... snowdrop lectin genes in transgenic tobacco plants Y. Shi1, MB Wang, KS Powell, E. Van Damme2... more ... snowdrop lectin genes in transgenic tobacco plants Y. Shi1, MB Wang, KS Powell, E. Van Damme2, VA Hilder, AMR Gatehouse, D. Boulter and JA Gatehouse3 ... EMBO Journal 10, 2635^*4. Hllder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D. 1987. ...
Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpu... more Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpus integrifolia), is not directed exclusively against the T-antigen disaccharide Galβ1,3GalNAc, lactose and galactose, but also against mannose and oligomannosides. Biochemical analyses based on surface-plasmon-resonance measurements, combined with the X-ray-crystallographic determination of the structure of a jacalin—α-methyl-mannose complex at 2Å resolution, demonstrated clearly that jacalin is fully capable of binding mannose. Besides mannose, jacalin also interacts readily with glucose, N-acetylneuraminic acid and N-acetylmuramic acid. Structural analyses demonstrated that the relatively large size of the carbohydrate-binding site enables jacalin to accommodate monosaccharides with different hydroxyl conformations and provided unambiguous evidence that the β-prism structure of jacalin is a sufficiently flexible structural scaffold to confer different carbohydrate-binding specificities...
The immune system consists of a complex network of cells and molecules that interact with each ot... more The immune system consists of a complex network of cells and molecules that interact with each other to initiate the host defense system. Many of these interactions involve specific carbohydrate structures and proteins that specifically recognize and bind them, in particular lectins. It is well established that lectin-carbohydrate interactions play a major role in the immune system, in that they mediate and regulate several interactions that are part of the immune response. Despite obvious differences between the immune system in animals and plants, there are also striking similarities. In both cases, lectins can play a role as pattern recognition receptors, recognizing the pathogens and initiating the stress response. Although plants do not possess an adaptive immune system, they are able to imprint a stress memory, a mechanism in which lectins can be involved. This review will focus on the role of lectins in the immune system of animals and plants.
Mededelingen (Rijksuniversiteit te Gent. Fakulteit van de Landbouwkundige en Toegepaste Biologische Wetenschappen), 2002
Ee-CBP, a hevein-type antimicrobial peptide was isolated from the bark of the spindle tree (Euony... more Ee-CBP, a hevein-type antimicrobial peptide was isolated from the bark of the spindle tree (Euonymus europaeus L.). This 4992.5 Da protein exhibited a very strong antifungal activity against five different fytopathogenic fungi that were tested. Concentrations required to inhibit the growth of Botrytis cinerea in agar diffusion assays and microtiterplate assays were 5 micrograms/ml and 1 microgram/ml, respectively. Comparative tests further indicated that Ee-CBP is a more potent antifungal protein than Ac-AMP2, an antimicrobial peptide from seeds of Amaranthus caudatus L. when tested with the same fungus.
The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Char... more The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Characterization of the protein and molecular cloning of the corresponding gene from banana demonstrated that the native protein consists of a single polypeptide chain of 200 amino acid residues. Molecular modelling further revealed that the banana thaumatin-like protein (Ban-TLP) adopts an overall fold similar to that of thaumatin and thaumatin-like PR-5 proteins. Although the banana protein exhibits an electrostatically polarized surface, which is believed to be essential for the antifungal properties of TLPs, it is apparently devoid of antifungal activity towards pathogenic fungi. It exhibits a low but detectable in vitro endo-beta-1,3-glucanase (EC 3.2.1.x) activity. As well as being present in fruits, Ban-TLP also occurs in root tips where its accumulation is enhanced by methyl jasmonate treatment of plants. Pulp of plantains (Musa acuminata) also contains a very similar TLP, which is even more abundant than its banana homologue. Our results demonstrate for the first time that fruit-specific (abundant) TLPs are not confined to dicots but occur also in fruits of monocot species. The possible role of the apparent widespread accumulation of fruit-specific TLPs is discussed.
The motility and the chemotactic response towards plant roots of Radopholus similis, after treatm... more The motility and the chemotactic response towards plant roots of Radopholus similis, after treatment with novel types of lectins, were examined in vitro by analysing movement tracks on agar plates. Six plant lectins belonging to five different lectin families and a banana thaumatin-like protein (BanTLP) were included in the experiment. A 1% concentration of Phaseolus vulgaris agglutinin (PHA) had an adverse effect on the motility of R. similis females: 63% showed no or very little movement on agar plates compared to an average of 33% for other lectins and 3% for the control treatment. A 0.05% concentration of PHA still reduced the motility of R. similis females by 75%. Concanavalin A and wheat germ agglutinin did not alter the chemotactic response towards plant roots, despite binding of both lectins to R. similis. In contrast, Galanthus nivalis agglutinin (GNA) reduced orientated movement of R. similis towards plant roots. Subsequently, secretions of R. similis were stained with Coo...
Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cot... more Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leaf-worm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good control agent for different pest insects. RSA at 10 mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8 days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3 days with 9 μM of RSA in the liquid diet. Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a primary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there was high fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells of S. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in the midgut cells. In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of 0.3 μM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-binding dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms of apoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of caspase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cells followed by LC-MS/MS allowed the identification of 5747 unique peptides, among which four putatively glycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated factor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data are discussed in relation to the physiological effects of RSA.
ABSTRACT In the last two decades plants have emerged as valuable alternatives to mammalian cells ... more ABSTRACT In the last two decades plants have emerged as valuable alternatives to mammalian cells for the production of pharmaceuticals and their potential as expression systems was shown by the commercial availability and acceptance of several plant made therapeuticals in clinical trials. Plants have many advantages over yeast, insect and bacterial expression systems such as the potential to properly fold the expressed proteins and the synthesis of more human-like N-glycans on the proteins. However, several constraints, such as expression yields, downstream processing and structural authenticity, currently limit the widespread use of plant expression systems. In this review, the focus is on the current limitations of plant systems for the production of pharmaceuticals and the possibilities to overcome these obstacles. A comparison is made with insect cell and yeast expression systems. Furthermore, the importance of glycosylation, in particular N-glycosylation for the biological function(s) of therapeutics in the human body will be discussed in detail and an overview of the state of art in the humanization of the N-glycosylation pathway in plants is provided.
... snowdrop lectin genes in transgenic tobacco plants Y. Shi1, MB Wang, KS Powell, E. Van Damme2... more ... snowdrop lectin genes in transgenic tobacco plants Y. Shi1, MB Wang, KS Powell, E. Van Damme2, VA Hilder, AMR Gatehouse, D. Boulter and JA Gatehouse3 ... EMBO Journal 10, 2635^*4. Hllder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D. 1987. ...
Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpu... more Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpus integrifolia), is not directed exclusively against the T-antigen disaccharide Galβ1,3GalNAc, lactose and galactose, but also against mannose and oligomannosides. Biochemical analyses based on surface-plasmon-resonance measurements, combined with the X-ray-crystallographic determination of the structure of a jacalin—α-methyl-mannose complex at 2Å resolution, demonstrated clearly that jacalin is fully capable of binding mannose. Besides mannose, jacalin also interacts readily with glucose, N-acetylneuraminic acid and N-acetylmuramic acid. Structural analyses demonstrated that the relatively large size of the carbohydrate-binding site enables jacalin to accommodate monosaccharides with different hydroxyl conformations and provided unambiguous evidence that the β-prism structure of jacalin is a sufficiently flexible structural scaffold to confer different carbohydrate-binding specificities...
The immune system consists of a complex network of cells and molecules that interact with each ot... more The immune system consists of a complex network of cells and molecules that interact with each other to initiate the host defense system. Many of these interactions involve specific carbohydrate structures and proteins that specifically recognize and bind them, in particular lectins. It is well established that lectin-carbohydrate interactions play a major role in the immune system, in that they mediate and regulate several interactions that are part of the immune response. Despite obvious differences between the immune system in animals and plants, there are also striking similarities. In both cases, lectins can play a role as pattern recognition receptors, recognizing the pathogens and initiating the stress response. Although plants do not possess an adaptive immune system, they are able to imprint a stress memory, a mechanism in which lectins can be involved. This review will focus on the role of lectins in the immune system of animals and plants.
Mededelingen (Rijksuniversiteit te Gent. Fakulteit van de Landbouwkundige en Toegepaste Biologische Wetenschappen), 2002
Ee-CBP, a hevein-type antimicrobial peptide was isolated from the bark of the spindle tree (Euony... more Ee-CBP, a hevein-type antimicrobial peptide was isolated from the bark of the spindle tree (Euonymus europaeus L.). This 4992.5 Da protein exhibited a very strong antifungal activity against five different fytopathogenic fungi that were tested. Concentrations required to inhibit the growth of Botrytis cinerea in agar diffusion assays and microtiterplate assays were 5 micrograms/ml and 1 microgram/ml, respectively. Comparative tests further indicated that Ee-CBP is a more potent antifungal protein than Ac-AMP2, an antimicrobial peptide from seeds of Amaranthus caudatus L. when tested with the same fungus.
The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Char... more The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Characterization of the protein and molecular cloning of the corresponding gene from banana demonstrated that the native protein consists of a single polypeptide chain of 200 amino acid residues. Molecular modelling further revealed that the banana thaumatin-like protein (Ban-TLP) adopts an overall fold similar to that of thaumatin and thaumatin-like PR-5 proteins. Although the banana protein exhibits an electrostatically polarized surface, which is believed to be essential for the antifungal properties of TLPs, it is apparently devoid of antifungal activity towards pathogenic fungi. It exhibits a low but detectable in vitro endo-beta-1,3-glucanase (EC 3.2.1.x) activity. As well as being present in fruits, Ban-TLP also occurs in root tips where its accumulation is enhanced by methyl jasmonate treatment of plants. Pulp of plantains (Musa acuminata) also contains a very similar TLP, which is even more abundant than its banana homologue. Our results demonstrate for the first time that fruit-specific (abundant) TLPs are not confined to dicots but occur also in fruits of monocot species. The possible role of the apparent widespread accumulation of fruit-specific TLPs is discussed.
The motility and the chemotactic response towards plant roots of Radopholus similis, after treatm... more The motility and the chemotactic response towards plant roots of Radopholus similis, after treatment with novel types of lectins, were examined in vitro by analysing movement tracks on agar plates. Six plant lectins belonging to five different lectin families and a banana thaumatin-like protein (BanTLP) were included in the experiment. A 1% concentration of Phaseolus vulgaris agglutinin (PHA) had an adverse effect on the motility of R. similis females: 63% showed no or very little movement on agar plates compared to an average of 33% for other lectins and 3% for the control treatment. A 0.05% concentration of PHA still reduced the motility of R. similis females by 75%. Concanavalin A and wheat germ agglutinin did not alter the chemotactic response towards plant roots, despite binding of both lectins to R. similis. In contrast, Galanthus nivalis agglutinin (GNA) reduced orientated movement of R. similis towards plant roots. Subsequently, secretions of R. similis were stained with Coo...
Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cot... more Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leaf-worm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good control agent for different pest insects. RSA at 10 mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8 days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3 days with 9 μM of RSA in the liquid diet. Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a primary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there was high fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells of S. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in the midgut cells. In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of 0.3 μM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-binding dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms of apoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of caspase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cells followed by LC-MS/MS allowed the identification of 5747 unique peptides, among which four putatively glycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated factor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data are discussed in relation to the physiological effects of RSA.
ABSTRACT In the last two decades plants have emerged as valuable alternatives to mammalian cells ... more ABSTRACT In the last two decades plants have emerged as valuable alternatives to mammalian cells for the production of pharmaceuticals and their potential as expression systems was shown by the commercial availability and acceptance of several plant made therapeuticals in clinical trials. Plants have many advantages over yeast, insect and bacterial expression systems such as the potential to properly fold the expressed proteins and the synthesis of more human-like N-glycans on the proteins. However, several constraints, such as expression yields, downstream processing and structural authenticity, currently limit the widespread use of plant expression systems. In this review, the focus is on the current limitations of plant systems for the production of pharmaceuticals and the possibilities to overcome these obstacles. A comparison is made with insect cell and yeast expression systems. Furthermore, the importance of glycosylation, in particular N-glycosylation for the biological function(s) of therapeutics in the human body will be discussed in detail and an overview of the state of art in the humanization of the N-glycosylation pathway in plants is provided.
Uploads