Antinutritional and Allergenic Proteins - ACS Publications - American

Chapter 4

Antinutritional and Allergenic Proteins H. Frøkiær, T. M . RJørgensen,A. Rosendal, M. C. Tonsgaard, and V. Barkholt

Downloaded by COLUMBIA UNIV on July 18, 2012 | http://pubs.acs.org Publication Date: April 1, 1997 | doi: 10.1021/bk-1997-0662.ch004

Department of Biochemistry and Nutrition, Building 224, Technical University of Denmark, DK-2800 Lyngby, Denmark

Immunologic and immunochemical methods are valuable tools in the investigation of food allergens and antinutritial proteins. Protease inhibitors and lectins are present in a broad spectrum of foods. They can retain their activity during passage through the digestive tract and induce different biological reactions, beneficial as well as harmful. The potential nutritional importance of legume inhibitors and lectins is evaluated from analyses of their concentration, biological activity and their stability during food processing conditions. Food allergens comprise a very varied group of proteins and glycoproteins with different sensitization routes for children and adults that are currently discussed. Food allergens' induction of oral tolerance was investigated by oral immunization of mice with milk or β-lactoglobulin.

A major part of our common protein sources may give rise to adverse reactions. Food components with general adverse effects are described as toxic or antinutritional, whereas adverse reactions towards components in amounts that do not normally give rise to adverse effects are described as either intolerance or allergy. Antinutritional Proteins Protease inhibitors as well as lectins are widely spread in nature and have essential importance for the availability of nutrients upon ingestion. They occur in most, if not all, plants and protect them against attack from microorganisms and insects through various low-molecular-weight compounds. Crops with a high protein content, e.g. members of the leguminosae family, contain especially high levels of antinutritional proteins. A common property of the antinutritional proteins is their resistance towards proteolytic degradation as well as a generally high stability towards heat treatment.

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© 1997 American Chemical Society In Antinutrients and Phytochemicals in Food; Shahidi, F.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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Lectins. Lectins occur in many plants and are characterized by their highly specific carbohydrate binding activity. However, lectins are not only present in plants but also in microorganisms and mammals, including humans. Although characterized by their common capability to bind carbohydrate, lectins form a very heterogeneous group of proteins with respect to their molecular structure, carbohydrate specificity and biological activity. Lectins were earlier named phytohemagglutinins as the first plant lectins discovered were able to agglutinate red blood cells (7). As plant lectins are not always capable of hemagglutination, a new definition has been introduced. According to this, all proteins possessing at least one non-catalytic domain that specifically binds monoor oligosaccharides reversibly are defined as lectins. Plant lectins can be divided into four groups: legume lectins, chitin-binding lectins, monocot mannose-binding lectins and type-2 ribosome-inactivating proteins (2). The two groups of lectins described below are most important in connection with food antinutrients. Legume Lectins. Proteins from the legume lectin group are structurally related lectins that occur exclusively in legumes. Most of these lectins are glycoproteins. In spite of their structural relationship, legume lectins show a broad variation in their specificity towards carbohydrates and thereby in their biological activities (3). Type-2 Ribosome-inactivating Lectins. Type-2 ribosome-inactivating lectins which includes ricin from castor bean bind to the surface of most eucaryotic cells and are taken up by the cells. Part of the lectin is able to attach to the 60 S ribosomal subunit and thereby halting the protein synthesis (3). Biological Effects of Lectins. Although the first recognized activity of lectins was the capability of these proteins to agglutinate red blood cells, this activity is not important with respect to most lectins found in plant seeds used for human consumption and animal feed. Lectins bind with high affinity to oligosaccharides which are absent in plant but are abundant in bacteria as polysaccharides of bacterial ceil walls and in animals especially as constituents of glycoproteins in cell membranes. Several membrane integrated glycoproteins function as receptors for hormones and cytokines or are directly involved in cell to cell contact. Binding of lectins to these glycoproteins may therefore trigger reactions such as cell division and growth (mitogenic effect), cell maturation and cell death (3). Responses in Small Intestine to Lectins. The generally high resistance of lectins towards proteolytic degradation enables them to pass through the digestive tract of humans and animals in the intact form (3). Furthermore, when the ingested lectin reaches the small intestine it may bind to certain glycoproteins on the surface of intestinal cells according to the carbohydrate specificity of the lectin. Plant lectins, specific for N-acetylgalartosarnine/galatose, e.g. soybean agglutinin (SB A) and wheat germ agglutinin (WGA), or specific for complex carbohydrate containing these

In Antinutrients and Phytochemicals in Food; Shahidi, F.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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ANTINUTRIENTS AND P H Y T O C H E M I C A L S IN F O O D

residues (e.g. kidney bean lectin, PHA), bind strongly to receptors on epithelium cells in the digestive tract and stimulate the growth of the small intestine and the pancreas as well as accumulation of polyamines in these sites (4). Lectins that bind less avidly to the epithelium cells, e.g. mannose specific lectins such as pea lectin (PSA), induce less prominent growth of the intestine and pancreas. As the glycosylation patterns of membranes of the epithelial cells differ with animal species and age of the animal, the effects of a lectin may vary widely. Except for a few incidences of accidental poisoning of humans with lectins from castor beans and red kidney beans, little is known about the toxicity of plant lectins to humans, either the acute toxicity or possible effects upon prolonged ingestion (5). However, experiments with the binding of various lectins to membrane of the human intestinal cancer cell line Caco2 have provided information of the binding activity of several legume lectins (5). P H A and SBA bound strongly to the cells and resulted in a shortening of the microvilli of the cells. PSA bound less avidly but to more sites on the cells and did not result in any decrease in the length of the microvilli. Moreover, these studies showed that the binding of lectins with high avidity for carbohydrate on the cell surface caused increased growth of the cells and destruction of the cytoskeleton (6). The effects of these and other lectins may either impair the capacity of the epithelial cells to absorb nutrients from the gut or increase the permeability of the mucosal barrier to macromolecules. Isolectins with different specificity. Many legumes contain two isolectins (e.g. kidney bean and castor bean) which show high sequence homology but differ in carbohydrate specificity and activity (7). Of the two different isoforms of kidney bean lectin, P H A - L and PHA-E, PHA-E binds to a larger extent to the surface of epithelial cells than PHA-L, whereas only PHA-L has been demonstrated to be mitogenic towards lymphocytes (8). Effects of Lectins on Cells of the Immune System. Independent of the capability of lectins to induce growth of intestinal cells, lectins may interfere with other cells of animals and humans. As lectin binds to the epithelium of the small intestine, it is absorbed by epithelium cells by endocytosis and taken up into systemic circulation in the intact form (2). Once the lectin reaches the circulatory system it may affect cells which are not present in the digestive tract. Animals fed soybean or red kidney bean develop a strong humoral antibody response towards the respective lectin (9,70), in contrast to other dietary proteins (77) which usually cause immunological tolerance when ingested. Furthermore, many lectins exhibit strong mitogenic effects on different subpopulations of lymphocytes. Lectins taken up systemically may therefore change the micro-environment in the lymphoid organs and thereby interfere with the immune status of the recipient. We have investigated the mitogenic effects on mouse lymphocytes from various lymph organs of four lectins from legume seeds extensively used for human consumption and animal feed, PHA-L, SBA, PSA, and peanut lectin (PNA). Of these lectins only SBA has been demonstrated to cause damage upon binding to epithelial



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cells in the small intestine while the other lectins are known to bind with less avidity to intestinal cells without causing damage to the epithelium (7,9). As shown in Figure 1, SBA does not significantly stimulate the growth of mouse lymphocytes in any lymph organ except for spleen cells. PHA-L and PSA demonstrated mitogenic activity towards lymphocytes of lymph nodes, spleen and to a lesser extent lymphocytes from Peyers' patches. P N A stimulated, exclusively, the growth of lymphocytes from Peyer's patches. The lymph nodes of the intestine, Peyer's patches (PP) are surrounded by specialized epithelial cells, M cells, with only slightly folded membranes compared to other enterocytes. M cells contain less lysosomes and are capable to transport dietary proteins, including lectins, to PP for presentation to the immune system (72). P N A is known to bind to immature B cells in the germinal centers which are constantly abundant in PP in contrast to other lymphoid organs due to the constant presentation of dietary proteins and microorganisms. Our results indicate that PNA, in addition to its ability to bind to B cells in germinal centers, has the ability to stimulate proliferation of the B cells. Therefore, small amounts of lectins, apparently harmless upon digestion, may change the interaction between lymphocytes in PP as well as in other lymphoid tissues because of their mitogenic effects. Long-term effects of lectins. As most of the observed effects of dietary lectins have been obtained through short-term experiments, it is not known whether exposure over a long period may have any effect. The fact that many apparently harmless dietary lectins are able to cross the epithelium of the intestine and exhibit mitogenic activity towards different cell types raises an important question as to whether even small amounts of lectins can induce uncontrolled cell growth in organs or tissues. It remains an important issue to investigate whether such effects could lead to induction of malignant or benign turmors or immune disorders. Analysis of lectins. As indicated by their earlier name, hemagglutinins, lectins can be detected by their agglutinating activities on red blood cells. This agglutinating activity has been used to detect active lectins from winged bean and jack bean in gut content and feces (3,74-76), whereas SBA activity was not found in similar experiments (77). These different results may be explained either by different stabilities or binding activities of the lectins or by differences in sensitivity of the hemagglutination assays. Hemagglutination assay is performed by a dilution series of lectins added to a fixed concentration of blood cells. The lowest dilution of lectin agglutinating the blood cells is visually determined. The sensitivity of hemagglutination analyses varies strongly depending of the actual lectin, the source of blood cells and the length of time since the blood was drawn. Some lectins are not capable of agglutinating blood cells at all and the sensitivity of hemagglutination assays for lectins capable of agglutination varies at least 1000 fold, typically in the range of 50 ng/ml to 50 //g/ml. Alternative to hemagglutination assays the most widespread methods to determine lectins are different immunochemical analyses (J8). Among the immunochemical analyses, ELIS A (enzyme linked immunosorbent assay) is the easiest



ANTINUTRIENTS AND PHYTOCHEMICALS IN FOOD

HSBA D PHA-L PSA

I

PNA

Spleen

Figure 1. Mitogic activity of four legume lectins towards mouse lymphocytes from different lymph organs. Proliferation of lymphocytes was measured as incorporation of HMabelled thymidine: Lymphocytes (2 • 10 cells/ml) were stimulated with lectins from soybean (SBA), kidney bean (PHA-L), pea (PSA) and peanut (PNA). L N : inguinal and popliteal lymph nodes, M L N : mesenteric lymph nodes; PP: Peyer's patches.

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to perform and usually also the most sensitive. Using a sandwich ELISA with two monoclonal antibodies against PSA, we were able to detect as little as 0.5 ng/ml of lectin is shown in Figure 2. Another type of assay is FLIA (functional lectin immuno assay), where lectin in the sample reacts with glycoproteins attached to the solid phase and is detected by a specific antibody. FLIA is not as sensitive as the sandwich ELISA but ensures that only the active lectin is detected. The FLIA method can be used to follow inactivation of lectin during food processing as illustrated in Figure 2, whereas the sandwich ELISA is useful to determine the amount of lectin absorbed from the intestine in feeding trials as this method is the most sensitive (unpublished results). Proteinase Inhibitors. Similar to lectins, inhibitors of proteolytic enzymes are able to resist proteolytic degradation and thus survive passage through the digestive tract. The inhibitors bind tightly to the proteolytic enzymes. Thereby the enzymes are inactivated and the resultant enzyme-inhibitor complex as well as unbound inhibitors pass undegraded through the digestive tract causing poor utility of the food and in addition loss of endogenous protein. Inhibitors of trypsin and chymotrypsin are abundant in seeds of most plants, but as for lectins the highest levels of trypsin inhibitors are found in the protein rich seeds of legumes {19), Two classes of trypsin inhibitors are found to dominate in legume seeds, the Kunitz soybean trypsin inhibitor (KSTI) and the Bowman-Birk inhibitor (BBI). These two inhibitors are present in high levels in soybean from where they were first isolated (Table I). Kunitz Soybean Inhibitor. KSTI is a 20.1 kD protein that binds very tightly to trypsin in a 1:1 molar ratio. This inhibitor consists of 180 amino acid residues, including 2 disulfide bonds, and is tightly folded due to a hydrophobic interior which makes the inhibitor very stable at pH values in the range of pH 1-12 (20). KSTI is only present in two species of the leguminosae family used for food and feed: soy bean and winged bean, but homologous proteins are present in many other plant seeds including other legumes that are not used for human consumption. Bowman-Birk Inhibitors. BBI inhibitors are small and very compact proteins with a molecular weight of approximately 8 kD containing 7 disulfide bonds per molecule which makes the inhibitor extremely heat and acid stable (27). This inhibitor usually exists in various isoforms in the plant seed (22,23). BBI contains a binding site for trypsin and a separate site for chymotrypsin, both proteases are secreted by the pancreas into the lumen of the upper small intestine. The B B I type inhibitors are present in the seeds of most legumes. European pea breeding programs have resulted in production of pea cultivars with a very low content of the BBI-type trypsin inhibitor (Table I). Trypsin inhibitors are generally rich in cysteine and, with sulfiir-containing amino acids being nutritionally limiting amino acids in legume proteins, cultivars with a low content of BBI-type inhibitors may have a lower nutritional quality than cultivars with a high inhibitor content if they can be utilized by humans and animals, e.g. after heat denaturation.



ANTINUTRIENTS AND P H Y T O C H E M I C A L S IN F O O D

B

100

80

O

60

a 00

40

Antinutritional and Allergenic Proteins - ACS Publications - American

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