E. J. M. Van Damme

Lectins as plant defense proteins.

Many plant species contain carbohydrate-binding proteins, which are commonly referred to as either lectins or agglutinins. Generally speaking, lectins are proteins that bind reversibly to specific monoor oligosaccharides. Since the initial discovery of a hemagglutinating factor in castor bean extracts by Stillmark in 1888, several hundred of these proteins have been isolated and characterized in some detail with respect to their carbohydrate-binding specificity, molecular structure, and biochemical properties. Lectins from different plant species often differ with respect to their molecular structure and specificity. It is important, therefore, to realize that all plant lectins are artificially classified together solely on the basis of their ability to recognize and bind carbohydrates. Moreover, the question arises whether proteins with a completely different structure and sugar-binding specificity fulfill the same physiological role. No conclusive answer can be given to this question as yet, for the simple reason that the role of most plant lectins is not known with certainty. There is, however, growing evidence that most lectins play a role in the plants defense against different kinds of plant-eating organisms. The idea that lectins may be involved in plant defense is not new. In an earlier review, Chrispeels and Raikhel (1991) critically assessed the defensive role of the phytohemagglutinin family and a number of chitin-binding proteins. During the last few years important progress has been made in the study of plant lectins in general and in the understanding of their effects on other organisms in particular. In this Update we summarize the recent developments that support the defensive role of plant lectins and, in addition, discuss earlier work in this field against the background of our present knowledge of this group of plant proteins.

Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.

Alpha-(1-3)- and alpha-(1-6)-D-mannose-specific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro.

The alpha-(1-3)-D-mannose- and alpha-(1-6)-D-mannose-specific agglutinins (lectins) from Galanthus nivalis, Hippeastrum hybrid, Narcissus pseudonarcissus, and Listera ovata inhibited infection of MT-4 cells by human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus at concentrations comparable to the concentrations at which dextran sulfate (molecular weight, 5,000 [DS-5000]) inhibits these viruses (50% effective concentration, 0.2 to 0.6 microgram/ml). Unlike DS-5000, however, the plant lectins did not inhibit the replication of other enveloped viruses, except for human cytomegalovirus (50% effective concentration, 0.9 to 1.6 microgram/ml). The plant lectins suppressed syncytium formation between persistently HIV-1- or HIV-2-infected HUT-78 cells and uninfected MOLT-4 (clone 8) cells at concentrations that were 5- to 10-fold lower than that required for DS-5000. Unlike DS-5000, however, the plant lectins did not inhibit HIV-1 binding to CD4+ cells. Combination of the plant lectins with DS-5000 led to a potent synergistic inhibition of HIV-1-induced cytopathogenicity in MT-4 cells and syncytium formation between HIV-infected HUT-78 cells and MOLT-4 cells. Our data suggest that alpha-(1-3)-D- and alpha-(1-6)-D-mannose-specific plant lectins interfere with an event in the HIV replicative cycle that is subsequent to the attachment of the virions to the cells (i.e., the fusion process).

Relationship between survival and binding of plant lectins during small intestinal passage and their effectiveness as growth factors

The effects on the small intestine and the growth of rats of six pure plant lectins: PHA (Phaseolus vulgaris); SBL (Glycine maxima); SNA-I and SNA-II (Sambucus nigra); GNA (Galanthus nivalis) and VFL (Vicia faba), covering most sugar specificities found in nature, were studied in vivo. Variable amounts, 25% (VFL) to 100% (PHA, GNA) of the lectins administered intragastrically, remained in immunochemically intact form in the small intestine after 1 h. All lectins, except GNA, showed binding to the brush border on first exposure, although this was slight with VFL. Thus, binding to the gut wall was not obligatory for resistance to proteolysis. Exposure of rats to lectins, except VFL, for 10 days, retarded their growth but induced hyperplastic growth of their small intestine. The two activities were directly related. PHA and SNA-II, whose intestinal binding and endocytosis was appreciable after 10 days of feeding the rats with diets containing these lectins and similar to that found on acute (1 h) exposure, were powerful growth factors for the small intestine. GNA, which did not bind at the start but was reactive after 10 days, and SNA-I, which behaved in the opposite way, induced changes in receptor expression in the gut. As they were bound to the brush border transiently, they were less effective growth factors. VFL was not bound or endocytosed, was non-toxic and did not promote gut growth.

Antinutritive effects of wheat-germ agglutinin and other N-acetylglucosamine-specific lectins

Incorporation of N-acetylglucosamine-specific agglutinins from wheat germ (Triticum aestivum; WGA), thorn apple (Datura stramonium) or nettle (Urtica dioica) rhizomes in the diet at the level of 7 g/kg reduced the apparent digestibility and utilization of dietary proteins and the growth of rats, with WGA being the most damaging. As a result of their binding and endocytosis by the epithelial cells of the small intestine, all three lectins were growth factors for the gut and interfered with its metabolism and function to varying degrees. WGA was particularly effective; it induced extensive polyamine-dependent hyperplastic and hypertrophic growth of the small bowel by increasing its content of proteins, RNA and DNA. Furthermore, an appreciable portion of the endocytosed WGA was transported across the gut wall into the systemic circulation, where it was deposited in the walls of the blood and lymphatic vessels. WGA also induced the hypertrophic growth of the pancreas and caused thymus atrophy. Although the transfer of the gene of WGA into crop plants has been advocated to increase their insect resistance, as the presence of this lectin in the diet may harm higher animals at the concentrations required to be effective against most pests, its use in plants as natural insecticide is not without health risks for man.

Structure-function relationship of monocot mannose-binding lectins.

The monocot mannose-binding lectins are an extended superfamily of structurally and evolutionarily related proteins, which until now have been isolated from species of the Amaryllidaceae, Alliaceae, Araceae, Orchidaceae, and Liliaceae. To explain the obvious differences in biological activities, the structure-function relationships of the monocot mannose-binding lectins were studied by a combination of glycan-binding studies and molecular modeling using the deduced amino acid sequences of the currently known lectins. Molecular modeling indicated that the number of active mannose-binding sites per monomer varies between three and zero. Since the number of binding sites is fairly well correlated with the binding activity measured by surface plasmon resonance, and is also in good agreement with the results of previous studies of the biological activities of the mannose-binding lectins, molecular modeling is of great value for predicting which lectins are best suited for a particular application.

The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from and mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.

The bark of Robinia pseudoacacia contains a complex mixture of lectins : characterization of the proteins and the cDNA clones

Two lectins were isolated from the inner bark of Robinia pseudoacacia (black locust). The first (and major) lectin (called RPbAI) is composed of five isolectins that originate from the association of 31.5- and 29-kD polypeptides into tetramers. In contrast, the second (minor) lectin (called RPbAII) is a hometetramer composed of 26-kD subunits. The cDNA clones encoding the polypeptides of RPbAI and RPbAII were isolated and their sequences determined. Apparently all three polypeptides are translated from mRNAs of approximately 1.2 kb. Alignment of the deduced amino acid sequences of the different clones indicates that the 31.5- and 29-kD RPbAI polypeptides show approximately 80% sequence identity and are homologous to the previously reported legume seed lectins, whereas the 26-kD RPbAII polypeptide shows only 33% sequence identity to the previously described legume lectins. Modeling the 31.5-kD subunit of RPbAI predicts that its three-dimensional structure is strongly related to the three-dimensional models that have been determined thus far for a few legume lectins. Southern blot analysis of genomic DNA isolated from Robinia has revealed that the Robinia bark lectins are the result of the expression of a small family of lectin genes.

Entomotoxic effects of fungal lectin from Rhizoctonia solani towards Spodoptera littoralis

The effects of the Rhizoctonia solani lectin (RSA) on the growth, development and survival of an economically important caterpillar in agriculture and horticulture, the cotton leafworm, Spodoptera littoralis were studied. The high lectin concentration present in the sclerotes of the soil pathogen R. solani allowed the purification of large amounts of the pure lectin for feeding experiments with cotton leafworm. Rearing of insects on a diet containing different concentrations of RSA exerted a strong effect on the larval weight gain. This effect was visible at the lowest concentration of 0.1 % RSA at day 8 and day 11. Interestingly with 1 % RSA, there was a dramatic reduction in larval weight of 89 % at the end of L6 which was followed by a high mortality rate of 82 % in the treated larvae. Furthermore, the other developmental stages of pupation and adult formation were also affected. In addition, the data demonstrated that the combination of RSA with Bt toxin yielded synergistic effects. For instance, 0.03 % RSA+0.005 % Bt toxin caused reduced growth rate and higher mortalities. These findings suggest that RSA is an interesting tool that can be used for bioengineering insect resistance in important agronomical crops.

Isolation, characterization, molecular cloning and molecular modelling of two lectins of different specificities from bluebell (Scilla campanulata) bulbs.

Two lectins have been isolated from bluebell (Scilla campanulata) bulbs. From their isolation by affinity chromatography, they are characterized as a mannose-binding lectin (SCAman) and a fetuin-binding lectin (SCAfet). SCAman preferentially binds oligosaccharides with alpha(1,3)- and alpha(1,6)-linked mannopyranosides. It is a tetramer of four identical protomers of approx. 13 kDa containing 119 amino acid residues; it is not glycosylated. The fetuin-binding lectin (SCAfet), which is not inhibited by any simple sugars, is also unglycosylated. It is a tetramer of four identical subunits of approx. 28 kDa containing 244 residues. Each 28 kDa subunit is composed of two 14 kDa domains. Both lectins have been cloned from a cDNA library and sequenced. X-ray crystallographic analysis and molecular modelling studies have demonstrated close relationships in sequence and structure between these lectins and other monocot mannose-binding lectins. A refined model of the molecular evolution of the monocot mannose-binding lectins is proposed.