around 22 micrograms per kilogram (1.76 mg for an average adult, around 1/228 of a standard aspirin tablet (0.4 g gross)) in humans if exposure is from injection or inhalation. Oral exposure to ricin is far less toxic and lethal dose can be up to 20–30 milligrams per kilogram.



Toxicity


Ricin is poisonous if inhaled, injected, or ingested, acting as a toxin by the inhibition of protein synthesis. It is resistant, but not impervious, to digestion by peptidases. By ingestion, the pathology of ricin is largely restricted to the gastrointestinal tract where it may cause mucosal injuries; with appropriate treatment, most patients will make a full recovery. Because the symptoms are caused by failure to make protein, they emerge only after a variable delay from a few hours to a full day after exposure. An antidote not yet tested on humans has been developed by the UK military, and a vaccine has been developed by the US military, and has had some human testing, and so far shown to be safe, and effective when lab mice were injected with ricin-antibody rich blood mixed with ricin.[5] Symptomatic and supportive treatment is available. Long term organ damage is likely in survivors. Ricin causes severe diarrhea and victims can die of shock. Death typically occurs within 3–5 days of the initial exposure. Abrin is a similar toxin, found in the highly ornamental rosary pea.
Deaths caused by ingestion of castor plant seeds are rare, partly because of the indigestible capsule, and partly because ricin can be digested (although it is resistant). The pulp from eight beans is considered toxic for an adult. A solution of saline and glucose has been used to treat ricin overdose. 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."






Overdosage


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. Victims often manifest nausea, emesis, diarrhea, tachycardia, hypotension and seizures persisting for up to a week. Blood, plasma or urine ricin concentrations may be measured to confirm diagnosis.




Biochemistry


Ricin is classified as a type 2 ribosome inactivating protein (RIP). Whereas Type 1 RIPs consist of a single enzymatic protein chain, Type 2 RIPs, also known as holotoxins, are heterodimeric glycoproteins. Type 2 RIPs consist of an A chain that is functionally equivalent to a Type 1 RIP, covalently connected by a single disulfide bond to a B chain that is catalytically inactive, but serves to mediate entry of the A-B protein complex into the cytosol. Both Type 1 and Type 2 RIPs are functionally active against ribosomes in vitro, however only Type 2 RIPs display cytoxicity due to the lectin properties of the B chain. In order to display its ribosome inactivating function, the ricin disulfide bond must be reductively cleaved.




Structure


The tertiary structure of ricin was shown to be a globular, glycosylated heterodimer of approximately 60-65 kDA. Ricin toxin A chain and ricin toxin B chain are of similar molecular weight, approximately 32 kDA and 34 kDA respectively.
Ricin A Chain (RTA) is an N-glycoside hydrolase composed of 267 amino acids. It has three structural domains with approximately 50% of the polypeptide arranged into alpha-helices and beta-sheets. The three domains form a pronounced cleft that is the active site of RTA.
Ricin B Chain (RTB) is a lectin composed of 262 amino acids that is able to bind terminal galactose residues on cell surfaces. RTB form 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.
Many plants such as barley have the A chain but not the B chain. People do not get sick from eating large amounts of such products, as ricin A is of extremely low toxicity as long as the B chain is not present.




Entry into the cytosol


The ability of ricin to enter the cytosol depends on hydrogen bonding interactions between RTB amino acid residues and complex carbohydrates on the surface of eukaryotic cells containing either terminal N-acetyl galactosamine or beta-1,4-linked galactose residues. Additionally, the mannose-type glycans of ricin are able to bind cells that express mannose receptors.Experimentally, RTB has been shown to bind to the cell surface on the order of 106-108 ricin molecules per cell surface.
The profuse binding of ricin to surface membranes allows internalization with all types of membrane invaginations. Experimental evidence points to ricin uptake in both clathrin-coated pits, as well as clathrin-independent pathways including caveolae and macropinocytosis. Vesicles shuttle ricin to endosomes that are delivered to the Golgi apparatus. The active acidification of endosomes are 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 offer little or no protection against ricin. 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).
For ricin to function cytotoxically, RTA must be reductively cleaved from RTB in order 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. 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. Roles for the ER chaperones GRP94  and EDEM  have been proposed prior to the 'dislocation' of RTA from the ER lumen to the cytosol in a manner that utilizes 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, 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. 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 cytosolic molecular chaperones that results in its folding to a catalytic conformation  that de-purinates ribosomes, thus halting protein synthesis.