The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively.

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. The first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N-terminal sequencing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence. In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12-kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASAII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.     

Developmental Regulation of Lectin and Alliinase Synthesis in Garlic Bulbs and Leaves

Using a combination of northern blot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a detailed study was made of the temporal and spatial regulation of garlic (Allium sativum L.) lectins and alliinase throughout the life cycle of the plant. The two bulb-specific lectins (ASAI and ASAII), which are the most predominant bulb proteins, accumulate exclusively in the developing garlic cloves and progressively disappear when the old clove is consumed by the plant. On the basis of these observations, ASAI and ASAII can be regarded as typical vegetative storage proteins. The leaf-specific lectin (ASAL), on the contrary, is specifically synthesized in young leaves and remains present until withering. Because ASAL is only a minor protein, it probably fulfills a specific function in the plant. Unlike the lectins, alliinase is present in large quantities in bulbs as well as in leaves. Moreover, intact alliinase mRNAs are present in both tissues as long as they contain living cells. The latter observation is in good agreement with the possible involvement of alliinase in the plant's defense against pathogens and/or predators.     

Ectopically expressed leaf and bulb lectins from garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) protect transgenic tobacco plants against cotton leafworm (<Emphasis Type="Italic">Spodoptera littoralis</Emphasis>)

The insecticidal activity of the leaf (ASAL) and bulb (ASAII) agglutinins from Allium sativum L. (garlic) against the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied using transgenic tobacco plants expressing the lectins under the control of the constitutive CaMV35S promoter. PCR analysis confirmed that the garlic lectin genes were integrated into the plant genome. Western blots and semi-quantitative agglutination assays revealed lectin expression at various levels in the transgenic lines. Biochemical analyses indicated that the recombinant ASAL and ASAII are indistinguishable from the native garlic lectins. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed ASAL and ASAII significantly (P < 0.05) reduced the weight gain of 4th instar larvae of S. littoralis. Further on, the lectins retarded the development of the larvae and their metamorphosis, and were detrimental to the pupal stage resulting in weight reduction and lethal abnormalities. Total mortality was scored with ASAL compared to 60% mortality with ASAII. These findings suggest that garlic lectins are suitable candidate insect resistance proteins for the control of S. littoralis through a transgenic approach.     

Effectiveness of garlic lectin on red spider mite of tea

Abstract

Red spider mite (Oligonychus coffeae) is one of the major pests of tea and damages 5–15% of the total crop every year. Mannose binding 25kDa lectins (ASAI, Allium sativum bulb agglutinin I and ASAII, A. sativum bulb agglutinin II), purified from bulbs of A. sativum (Garlic), was analyzed through SDS-PAGE and studied for its agglutination property using rabbit erythrocytes. Cross reactivity of the purified lectin was verified through western blot using anti-ASA antibody. ASAI was found to be a dimer built up of two heteromeric subunits whereas the ASAII is a homodimer. The insecticidal activity of the mixture of ASA lectins was tested against red spider mite in an artificial diet. The LC₅₀ values for red spider mite was determined to be 12.4±1.918µg/ml. This finding opens up a possibility of using the ASA genes against red spider mite through tea transgenic approach.     

Comparative study of the post-translational processing of the mannose-binding lectins in the bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.)

The biosynthesis and processing of the homodimeric and heterodimeric lectins from the bulbs of garlic (Allium sativum) and ramsons (wild garlic;Allium ursinum) were studied using pulse and pulse-chase labelling experiments on developing bulbs. By combining the results of thein vivo biosynthesis studies and the cDNA cloning of the respective lectins, the sequence of events leading from the primary translation products into the mature lectin polypeptides could be reconstructed. From this it is demonstrated that garlic and ramsons use different schemes of post-translational modifications in order to synthesize apparently similar lectins from totally different precursors. Both the homomeric garlic lectin (ASAII) and its homologue in ramsons (AUAII) are synthesized on the endoplasmic reticulum (ER) as nonglycosylated 13.5 kDa precursors, which, after their transport out of the ER are converted into the mature 12.0 kDa lectin polypeptides by the cleavage of a C-terminal peptide. The heterodimeric garlic lectin ASAI is synthesized on the ER as a single glycosylated precursor of 38 kDa, which after its transport out of the ER undergoes a complex processing which gives rise to two mature lectin subunits of 11.5 and 12.5 kDa. In contrast, both subunits of the heterodimeric ramsons lectin AUAI are synthesized separately on the ER as glycosylated precursors, which after their transport out of the ER are deglycosylated and further processed into the mature lectin polypeptides by the cleavage of a C-terminal peptide.     

Antimetabolic effect of phytohemagglutinin to the grain aphid Sitobion avenae fabricius

The insecticidal activity of plant lectins against a wide range of insect species have been intensively studied. Understanding the mechanism of the toxicity of lectins is one of the studied aspects. In the present research, the first step was determine the effect of phytohemagglutinin (PHA) on the development, fecundity and mortality of grain aphid. Next, the effect of PHA lectin on the activity of such enzymes as: α- and β-glucosidases, alkaline (AkP) and acid (AcP) phosphatases, aminopeptidase N and cathepsin L involved in the metabolism of sugar, phosphorus and proteins of an adult apterae aphids was investigated. The PHA lectin added into the liquid diet increased the pre-reproductive period, mortality of Sitobion avenae, the time of generation development and decreased its fecundity and the intrinsic rate of natural increase. In addition, activity of α-glucosidase, alkaline phosphatase and aminopeptidase N of adult apterae exposed to PHA were reduced. The results indicate that the insecticidal activity of PHA on S. avenae may involve changes in activity of the enzymes in the midgut and it may be part of its toxicity.     

Toxicity of lectins and processing of ingested proteins in the pea aphidAcyrthosiphon pisum

Acute toxicity of thirty lectins was tested against the pea aphidAcyrthosiphon pisum (Harris) (Homoptera, Aphididae: Macrosiphini). Activity was measured on artificial diets containing moderate concentrations of lectins (10–250 μg/ml) by scoring mortality and growth inhibition over the whole nymphal period (7 days at 20°C). Most of the proteins tested exhibited low toxicity, but some induced significant mortality; these included the lectins from jackbean (Concanavalin A), amaranth, lentil and snowdrop. There was no direct correlation between toxicity and sugar specificity of the lectin; however, many mannose-binding lectins were toxic towardsA. pisum. Concanavalin A was also tested on five other aphid species (Aphis gossypii, Aulacortum solani, Macrosiphum euphorbiae, Macrosiphum albifrons andMyzus persicae) at concentrations between 10–1500 μg/ml. Mortality was very variable from one species to another. Strong growth inhibition invariably occurred within this concentration range, although dose-response curves differed substantially between aphid species. The peptidase complement ofA. pisum’s digestive tract was also investigated, as well as the oral toxicity of some protease inhibitors (PIs) to this aphid. Most protein PIs were inactive, and no part of the digestive tract contained detectable amounts of endo-protease activity. This is in contrast to the strong amino-peptidase activity which was shown to occur predominantly in the midgut and crop portions of the digestive tract. The potential of lectins in transgenic crops to confer Host-Plant Resistance to aphids is discussed.     

Production and characterization of the B chains of mistletoe toxic lectins

Mistletoe toxic lectins consist of two polypeptide chains: an enzymatically active A chain, which is a toxic component, and a disulfide-bonded B chain, which confers the lectin properties on the total molecule. Mistletoe leaves contain three toxic lectins encoded by three genes. The B chains of these lectins were overproduced in Escherichia coli in a soluble form. The recombinant proteins bound with asialofetuin, but had substantially lower affinity for simple sugars D-galactose and N-acetyl-D-galactosamine as compared with the natural proteins. The functional properties of the B chains strongly depended on the storage conditions (salt concentration and the presence of galactose); the dependence was explained by structural instability of nonglycosylated recombinant proteins. The lectin activity of one of the recombinant B chains was close to that of the native protein, which was attributed to the lack of N-glycosylation sites in the latter.     

Identification of receptors responsible for binding of the mannose specific lectin to the gut epithelial membrane of the target insects

The sap-sucking homopteran insects, commonly known as aphids and leafhoppers are responsible for a huge amount of lost productivity of mustard, chickpea, cabbage, rice and many other important crops. Due to their unique feeding habits and ability to build up a huge population in a very short time, they are very difficult to control. The objective of the ongoing program is to develop insect-resistant crop species through genetic engineering techniques to combat the yield losses, which necessitates the identification of appropriate control elements. In this direction, mannose-binding 25 kDa lectins have been purified from leaves of garlic, Diffenbachia sequina and tubers of Colocasia esculanta. The purified lectins have been analyzed in SDS-PAGE. The effectiveness of these lectins against chickpea aphids, mustard aphids and green leaf hoppers of rice have been tested. The LC₅₀ value of each lectin against different insects had been monitored [1,2]. Through immunolocalization analysis, the binding of the lectin had been demonstrated at the epithelial membrane of the midgut of the lectin-treated insects [1]. Receptor proteins of brush border membrane vesicle (BBMV) of the target insects, responsible for binding of the lectin to the midgut of the epithelial layer have been purified and analyzed through ligand assay. Biochemical studies have been undertaken to investigate the lectin-receptor interaction at molecular level. Published in 2004..     

Identification of receptors responsible for binding of the mannose specific lectin to the gut epithelial membrane of the target insects

The sap-sucking homopteran insects, commonly known as aphids and leafhoppers are responsible for a huge amount of lost productivity of mustard, chickpea, cabbage, rice and many other important crops. Due to their unique feeding habits and ability to build up a huge population in a very short time, they are very difficult to control. The objective of the ongoing program is to develop insect-resistant crop species through genetic engineering techniques to combat the yield losses, which necessitates the identification of appropriate control elements. In this direction, mannose-binding 25 kDa lectins have been purified from leaves of garlic, Diffenbachia sequina and tubers of Colocasia esculanta. The purified lectins have been analyzed in SDS-PAGE. The effectiveness of these lectins against chickpea aphids, mustard aphids and green leaf hoppers of rice have been tested. The LC(50) value of each lectin against different insects had been monitored [1,2]. Through immunolocalization analysis, the binding of the lectin had been demonstrated at the epithelial membrane of the midgut of the lectin-treated insects [1]. Receptor proteins of brush border membrane vesicle (BBMV) of the target insects, responsible for binding of the lectin to the midgut of the epithelial layer have been purified and analyzed through ligand assay. Biochemical studies have been undertaken to investigate the lectin-receptor interaction at molecular level.     

Snowdrop and wheatgerm lectins and avidin as antimetabolites for the control of sugarcane whitegrubs

Snowdrop and wheatgerm lectins were found to be insecticidal and growth inhibiting dietary proteins for larvae of the sugarcane whitegrub Antitrogus parvulus. At concentrations as low as 0.5 mg of snowdrop lectin per gram of semi-artificial diet, growth was inhibited by 21 days of feeding and significant mortality was apparent by 28 days. Wheatgerm lectin was active at similar concentrations, although expression of the effects was slower. Avidin was found to be a growth inhibiting dietary protein for larvae of Antitrogus consanguineus. At levels as low as 0.01 mg g^-1 of diet, growth was inhibited by 28 days of feeding. Avidin caused no significant mortality after 35 days of feeding. Snowdrop and wheatgerm lectins and avidin are insect growth-inhibiting proteins whose genes potentially could be manipulated into sugarcane and improve host-plant resistance to whitegrubs.     

Bioengineering of symbiotic systems: creation of new associative symbiosis with the use of lectins on the example of tobacco and colza

"Barbate roots" in tobacco and colza transgenic on lectin gene were obtained with the use of a wild strain of Agrobacterium rhizogenes 15834 transformed with pCAMBIA1305.1 plasmid containing the full-size lectin gene (psl) from the Pisum sativum. Influence of expression oflectin gene on colonization oftransgenic roots with symbiont of pea (Rhizobium leguminosarum) was investigated. The number of adhered bacteria onto the roots transformed with lectin gene was 14-fold and 37-fold higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of tobacco and colza. The developed experimental approach, based on the simulation of recognition processes and early symbiotic interactions with lectins of pea plants, may, in perspective, be used for obtaining stable associations of economically valuable, nonsymbiotrophic plant species with rhizobia.     

Expression of isolated C-type carbohydrate recognition domains

A galactose-binding lectin from the venom of the snake Trimeresurus stejnegeri consists of isolated carbohydrate recognition domains, belonging to group VII of the C-type animal lectins. As a first step toward determining the tertiary structure of the galactose-specific lectin, we produced the lectin in Escherichia coli. By in vitro refolding and affinity chromatography, modest amounts (8 mg/liter) of active recombinant proteins were obtained. The recombinant protein was homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. Its amino acid sequence without the initiated methionine at the N-terminus was also characterized by mass spectrometry. The data of hemagglutination and enzyme-linked lectin binding assays demonstrated that the recombinant lectin showed similar sugar-binding activity as the native protein. In addition, fluorescence spectroscopy and circular dichroism also showed obviously their structural similarity.     

Effect of a fungal lectin from Xerocomus chrysenteron (XCL) on the biological parameters of aphids

Aphids are important pests of crop plants in Europe. Increasing resistance of aphids to insecticides and their side effects on the environment and non target organism's including human's stimulated research on alterative methods of aphid control, including the use of entomotoxic proteins. Lectins are carbohydrate binding proteins that are widely distributed in nature; they have been isolated from microorganisms, fungi, plants and animals. Several of these proteins were tested for their potential biocide effect on plenty of pests. A fungal lectin, namely Xerocomus Chrysenteron lectin (XCL) was previously purified and was shown to be toxic for several pests including aphids. XCL was clearly the most toxic lectin against M. persicae. In this work, bioassays using artificial diets incorporating a broad range of XCL concentrations (from 10 microg x ml(-1) to 5000 microg x ml(-1)) were developed to assess the negative effects of XCL on the biological parameters (development duration, weight and fecundity) of M. persicae a polyphagous aphid found on more than 400 host plant species and transmitting more than 100 viral diseases. A significant mortality of aphids was observed, corresponding to the LC50 and LC90 of 0, 46 and 6, 02 mg/ml respectively after 24hrs. Significant differences of M. persicae weight, development duration and fecundity (P < 0.05) was observed between the tested XCL concentrations. Conavalia ensifomris lectin (ConA) was included as lectin reference on the bioassay experiments and was shown to be less toxic and induced lower negative changes in M. persicae biological parameters when compared with XCL.     

Lectins and the problem of phitopatogene recognition by a host plant

Under consideration are some questions concerning participation of lectins in the plant pathogenesis, including their role in the recognition of microbes and elicitors, and as a protective agent limiting pathogenic growth and displacements. "Classical" lectins also probably play an important role in these processes along with lectin-like receptor kinases. The principal features of those "classical" lectins are their relativly high concentration in the plant tissues, monosaccharide specificity, and limited number of the isolecin forms. Therefore, in supposing their participation in the biological recognition, it is needed to clarify how does a limited number of lectins with a limited number of carbohydrate groups can provide recognition of a potentially huge number of pathogens. This task can be fulfilled by recognition of carbohydrate residues peculiar to a particular microbe group by the "classical" lectins. These recognition processes are similar to acivity of the animal inherited immune system responsible for a rapid primary protection even in animals with well developed antibody system. A mechanism widening the carbohydrate specificity of the carbohydrate-binding center includes interaction with hydrophobic substituents in a carbohydrate residue, as well as lectin modular organization allowing for regulation of lectin binding with oligo- and polysaccharides. The free lectins effect on the microbe growth in both plants and animals. Such an action may be inhibiting in pathogenesis, while in the case of symbiotic relations, the lectin can bear signal that readdresses metabolism of a future symbiont. So, lectins seem to serve as natural deciphering device for information contained in the carbohydrate polymers, and reading of this information is the main lectin function in the cell.     

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.     

A novel method for the purification of recombinant subunit I of theDolichos biflorus seed lectin

Lectins are carbohydrate-binding proteins that are ubiquitous in nature. Their ability to specifically bind carbohydrates has been used as a means of purification mainly through affinity chromatography techniques. Plant lectins are one of the most thoroughly studied class of lectins, however, details of theirin situ function remains elusive. Recent advances in recombinant DNA techniques have been used in several laboratories to study the function of these lectins by heterologous over-expression. The larger subunit of theDolichos biflorus seed lectin was described by Chao et al. in 1994 and purification through affinity chromatography techniques was described. Here we report on a new method for the purification of this recombinant protein with techniques that are not dependent on the ability of the lectin to bind sugars. This method may have uses in the purification of mutant proteins that may not bind carbohydrates. Characterization of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy shows that the lectin is over 99% pure with a molecular weight of 27,090±16.17 Da, and hemagglutination assays confirm that the lectin retains its biological activity.     

A simple strategy for the creation of a recombinant lectin microarray

Glycomics, i.e. the high-throughput analysis of carbohydrates, has yet to reach the level of ease and import of its counterparts, genomics and proteomics, due to the difficulties inherent in carbohydrate analysis. The advent of lectin microarray technology addresses many of these problems, providing a straightforward approach for glycomic analysis. However, current microarrays are limited to the available lectin set, which consists mainly of plant lectins isolated from natural sources. These lectins have inherent problems including inconsistent activity and availability. Also, many plant lectins are glycosylated, complicating glycomic evaluation of complex samples, which may contain carbohydrate-binding proteins. The creation of a recombinant, well-defined lectin set would resolve many of these issues. Herein, we describe an efficient strategy for the systematic creation of recombinant lectins for use in microarray technology. We present a small panel of simple-to-purify bacterially-derived lectins that show reliable activity and define their binding specificities by both carbohydrate microarray and ELISA. We utilize this panel to create a recombinant lectin microarray that is able to distinguish glycopatterns for both proteins and cell samples. This work opens the door to the establishment of a vast set of defined lectins via high-throughout approaches, advancing lectin microarray technology for glycomic analysis.     

Recombinant lectins: an array of tailor-made glycan-interaction biosynthetic tools

Lectins are a heterogeneous group of proteins found in plants, animals and microorganisms, which possess at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharides. The range of lectins and respective biological activities is unsurprising given the immense diversity and complexity of glycan structures and the multiple modes of interaction with proteins. Recombinant DNA technology has been traditionally used for cloning and characterizing newly discovered lectins. It has also been employed as a means of producing pure and sequence-defined lectins for different biotechnological applications. This review focuses on the production of recombinant lectins in heterologous organisms, and highlighting the Escherichia coli and Pichia pastoris expression systems, which are the most employed. The choice of expression host depends on the lectin. Non-glycosylated recombinant lectins are produced in E. coli and post-translational modified recombinant lectins are produced in eukaryotic organisms, namely P. pastoris and non-microbial hosts such as mammalian cells. Emphasis is given to the applications of the recombinant lectins especially (a) in cancer diagnosis and/or therapeutics, (b) as anti-microbial, anti-viral, and anti-insect molecules or (c) in microarrays for glycome profiling. Most reported applications are from recombinant plant lectins. These applications benefit from the tailor-made design associated with recombinant production and will aid in unraveling the complex biological mechanisms of glycan-interactions, bringing recombinant lectins to the forefront of glycobiology. In conclusion, recombinant lectins are developing into valuable biosynthetic tools for biomedical research.     

Detection of a high affinity binding site in recombinant <Emphasis Type="Italic">Aleuria aurantia</Emphasis> lectin

Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K (d) values in the micromolar range. This would make mushroom lectins ideal candidates to study protein-carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K (d) values in the nanomolar range. This site could bind to oligosaccharides with fucose linked alpha1-2, alpha1-3 or alpha1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with alpha1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.