Modeccin is a toxic lectin, a group of glycoproteins capable of binding specifically to sugar moieties. Modeccin is found in the roots of the African plant Adenia digitata. These roots are often mistaken for edible roots, which has led to some cases of intoxication. Sometimes the fruit is eaten, or a root extract is drunk as a manner of suicide.
Structure and reactivity Modeccin consists of two subunits, bound by a disulfide linkage. The intact protein has a molecular weight of approximately 57-63 kDa. When treated with mercaptoethanol, the chains can be dissociated into two subunits, subunit A (25-28 kDa) and subunit B (31-35 kDa). The A-chain, or effectomer, possesses ribosomal-inactivating properties. The B-chain, or haptomer, contains the carbohydrate binding site. While the intact toxin molecules have potent cytotoxic effects on cells, they exhibit no ribosomal inactivating activity on ribosomes in a cell-free system. Reduction of the toxin with a disulfide reducing agent creates the opposite effects. Reduced, dissociated toxin subunits inhibit ribosomal activity in cell-free systems, but have no effect on intact cells. These properties are due to the toxin's mode of action. Toxin molecules bind through saccharide recognition sites on the B-chain to particular β-galactosyl-containing glycoprotein or glycolipid components on the surface of cell membranes. In animals that are sensitive to these toxins these polysaccharides are present in virtually all cell types. The toxin binds to cell-surface polysaccharide receptors with a high affinity (Ka in the range of 107–108/M). When the toxin binds to the cell, the A-chain enters through either active transport or endocytosis. Once inside the cell, the A-chain enters the cytoplasmic space, binds to the 60S ribosomal subunit and enzymatically inactivates it. The mechanism is catalytic because of this one toxin molecule is enough to disrupt protein synthesis and kill the target cell.
Related toxins Cytotoxic lectins including modeccin act in a similar manner as ricin, a well understood toxic lectin, though each one has a different saccharide binding specificity. Cytotoxic lectins include ricin, abrin, modeccin, volkensin (least toxic, 10 and 40 times less cytotoxic than ricin and modeccin respectively.) and viscumin (10 times less cytotoxic than ricin). Comparison of the parenteral lethality of ricin and related toxins in laboratory mice
Hybrids Synthetically produced toxins and genetically engineered toxin chimeras are areas of emerging interest due to their possible application as new medical modalities (e.g., IgTs) and powerful research tools, as well as their potential misuse as toxin weapons to confuse traditional medical countermeasures. Hybrid molecules were prepared from the A- and B-chains of the toxic lectins ricin and modeccin by dialyzing mixtures of isolated chains to allow a disulfide bridge to be formed between them. Whereas the hybrid consisting of ricin A-chain and modeccin B-chain was non-toxic, the converse hybrid, modeccin A-chain/ricin B-chain, was even more toxic than parent toxins, native ricin and modeccin.
Extraction Extraction of modeccin from the roots of Adenia digitata follows the following procedure. It is firstly chopped into small pieces and then soaked in a sodium chloride solution overnight. It is then homogenised in a Waring Blender and left standing overnight. A cheesecloth is used to wring out all moisture from the mixture. The extract was left standing again overnight, and the remaining supernatant was obtained through centrifugation. The dissolved proteins were precipitated by saturation of the supernatant using ammonium sulfate. The proteins were obtained through centrifugation, redissolving in H2O and dialysis against running water for two days. Finally, the precipitate was removed via low speed centrifugation and the supernatant was freeze-dried. The extract was fractioned using a Sephadex column. The different fractions were assayed for toxicity to mice, to which the most toxic fraction was pooled and subsequently applied to a DEAE-(diethylaminoethyl)-column. The bound proteins, including modeccin, were eluted and fractionised using a gradient of sodium chloride in solution. Again, the fractions were assayed for toxicity to mice, to which the most toxic fraction was pooled and analysed by Gel Electrophoresis. To better follow the protein during further purification, labelling using 125I is done via the lactoperoxidase method. This does not affect toxicity. Further purification using affinity chromatography with immobilised glycoproteins. Affinity was increased by glycoprotein-treatment with neuraminidase, enzymes that cleave glycosidic linkages of neuraminic acids. Elution of the proteins from the column was effectuated using lactose. Dialysis of the eluted proteins removed the lactose. The different fractions were assayed for toxicity to mice. Gel electrophoresis of the fractions was carried out on a polyacrylamide gel. Samples were made containing sodium dodecyl sulphate, and in some cases small amounts of mercapto- ethanol. A molecular weight of approximately 63 kDa is found in the protein fractions, with traces of 38 kDa and 28 kDa. These trace-fractions are more apparent in mercaptoethanol treated protein fractions. This confirms that modeccin consists of two protein chains of 28 and 38 kDa respective, linked via a disulfide bond.
Mechanism of action Modeccin inhibits protein synthesis by inactivating the 60S ribosomal subunit. The toxin inhibits both initiation as elongation of peptide chains. The toxin only attacks eukaryotic ribosomes, as bacteria are resistant.
The B-chain The B-chain has saccharide recognition sites for particular ß-galactosyl-containing glycopro-teins or glycolipid compounds on the cell membrane. Modeccin B-chain enters the cytosol after a delay, since most of the time it is present in intracellular vesicles. Without the Golgi complex, the B-chain cannot enter the cytosol and therefore loses its toxicity. It only enters the cytosol after it has reached the Golgi complex. Modeccin requires a low pH for entry into the cell. Below pH of 6.0, modeccin can't enter the cell via endocytosis. It is also known that entry to the cytosol require Ca2+-ions.
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