Lectins are proteins or glycoproteins, mainly of plant origin, which bind specifically to monosaccharide or oligosaccharide sugars. Owing to the abundance of these proteins in plant seeds and their wide range of specificities for complex carbohydrates, lectins are extensively used as tools in different areas of scientific research and have a broad range of potential applications in biomedicine and biotechnology. Certain plant lectins bind to sugars expressed by cells of the gastrointestinal tract and consequently these lectins have received interest as targeting agents for oral delivery of vaccines and drugs. Many plant lectins have been shown to be toxic to animal cells and certain of these proteins are known to modulate or change important immune mechanisms such as inflammatory responses. The aim of this project is to examine the cytotoxic and immunostimulatory effects of a range of plant lectins on immune cells and to determine if there is any correlation between toxicity and immunomodulatory effects. Macrophages and dendritic cells are two innate cell types which are important in the initial immune response to foreign material, and these cells are crucial in immunosurveillance to invading pathogens and in responding to inflammatory signals. This project will firstly examine the cytotoxic effects of a range of plant lectins on macrophage and dendritic cell lines. Macrophages and dendritic cells, as part of their important role in the initiation of innate immunity, are also potent producers of immune mediators or signalling molecules termed cytokines. Several cytokines, such as interleukin(IL)-6 and IL-1beta, may be released by innate cells upon stimulation with an inflammatory stimulus. Therefore, this project will also assay cytokine (IL-6 and IL-1beta) secretion by innate cells stimulated with plant lectins, to examine the inflammatory responses induced.
Main Research Questions:
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It is possible to examine the cytotoxic and immunostimulatory effects of a range of plant lectins on immune cells and to determine if there is any correlation between toxicity and immunomodulatory effects.
To examine the cytotoxic and immunostimulatory effects of a range of plant lectins on immune cells
To develop a standard, reliable system to examine the cytotoxic effects of a range of plant lectins on macrophage and dendritic cell lines.
To examine the inflammatory responses induced by assaying cytokine (IL-6 and IL-1beta) secretion by innate cells stimulated with plant lectins.
Contribution to Knowledge of Discipline Area:
Development of examination of the cytotoxic effects of a range of plant lectins on macrophage and dendritic cell lines
Investigate the relationship between toxicity and immunomodulatory effects
Investigate the inflammatory responses induced by assaying cytokine (IL-6 and IL-1beta) secretion by innate cells stimulated with plant lectins
Background of Plant Lectins
Lectins are proteins or glycoproteins, mainly of plant origin, which bind specifically to monosaccharide or oligosaccharide sugars. Most plants contain at least one lectin. Due to the abundance of these proteins in plant seeds and their wide range of specificities for complex carbohydrates, lectins are extensively used as tools in different areas of scientific research and have a broad range of potential applications in biomedicine and biotechnology although the function of plant lectins are just beginning to be understood. And most lectins are mutivalent and aggulutinating cells. Furthermore, since lectins can recognize the difference kinds of carbohydrates to bind with, they are applied in the characterization of glycoconjugates.
Lectins are usually discovered in the cell, membrane of different kinds of organisms and tolerate a degree of binding with carbohydrates. Carbohydrates are a diverse group of molecules , which include more molecules than proteins. Beyond the variation found in the carbon backbone length of the monomer, anomericity, side group orientation and substitution, the carbohydrate assembly often branches, further complicating structure. And due to these large differences, there is a characteristic carbohydrate coat every cell, membrane or intracelluar organelle which can be recognized by lectins. Carbohydrate residues on the exterior of a cell are often bound by a specific lectin or a small group of lectins, and as such lectins have been used as histochemicals reagents to label mammalian cells. In addition
History of Plant Lectins
The plant Lectin was discovered by biochemist J.B. Sumner in 1919. He crystallized the lectin called concanavalin A which comes from the common jackbean Canavalia ensiformis. And later he discovered that ConA can bind and precipitate some polysaccharides such as starch and glycogen in 1926. And the term lectin was not used until1954. At that time, the term lectin was limited to soluble, multivalent proteins capable of agglutination. But now the term lectin is widely used as all types of carbohydrate-binding proteins which do not catalyze reactions with their ligands. And nowadays thousands of different plant lectins have been discovered and identified. (1)
Classification of Plant lectins
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Basically there are seven types of plant lectins have been discovered so far. They are the Leguminosae such as ConA, soybean aggltinin, and lentil lectin, amaranthins, Cucurbitaceae phloem lectins, lectins with hevein domains, jacalin related lectins, legume lectins, mannose-binding lectins from monocots and type-2 ribosome-inactivating proteins. And the Leguminosae is the best-characterized family. In addition, some scientists also classify the lectins according to the small carbohydrate hapten they recognize such as galactosebinding lectins or GlcNAc-binding lectins. (2-4)
Toxicity of Plant Lectins
Most plant lectins are toxic to animal cells but not all of them. Owing to lectins can be denatured by the cooking and proteolytically digested upon consumption, lectins can become harmless. This point reminds us of avoiding the plant lectins from the high temperature in the lab. And raw lectins can be harmful because uncooked lectins are stable to proteases. For instance, plant lectins from the raw navy beans can change the intestinal microflora and results in gastrointestinal dysfunction. And Raw
soybean lectin and wheat-germ agglutinin can lead to releasing of cholecystokinin which have effects on gastrointestinal function and growth. Moreover, some plant lectins can be extremely toxic which result in death of cells in culture and animals. And this type of lectins is usally heterodimeric and contains one subunit for binding to carbohydrate ligands and a second subunit that has an enzyme binding activity instead of carbohydrate-binding activity. For example, ricin from Ricinus communis is a heterodimeric protein containing an A chain and B chain. The B chain is the carbohydrate-binding subunit and the A chain is an enzyme with adenosine-N-glycosidase activity that can inactivate ribosomes. A molecule of A subunit can completely block protein synthesis. These toxic plant lectin subunits are now being tested as treatments for cancer. (5-8)
Uses of Plant Lectins
Although the natural functions of plant lectins are just now being investigated, scientists have discovered many applications of plant lectins. In the past, lectins were used to distinguish the blood typing because of the ability of recognization of carbohydrate determinants in blood cells. And lectins are useful in immunological studies, which are mainly involved in this project. At low concentrations lectins such as ConA, pokeweed mitogen and LPHA are mitogenic to peripheral blood lymphocyes. And high concentrations lectins such as ConA and LPHA are very cytotoxic to cells and have been used to select for cell lines mutated in glcosylation pathways. So this suggest the student to establish the dose-dependent standard curve of lectins to access the cytotoxic effects for immune cells. Moreover, lectins are also applied in histochemical studies and in lectin-blotting assays on elecrophoretically separated glycoproteins.(9-15)
Mechanism of 1L-1b secretion
The cytokine interleukin-1b(1L-1b) is the key mediator of the inflammatory response. It is significant for resistance to pathogens and host-response. In addition, it can make more damage during chronic disease and acute tissue injury. However, it is hard to find out the mechanism of 1L-1b release. It does not follow the conventional ER-Golgi route of secretion. But it is also the best characterized and most researched of the 11 1L-1 family, which is good for search others works to compare with. Its production within cells of the innate immune system such as monocytes and macrophages had been investigated a lot. An inactive 31 kDA precursor, termed pro-1L-1b is produced in response to molecular motifs carried by pathogens called PAMPs(pathogen asssociated molecular patterns). PAMPs act by PRRs(pattern recognition receptors) on macrophages to regulate pathways of controlling gene expressions. Induction of pro-1L-1beta expression is generally referred to as a priming step, and is and inefficient secretion stimulus. The primed cell must now encounter a further PAMP, or DAMP to lead to the processing and secretion of an active 1L-1b molecule. And the pro-inflammatory protease caspase-1 can cleave the pro-1L-1beta. And the caspase-1 is activated by recruitment to a multi-protein complex termed the inflammasome, a molecular scaffold composed of adaptor molecules, a cytosolic pattern recognition receptor, and pro-caspase-1. The cytosolic PRR NLRP3 can form the best characterised in flammasome. And the mechanism of secretion can be influenced by the type and strength of stimulus.(16)
Outcomes of Project
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We propose to examine the cytotoxic and immunostimulatory effects of a range of plant lectins on immune cells and there is correlation between toxicity and immunomodulatory effects. This project will firstly examine the cytotoxic effects of a range of plant lectins on macrophage and dendritic cell lines. At low concentrations lectins will be mitogenic to immune cells. And high concentrations lectins will be very cytotoxic to cells. The inflammatory responses induced were examined by assaying cytokine (IL-6 and IL-1beta) secretion by innate cells stimulated with plant lectins.