2012 Biochemistry Protein Projects


Biochemistry CHM 345
Fall Semester
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Contact: Dr. Matthew Saderholm
CPO 2191
Berea College
Berea KY 40404
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Ricin

Introduction:

Ricin is a protein composed of an A and B chain, with the single chains being found in barley and most wheat plants. What is commonly assumed when referencing “ricin” is the combination of A and B chains; this occurs naturally only in Castor beans. Ricin, in this form, is highly toxic and has been used extensively as a biological weapon. Ricin’s structure has a twofold impact, the B chain gains entrance into the cell allowing the A chain to mutate the rRNA by inactivating ribosomes (1). Extensive research has been launched to gain a better understanding of ricin’s structure and pathways towards toxicity, this is in the hope that better antibodies can be produced, reducing the toxicity of ricin in humans (2).

Ricin poses such a great threat, because of the miniscule dose required to be toxic. Ricin is so toxic because a one molecule of ricin in a cell’s cytosol can depurinate over 1500 ribosomes per minute, thusly resulting in the stop of protein synthesis. (1) The consequence of stopped protein synthesis is cell death, which can lead to abdominal pain and dehydration due to vomiting and diarrhea.

Background

The castor bean plant is an ornamental plant found in warm climates around the world. Ricin is found naturally in the castor bean, as they are a defense for the castor bean plant. Ricin is inadvertently produced during the extraction of castor oil from castor beans. The extraction process of castor oil begins with the washing and dehydration of the beans to 5% of their original moisture content. The beans are then crushed to weaken and rupture cell walls to aid in the release of the castor fat for extraction. The crushed beans are then loaded into a mechanical press and the oil is extracted. The mechanical process results in about 45% recovery of oil. The other extraction method is solvent extraction, in which non-polar solvent and crushed castor beans are placed in a commercial extractor. The solvent extraction method heats the crushed seeds, thereby denaturing all of the ricin present (3).

Castor oil has many uses, including medicinal use as a laxative and industrial uses as lubricants and as an ingredient in plastics, waxes, soaps, dyes, and paints. The castor bean mash, once denatured, can be used as animal feed (1). Ricin is toxic to humans and animals in extremely low doses. Improperly processed castor bean mash can result in the death of livestock. Ingestion of one castor bean can kill a child and several castor beans can kill an adult.

The mechanical extraction does not involve heating the seed mash, therefore leaving the ricin non-denatured by the process of mechanical extraction. From the seed mash, ricin can be extracted and purified via chromatography. The ricin is extracted from the seed mash with dilute acid, precipitated out of solution with ammonium sulfate, and then purified via column chromatography.

Molecular Structure as it pertains to Toxicity Pathways

Figure 1: Ricin. The A chain is the top right protein and the B chain is the bottom left protein. Note that the pairs of spheres in B chain represent two lactose moieties bound to B chain (4)(5).

As previously stated, an A and B chain comprise ricin. The A chain is an N-glycoside hydrolase with three domains that form an active site cleft (as seen around residue 50 in Figure 1). Glycoside hydroloases, a process of breaking large sugar molecules into smaller ones and then releasing them, catalyze the hydrolysis of glycosidic linkage. In this case it cleaves a specific adenine residue from 28S rRNA. The A chain bonds in the cleft then breaks the single adenine disulfide bond to a galactose. The N-glycoside hydrolase is composed of 267 amino acids, arrange primarily into α-helices and β-sheets (6). The B chain is a lectin composed of 262 amino acids, with lobes that contain three sub-domains that act as a sugar-binding pocket. The structure of the B chain allows ricin to bind to the terminal galactose on the cell. The B chain is lacking both α-helices and β-sheets; its structure, instead, forms a clover-shape. The B chain enters the cell by binding to the terminal galactose on the cell’s surface, the reducing atmosphere then cleaves the disulfide bond linking chains A and B. The A chain of ricin cleaves adenine from an adenosine nucleotide inside 28s rRNA. When A chain cleaves off the adenine, the ribosome is rendered inactive, thus having undergone depurination. The ribosome cannot bind to elongation factors when inactive (1). This covalent modification of ribosomal RNA is why ricin is toxic.

Figure 2: The Toxicity Pathway for Ricin in mammalian cells (7).

As seen in Figure 2, ricin enters the cell via clathrin mediated endocytosis (7). Endocytosis is how cells internalize molecules and clathrin mediated endocytosis is the inward budding of plasma membranes containing receptor sites specific for the molecule to be internalized. Clathrin, a protein found in the receptor sites, does not have specificity towards ricin or other toxins, but it can mediate the entrance of such toxins into the cell.

Once through the plasma membrane, the ricin travels to endosomes. From the endosomes, most of the ricin will be either recycled back to the surface or degraded in lysosomes. A small portion of the ricin will be transported to the trans golgi network (TGN) (8). The TGN is a complex network of membranes and vesicles that sorts and ships proteins to where they need to go based upon the molecular marker that they carry. From the TGN, ricin travels via the Golgi cisternae to the endoplasmic reticulum (ER). When ricin finally gets to the ER, the A chain crosses the membrane and depurinates ribosomes (9). Ricin and other toxins that follow this toxicity pathway are extremely potent because most of the toxin gets degraded or recycled to the surface, leaving only a small fraction to terminate the cell (10)(7).

Conclusion

Ricin is toxic in very small doses and is relatively easy to manufacture, as the sale and cultivation of castor beans is not prohibited. Ricin is an extremely potent toxin, utilizing both its A and B chains to infiltrate and eviscerate the cell by depurinating ribosomes, preventing elongation.

References

  1. Audi, J., Belson, M., Patel, M., Schier, J. Osterloh, J., Ricin Poisoning: A Comprehensive Review J. Am. Med. Assoc. 2005, 294 (18), 2342 – 2351.
  2. Endo, Y., Tsurugi, K., RNA N-Glycosidase Activity of A-chain, J. Biol. Chem.1987, 17(262), 8128 – 8130.
  3. Montfort, W., et al. The Three-dimensional Structure of Ricin at 2.8 Å, J. Biol. Chem.1987, 262(11), 5398 – 5403.
  4. Ho, M.C. , Sturm, M.B. , Almo, S.C. , Schramm, V.L. Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins, Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 20276 – 20281.
  5. Dai, J., Zhao, L., Yang, H., Guo, H., Fan, K., Wang, Huaizu, Qian, W., Zhang, D., Li, B., Wang, Hao, Guo, Y. Identification of a Novel Functional Domain of Ricin Responsible for its Potent Toxicity, J. Biol. Chem. 2011, 289, 12166 – 12171.

6.      Olsnes, S., The History of Ricin, Abrin, and related toxins, Toxicon 2004, 4(44), 361 – 370.

  1. Lord, J.M., Roberts, L.M., Robertus, J.D., Ricin: structure, mode of action, and some current applications, FASEB J. 1994, 2(8), 201-208.
  2. Chen, X.Y. , Link, T.M. , Schramm, V.L. Ricin A-Chain: Kinetics, Mechanism, and RNA Stem-Loop Inhibitors, Biochemistry 1998, 37, 11605 – 11613.
  3. Lord, J.M., Roberts, L.M., Retrograde transport: Going against the flow, Curr. Biol.,1998, 8(2), R56 – R58.
  4. Ricin Toxin from Castor Bean Plant, Ricinus communis, 2009. Plants Poisonous to Livestock on Cornell University: Dept. of Animal Science. http://www.ansci.cornell.edu/plants/toxicagents/ricin.html (Accessed on November 23, 2011)