PLANT LECTINS
The high toxicity of castor beans was recognized during the last century when the extract was found to agglutinate a suspension of erythrocytes of different animal species. Since then, lectins were studied and extracted from plants, including fungus and lichens, as well as in animals.
Lectins are glycoproteins of 60,000-100,000 MW that are known for their ability to agglutinate (clump) erythrocytes in vitro. There are over 400,000 estimated binding sites for kidney bean agglutinin on the surface of each erythrocytes. Lectins are found in most types of beans, including soybeans. Reduced growth, diarrhea, and interference with nutrient absorption are caused by this class of toxicants. Different lectins have different levels of toxicity, though not all lectins are toxic, though no all are toxins. The bright scarlet seeds of precatory bean Abrus precatorius contain the highly toxic glycoprotein, abrin. Less toxic lectins can be fatal if ingested in high amounts. Some of such lectins is concanavalin A from Concanavalia ensiformis (jack bean). Others may exhibit no hemagglutinating activity as in the case of ricin from castor bean and yet it is one of the most toxic substances.
The terms phytohemagglutinins, phytagglutinins, and lectins are used interchangeably. Lectins-containing plants have been found in many botanical groups including mono- and dicotyledons, molds and lichens, but most frequently they have been detected in Leguminoseae and Euphorbiaceae. They may exist in various tissues of the same plant and have different cellular localizations and molecular properties.
Lectins interaction with certain carbohydrate is very specific. This interaction is as specific as the enzyme-substrate, or antigen-antibody interactions. Lectins may bind with free sugar or with sugar residues of polysaccharides, glycoproteins, or glycolipids which can be free or bound (as in cell membranes). The term lectin refers to the specificity of the reaction (legere = to choose).
One of the major interest in this class of glycoproteins is the therapeutic use against HIV-1. Jacalin, a plant lectin, is found to completely block human immunodeficiency virus type 1 in vitro infection of lymphoid cells. This activity of the jacalin is attributed to its ability to specifically induce the proliferation of CD4+ T lymphocytes in human.
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The ability to agglutinate human erythrocytes or representatives of human indigenous microflora was detected in 29 of 88 food items. Many foods contained substantial amounts of agglutinating activity, and lectins extracts could be diluted several folds and still produce agglutination. Great variation was observed in agglutination activity in the same food item purchased from different stores or from the same store on different days. Sometimes a food that possessed substantial activity on one day was found to have little or even no activity on other day.
A survey of the fresh and processed foods found lectins in about 30% of the food stuffs tested, including such common foods as salad, fruits, spices, dry cereals, and roasted nuts. However, dry heat may not completely destroy lectin activity. Hemagglutinating activity is found in the processed wheatgerm, peanuts, and dry cereals. Several lectins are resistant to proteolytic digestion e.g., wheatgerm agglutinin, tomato, lectin, and navy bean lectin.
Not much is known about the functions of lectins in the organism they are formed. There is evidence that lectins may be involve in the recognition between cells or cells and various carbohydrate- containing molecules. This suggests that they may be involved in the regulating physiological functions. They seem to play an important role in the defence mechanisms of plants against the attack of microorganisms, pests, and insects. Fungal infection or wounding of the plant seems to increase lectins. In legumes, the role of lectins in the recognition of nitrogen-fixing bacteria Rhizobium genus, which have sugar-containing substances, has received a special attention.
Binding Nitrogen-Fixing Bacteria to legume Roots: among the possible functions of plant lectins is their participation in binding nitrogen-fixing bacteria to legume roots. In this reaction bacteria of the genus Rhizobium adhere to the surface of differentiated roots cells and are then internalized into the root hair to form nitrogen-fixing nodules. The symbiosis is specific in that certain species of Rhizobium can only associate with a particular species of legume. Such observations were made from the studies that were done on soybean Rhizobium japonicum and the clover Rhizobium trifolii. Other functions of lectins in plants may include:
- Enzymes (but unknown substrate)
- Storage of proteins
- Defense mechanism
- Cell wall extension
- Mitogenic stimulation
- Transport of carbohydrates
- Packaging and/or mobilization of storage materials
One major property of lectins is their specific saccharide- binding sites. Some lectins are composed of subunits with different binding sites. These include the lectin from the red kidney bean, Phaseolus vulgaris. It is composed of two different subunits combined into five different forms of noncovalently bound tetramers. Since subunits have very different specificities for cell surface receptors, each combination is considered to have a different function. The specificity of the binding sites of the lectins suggest that there are endogenous saccharide receptors in the tissues from which they are derived or on other cells or glycoconjugates with which the lectin is specialized to interact.
Metal Binding Sites
Biological activity of the lectins may be attributed to the metal ions which are the essential part of the native structure of most leguminous lectins. The most studies and fully sequenced lectin is concanavalin A. The metal binding sites of the concanavalin A are situated in the amino terminal part of the polypeptide chain. In this lectin, each subunit has aspartic 10 and 19, asparagine 14, histidine 24, serine 34, glutamic acid 8, and tyrosine 12 that are involved in the binding to one calcium and one magnesium ion.
Lectins of soybean, peas, faba bean, lentils, and sainfoin have amino acids that are involved in metal binding, which are conserved. The exception is of the tyrosine residue at position 12 of concanavalin A which is replaced with by phenylalanine in the other legume lectins.
Hydrophobic Sites
The stability of the native structure of most lectins is thought to be caused by the hydrophobic interactions. Such hydrophobic sites, forming cavities in the lectins structure, may play an important biological role. The hydrophobic binding sites of auxins, or cytokinin and adenine, for instance, by concanavalin A may enhance the functions of lectins on the plant life cycle.
Glycosylation Sites
Despite the generalization that most lectins are considered glycoproteins, concanavalin A, lentils lectin, and wheatgerm agglutinin contain no covalently attached carbohydrates. However, non-glycoprotein lectins are believed to be synthesized as glycosylated precursors. This is supported by the following observation:
- pro-concanavalin A is an inactive glycoprotein from which the glycosidic side chain is removed during post-translational processing.
- Non-glycoprotein wheatgerm agglutinin molecules are produced by removing a carboxyl terminal glycopeptide from the glycosylated precursor during post-translational processing.
All glycoprotein lectins contain a peptide sequence: asparagine- X-threonine/serine, which is characteristic of glycosylation sites. These sequences are different in the non-glycoprotein lectins. Also, peptide sequences, which in one glycoprotein lectin contain the glycosidic side-chains, are not necessarily conserved in another glycoprotein lectin. This may suggest that the biological activity of the lectins may not be determined by carbohydrate part of their structure.
Carbohydrate Binding Sites
Lectins differ markedly in their sugar-binding specificity. A sequence participating in carbohydrate-binding site of concanavalin A, for instance, are poorly conserved in other lectins.
The three dimensional structures of lectins can be used to show the structural similarities and homologies of legume lectins. When the secondary structure of Vicieae lectins, for instance, is compared with those concanavalin A, an identical or homologous B- turn structures are found in these lectins. Hydropathic profiles of two chains and single-chain lectins are superimposed, and concanavalin A has three domains that appear in some two-chain lectins of Vicieae.
The two-chain and single-chain legume lectins exhibit high homology in the primary sequences and three-dimensional structures. Therefore evolution seems to have imposed only slight modification in the genetic coding for these lectins. This fact suggests the possibility of using lectins as an acceptable phylogenetic markers.
Stereodiagram of monomer-monomer interface in pea lectin.
Stereodiagram of pea lectin monomer.
Lectins in soybean plant are different from those in seeds. Dolichos bifluorus's lectins were studies in both the leaves and seeds. Lectins found to be produced with low level and constant for the period between 2-8 weeks after germination, then increased several folds in the next ten weeks. At this time extracts of stems and leaves contain several nanograms of lectin- like material per microgram of nitrogen, whereas mature seeds have about 1000 ng lectin per microgram. It is notable that in developing seeds the amount of lectin rises very abruptly. It is undetectable during the first 26 days after flouring but reaching a maximal level by day 28. Lectins from the stems and leaves don not agglutinate erythrocytes that could be agglutinated by seeds lectins. Stems and leaves lectin is a dimer of a molecular weight of 68,000, in contrast to the tetrameric seed lectin with a molecular weight of 110,000.
Pathological lesions occur in animals injected with kidney beans extracts. Various tissues suffer from parenchymatous, fatty degeneration, and edema. In the liver local necrosis and fatty changes can be observed. Hemorrhages are observed in the stomach, the intestinal wall, and other organs. Distentions of capillary vessels may present in the Kidney and myocard with numerous thrombi. Morphological changes in rats fed navy beans include: increased weight of kidney and heart, pancreatic acinar atrophy, and fatty metamorphosis of the liver. Such changes may be attributed to the low availability of essential amino acids and low food intake of the animals consuming the raw bean diet. For example, rats fed raw kidney beans develop multiple histological lesions. Also, lectins from red kidney beans are found to induce small intestinal epithelial growth, crypt cell hyperplasia and DNA synthesis. Small amounts of isolated black bean agglutinin show low food absorption and nitrogen retention in rate.
The absorption of glucose from a ligated intestinal loop in anesthetized rats, previously fed a bean diet or given the black bean agglutinin by stomach tube, was much decreased. Raw kidney beans were found to interfere with vitamin E utilization in chicks. The hypoglycemia observed in rats fed a bean diet may indicate a reduced intestinal absorption of glucose.
Diet rich in raw soybean has a goitrogenic effect. This is indicated by the fact that fecal loss of thyroxine from the gut is higher in animals fed raw soybeans than in the controls. Raw soybean meals reduces fat and fatty acids absorption (not soybean trypsin inhibitor) in young chicks. Such meals also depresses the utilization of vitamin D in turkey. These effects are not found when the meals include heated soybean.