- 3-dimensional ribbon drawing of ricin, modeled from X-ray crystallography data.
- The dotted, upper right half ribbon, is the A chain (RTA).
- The solid, lower half ribbon is the B chain (RTB).
Ricin Utilizes a Two-Component Entry Pathway
to Exert its Cytoxicity in Eukaryote Cells
By, Bridget Morse-Rohn
ABSTRACT
Ricin follows a characteristic pathway to enter a cell and cause cellular death. Some mechanisms of this pathway have been clearly demonstrated through laboratory studies, while other pathway mechanisms are not well understood. The route of exposure (i.e., inhalation, ingestion, or injection) and dose greatly affects how much and how fast the toxin reaches a cell. Although exposure routes differ, the mechanism and pathway ricin follows to attack and kill a cell is similar from cell to cell, with possible variance among different cell types. Ricin can only exert its toxic effect if acting as a two-part molecule containing a catalytic polypeptide (the A-chain or RTA, the toxifying agent) and a cell-binding polypeptide (the B-chain or RTB, the cell binding agent). These components bound together are referred to as Type II among plant-derived ribosome-inactivating proteins (RIPs); whereas Type I RIP refers to a RTA alone. RTB must first bind to the cell membrane, which then allows RTA to enter the cell. Once inside, RTA undergoes retrograde transport by a mechanism called receptor-mediated endocytosis, resulting in the toxin being reduced and transported through the endoplasmic reticulum (ER), into the cytosol. The specifics by which the toxin is transported through the ER into the cytosol is poorly understood, however there are several theories. Once in the cytosol, the reduced toxin inactivates ribosomes, rendering the ribosome unable to bind and undergo protein synthesis. Reduction in protein synthesis eventually causes cellular death.
INTRODUCTION
Ricin is highly toxic to humans, animals and insects, and can easily be obtained by purifying large quantities of the castor bean plant, Ricinus communis. Its toxic characteristics allow for many uses:
1) Medicine (i.e., therapeutic application / selective injection to target and kill cancer cells)
2) Scientific study (i.e., model systems to characterize intracellular transport pathways; study endocytosis)
3) Biological weapon (i.e., worldwide availability; heat stability)
Like many other toxins, ricin must follow a typical pathway in order to exert its toxic effect. For example, an organism must first be exposed; once exposed, ricin must travel from the point of exposure to a location where it has an opportunity to interact with a potential “target” cell. Once reaching a potential cell, ricin must physically infiltrate the cell and undergo specific mechanisms that result in cell death.
There are no specific antidotal therapies, or methods of testing biologic fluids from a possibly exposed organism, for presence of ricin. Diagnosis is based on symptoms, body response, and knowledge of suspected exposure. Molecular biology studies using ricin yield an increased understanding in cellular knowledge, and the possibility for treatment in ricin toxicity in the near future.
EXPOSURE and SYMPTOMS
Severity of ricin toxicity depends on dose (concentration and quantity) and exposure route. Exposure routes include inhalation, ingestion, and injection. Assuming that a dose is constant, and knowing through laboratory experiments that ricin undergoes rapid uptake through cell surface carbohydrates, aerosol inhalation of ricin is the most toxic route of exposure.
Classic symptoms of ricin exposure may include:
Inhalation – Initial: nasal / throat congestion; chest tightness and cough; dyspnea; nausea; pulmonary necrosis; edema. Onset: severe respiratory distress; continued inflammation and pulmonary distress; death.
Ingestion – Initial: nausea; vomiting; diarrhea; abdominal cramping / pain. Onset: hemorrhage; hepatic, splenic, and renal necrosis.
Injection – Initial: flu-like symptoms such as general weakness, muscular pain, and fever; vomiting. Onset: hypertension; necrosis of muscle and lymph nodes; multi-organ failure.
RICIN BASICS
Ricin is a highly toxic ribosome–inactivating protein (RIP).
Two types of RIP’s exist, Type I and Type II.
Type I RIP’s
Consist only of the ricin toxin A-chain (RTA).
Unable to bind and enter a cell; thus considered harmless when alone.
Examples of plants containing only Type I RIP’s: wheat and barley.
Type II RIP’s
Consist of the ricin toxin A-chain (RTA) and ricin toxin B-chain (RTB) bound together.
The chains work together; one chain binds to the cell membrane, allowing the other chain to enter the cell and cause toxicity.
Example of plants containing only Type II RIP’s: ricin.
RICIN BASICS (2)
Structurally, ricin consists of two polypeptide chains, an A-chain and B-chain, linked together by a disulfide bond.
A-chain, or RTA
RTA is the catalytic, toxic half of the ricin molecule. It is a globular protein that utilizes enzymatic mechanisms to inactivate 28S ribosomal RNA, after undergoing reduction and transport into the cytosol.
B-chain, or RTB
RTB is the receptor-binding half of the ricin molecule. Its lectin properties allow for rapid uptake of RTA after RTB binds to cell surface carbohydrates, or ricin receptors.
The RTA and RTB in ricin act together to allow the toxin to enter a cell and cause a toxic effect.
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STEPS TO TOXICITY
There are 4 essential events or steps that lead to toxicity by ricin:
1) Receptor-mediated endocytosis
Some of the internalized ricin is transported back to the cell surface to undergo exocytosis; delivered to lysosomes and degraded; or undergoes retrograde transport.
2) Retrograde transport into lumen of the endoplasmic reticulum (ER)
3) Passage through the ER membrane, into the cytosol
4) Catalytic inactivation of target substrate (ribosomes) within the cytosol
STEP 1: Receptor-Mediated Endocytosis
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Studies indicate that binding of RTB triggers endocytotic signals, leading to the uptake of RTA into the cell. The transport vessel containing RTA (endosome) makes its way toward the Golgi apparatus. |
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STEP 2: Retrograde transport into lumen of the ER
The Golgi apparatus (Golgi) typically transports materials from the ER, to the cellular environment or cytosol.
As part of normal cellular activity, retrograde transport may also occur. Retrograde transport is the movement of materials from the Golgi to the ER.
An example of this activity is when ER proteins escape into the Golgi, and the Golgi acts to return the escaped proteins back to the ER.
Toxins such as ricin, can also utilize this retrograde transport pathway from the Golgi, into the ER.
Retrograde transport may occur with dependence on, or independence from many factors, typically protein complexes. A common retrograde pathway utilizes a coat protein I (COPI).
COPI is a large protein complex found primarily on vesicles budding from the Golgi membrane. One function of COPI is to select other proteins in the Golgi for retrograde transport to the ER (i.e., escaped ER proteins). Selection of proteins for transport is based on particular amino acid signals that some proteins carry.
The COPI recognizes the specific amino acid signal, puts the selected protein into a coated vessel, and sends it to the ER.
It was once believed that ricin underwent retrograde transport by COPI, or similar pathway. Laboratory research demonstrates that ricin does not have COPI required amino acid signals, and thus undergoes transport independent of COPI, as well as other protein pathways such as Rab6A.
Current research tries to isolate other pathways that ricin may be dependent on for retrograde transport. Many believe ricin utilizes multiple pathways so that if one pathway is inactivated, the toxin may use another pathway for transport.
Although the specific pathway(s) are unclear, it is clear that ricin undergoes retrograde transport from the Golgi, into the ER.
STEP 3: Passage through the ER membrane, into the cytosol
The specifics by which RTA is transported through the ER, undergoes translocation, and moves into the cytosol is poorly understood.
The most accepted theory is that the toxin undergoes glycosylation and imitates a mis-folded protein, or other substrate identified for the ER associated protein-degrading (ERAD) pathway, to the Sec61 complex.
Glycosylation is a chemical reaction in which glycosyl groups are added to a protein to produce a glycoprotein. In the case of ricin, the toxin most likely interacts with the lumenal surface of the ER membrane, triggering glycosylation and structural changes that permit it to be recognized as a “mis-folded” protein.
Once recognized as a “mis-folded” protein, the toxin is readily transported to the Sec61 complex via ERAD pathway. The Sec61 complex imports proteins into the ER, and also delivers truly mis-folded proteins out of the ER, into the cytosol to be degraded or recycled.
Once in the cytosol, the “mis-folded” protein (or toxin) manages to avoid being detected and broken down like most mis-folded proteins.
STEP 4: Inactivation of Ribosomes
The “mis-folded” protein (or toxin) may manage to avoid being detected and broken down like most mis-folded proteins by many means. One may be because it lacks receptors that lysosomes use to recognize material as mis-folded or abnormal.
The intact, activated (reduced) RTA then enzymatically attacks and removes a specific adenine residue of the 28S rRNA.
This action renders the ribosome incapable of binding, thus blocking protein synthesis.
Cells not capable of protein synthesis eventually die.
CONCLUSION
Ricin may enter an organism through various routes of exposure. Once the toxin enters a cell, cell death is almost inevitable in this day and age, leading to initial indicators such as flu-like symptoms and mild cell necrosis; more serious symptoms, such as severe cell necrosis and organ failure, may follow depending on the dose and route of exposure.
Direct evidence shows that ricin undergoes a type of retrograde transport through Golgi, and translocation through ER into the cytosol, but this evidence does not exclude the possibility that more efficient translocation routes exist. Some current studies investigate:
1) Whether translocation may occur from endosomes rather than through the ER.
2) Whether transport through Golgi is required.
Note: This information was researched and prepared for a presentation in my environmental toxicology graduate course. If you have any questions or would like information on sources I used, please contact me at rohnbri@yahoo.com. Thank you.