Enzyme-linked Lectinosorbent Assays Research Articles

PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF A T/TN SPECIFIC LECTIN FROM LEPECHINIA BULLATA SEEDS (LAMIACEAE)

Objective: This study focused on purifying and characterizing a lectin from Lepechinia bullata (L. bullata) seeds, and determining its specificity towards tumour-associated carbohydrate-antigens.Methods: Pigments were removed by washing the seeds with NH4OH 0.1 M pH 9.4 and treating the crude extracts with Pectinex®. The purification procedure consisted of anion exchange chromatography on diethylaminoethyl (DEAE)-Sephadex followed by affinity chromatography. For the characterization, the phase was used polyacrylamide gel electrophoresis-sodium dodecyl sulphate (SDS-PAGE), isoelectric focusing, hemagglutination assays, enzyme-linked lectinosorbent assay (ELLA) and thermal shift assay (TSA).Results: 6.2 mg of lectin were obtained from 100 g of seeds. It was able to agglutinate enzymatically treated erythrocytes with a minimal required lectin concentration of 7 μg. ml-1. Strong binding to asialo bovine submaxillary mucine (aBSM) was determined, corroborating Tn recognition.The isoelectric focusing showed a unique band at pH 8.5. Lectin pure shown bands at 28, 48 and 93 kDa by SDS-PAGE, with an incomplete dissociation of the last species despite trying several reduction conditions. By preparative electrophoresis under different conditions, three species were observed too, in all fractions one band at 28 kDa on Tricine-PAGE in reducing and no reducing conditions were found.Amino acid composition, carbohydrate content, thermal stability and Ca2+and Mn2+requirements were determined. N-acetylgalactosamine (GalNAc) and desialylated mucins inhibited the agglutinant activity on human cells. Fetuin inhibited hemagglutination of rabbit erythrocytes.Conclusion: A new lectin was isolated and characterized from L. bullata seeds, it recognizes T/Tn antigen and shows some similarities with other Lamiaceae lectins.

Enzyme-linked lectinosorbent assay of Escherichia coli and Staphylococcus aureus

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 106 and 5 × 105 cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.

Aeromonas punctata derived depolymerase improves susceptibility of Klebsiella pneumoniae biofilm to gentamicin.

BackgroundTo overcome antibiotic resistance in biofilms, enzymes aimed at biofilm dispersal are under investigation. In the present study, applicability of an Aeromonas punctata derived depolymerase capable of degrading the capsular polysaccharide (CPS) of Klebsiella pneumoniae, in disrupting its biofilm and increasing gentamicin efficacy against biofilm was investigated.ResultsIntact biofilm of K. pneumoniae was recalcitrant to gentamicin due to lack of antibiotic penetration. On the other hand, gentamicin could not act on disrupted biofilm cells due to their presence in clusters. However, when depolymerase (20 units/ml) was used in combination with gentamicin (10 μg/ml), dispersal of CPS matrix by enzyme facilitated gentamicin penetration across biofilm. This resulted in significant reduction (p < 0.05) in bacterial count in intact and disrupted biofilms. Reduction in CPS after treatment with depolymerase was confirmed by confocal microscopy and enzyme linked lectinosorbent assay. Furthermore, to substantiate our study, the efficacy of bacterial depolymerase was compared with a phage borne depolymerase possessing similar application against K. pneumoniae. Although both were used at same concentration i.e. 20 units/ml, but a higher efficacy of bacterial depolymerase particularly against older biofilms was visibly clear over its phage counterpart. This could be explained due to high substrate affinity (indicated by Km value) and high turnover number (indicated by Kcat value) of the bacterial depolymerase (Km = 89.88 μM, Kcat = 285 s−1) over the phage derived one (Km = 150 μM, Kcat = 107 s−1).ConclusionOverall the study indicated that, the A. punctata derived depolymerase possesses antibiofilm potential and improves gentamicin efficacy against K. pneumoniae. Moreover, it can serve as a potential substitute to phage borne depolymerases for treating biofilms formed by K. pneumoniae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0455-z) contains supplementary material, which is available to authorized users.

α1,3-Galactosyltransferase Deficiency in Germ-Free Miniature Pigs IncreasesN-Glycolylneuraminic Acids As the Xenoantigenic Determinant in Pig–Human Xenotransplantation

In this study, we examined whether Hanganutziu-Deicher (H-D) antigens are important as an immunogenic non-α1,3-galactose (Gal) epitope in pigs with a disrupted α1,3-galactosyltransferase gene. The targeting efficiency of the AO blood genotype was achieved (2.2%) in pig fibroblast cells. A total of 1800 somatic cell nuclear transfer (SCNT) embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. The α1,3-galactosyltransferase activity in lung, liver, spleen, and testis of heterozygote α1,3-galactosyltransferase gene knockout (GalT-KO) pigs was significantly decreased, whereas brain and heart showed very low decreasing levels of α1,3-galactosyltransferase activity when compared to those of control. Enzyme-linked lectinosorbent assay showed that the heterozygote GalT-KO pig had more sialylα2,6- and sialylα2,3-linked glycan than the control. Furthermore, the heart, liver, and kidney of the heterozygote GalT-KO pig had a higher N-glycolylneuraminic acid (Neu5Gc) content than the control, whereas the lung of the heterozygote GalT-KO pig had Neu5Gc content similar to the control. Collectively, the data strongly indicated that Neu5Gc is a more critical xenoantigen to overcoming the next acute immune rejection in pig to human xenotransplantation.

Recognition roles of the carbohydrate glycotopes of human and bovine lactoferrins in lectin– N-glycan interactions

Background Lactoferrin is an iron-binding protein belonging to the transferrin family. In addition to iron homeostasis, lactoferrin is also thought to have anti-microbial, anti-inflammatory, and anticancer activities. Previous studies showed that all lactoferrins are glycosylated in the human body, but the recognition roles of their carbohydrate glycotopes have not been well addressed. Methods The roles of human and bovine lactoferrins involved in lectin– N-glycan recognition processes were analyzed by enzyme-linked lectinosorbent assay with a panel of applied and microbial lectins. Results and conclusions Both native and asialo human/bovine lactoferrins reacted strongly with four Man-specific lectins — Concanavalia ensiformis agglutinin, Morniga M, Pisum sativum agglutinin, and Lens culinaris lectin. They also reacted well with PA-IIL, a LFuc>Man-specific lectin isolated from Pseudomonas aeruginosa. Both human and bovine lactoferrins also recognized a sialic acid specific lectin- Sambucus nigra agglutinin, but not their asialo products. Both native and asialo bovine lactoferrins, but not the human ones, exhibited strong binding with a GalNAc>Gal-specific lectin- Wisteria floribunda agglutinin. Human native lactoferrins and its asialo products bound well with four Gal>GalNAc-specific type-2 ribosome inactivating protein family lectins-ricin, abrin-a, Ricinus communis agglutinin 1, and Abrus precatorius agglutinin (APA), while the bovine ones reacted only with APA. General significance This study provides essential knowledge regarding the different roles of bioactive sites of lactoferrins in lectin– N-glycan recognition processes.

Specificity analysis of lectins and antibodies using remodeled glycoproteins

Due to their ability to bind specifically to certain carbohydrate sequences, lectins are a frequently used tool in cytology, histology, and glycan analysis but also offer new options for drug targeting and drug delivery systems. For these and other potential applications, it is necessary to be certain as to the carbohydrate structures interacting with the lectin. Therefore, we used glycoproteins remodeled with glycosyltransferases and glycosidases for testing specificities of lectins from Aleuria aurantia (AAL), Erythrina cristagalli (ECL), Griffonia simplicifolia (GSL I-B 4), Helix pomatia agglutinin (HPA), Lens culinaris (LCA), Lotus tetragonolobus (LTA), peanut ( Arachis hypogaeae) (PNA), Ricinus communis (RCA I), Sambucus nigra (SNA), Vicia villosa (VVA), and wheat germ ( Triticum vulgaris) (WGA) as well as reactivities of anti-carbohydrate antibodies (anti-bee venom, anti-horseradish peroxidase [anti-HRP], and anti-Lewis x). After enzymatic remodeling, the resulting neoglycoforms display defined carbohydrate sequences and can be used, when spotted on nitrocellulose or in enzyme-linked lectinosorbent assays, to identify the sugar moieties bound by the lectins. Transferrin with its two biantennary complex N-glycans was used as scaffold for gaining diverse N-glycosidic structures, whereas fetuin was modified using glycosidases to test the specificities of lectins toward both N- and O-glycans. In addition, α 1-acid glycoprotein and Schistosoma mansoni egg extract were chosen as controls for lectin interactions with fucosylated glycans (Lewis x and core α1,3-fucose). Our data complement and expand the existing knowledge about the binding specificity of a range of commercially available lectins.

Mucus Secretion and Cytoskeletal Modifications in Cultured Nasal Epithelial Cells Exposed to Wall Shear Stresses

The nasal epithelium is continuously subjected to wall shear stresses (WSS) induced by respiratory airflows. An in vitro experimental model was developed to expose nasal epithelial cells cultured under air-liquid interface conditions to steady airflow-induced WSS. Mucus secretion from epithelial goblet cells was quantified using an enzyme-linked lectinosorbent assay, and modifications of the cytoskeletal structure were qualitatively evaluated from fluorescent stains of actin and β-tubulin fibers. The results show increased mucus secretion from cells subjected to WSS of 0.1 and 1.0 dyne/cm2 for more than 15min in comparison with unstressed cells. The integrity levels of β-tubulin fibers were significantly lower in cells subjected to WSS than in unstressed cells. The increased mucus secretion in response to WSS was approximately the same in Taxol-free and Taxol-treated cultures, which indicates that there is no direct connection between β-tubulin fragmentation and mucus secretion. The stressed cells regained their normal cytoskeletal appearance 24h after the exposure to WSS. The results of this study suggest that WSS have an important role in the mechanical regulation of the nasal surface epithelium function.

Lectins as tools in glycoconjugate research

Lectins are ubiquitous proteins of nonimmune origin, present in plants, microorganisms, animals and humans which specifically bind defined monosugars or oligosaccharide structures. Great progress has been made in recent years in understanding crucial roles played by lectins in many biological processes. Elucidation of carbohydrate specificity of human and animal lectins is of great importance for better understanding of these processes. Long before the role of carbohydrate-protein interactions had been explored, many lectins, mostly of plant origin, were identified, characterized and applied as useful tools in studying glycoconjugates. This review focuses on the specificity-based lectin classification and the methods of measuring lectin-carbohydrate interactions, which are used for determination of lectin specificity or for identification and characterization of glycoconjugates with lectins of known specificity. The most frequently used quantitative methods are shortly reviewed and the methods elaborated and used in our laboratories, based on biotinylated lectins, are described. These include the microtiter plate enzyme-linked lectinosorbent assay, lectinoblotting and lectin-glycosphingolipid interaction on thin-layer plates. Some chemical modifications of lectin ligands on the microtiter plates and blots (desialylation, Smith degradation, beta-elimination), which extend the applicability of these methods, are also described.

Analysis of aggregative behavior of Pseudomonas sp. 30-3 isolated from Antarctic soil

Pseudomonas sp. 30-3, a toluene degrading microorganism isolated from oil-contaminated Antarctic soils, was shown to form aggregated flocs of cells when exposed to temperatures of 22 and 4 °C, with an increase in aggregation at 4 °C. This was speculated to be due to the secretion of an extracellular polymeric substance (EPS), thus protecting the organism from cold or frost damage. The flocs of cells were stained with the Live/Dead BacLight Bacterial Viability kit and found to be viable cells. The EPS was identified by lectin binding analysis to consist of N-acetyl- d-glucosamine and N-acetylneuraminic acid. An enzyme-linked lectinosorbent assay was also carried out to quantify the amount of EPS produced at 37, 22 and 4 °C. Results showed that at 37 °C the amount of EPS secreted was low, but there was little difference in the amount of EPS secreted at 22 and 4 °C by Pseudomonas sp. 30-3.

Isolation and characterisation of a Salvia bogotensis seed lectin specific for the Tn antigen

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18–20 mg/100 g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37 mM GalNAc, 0.019 mM ovine submaxillary mucin (OSM) or 0.008 mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin’s monomer MW (38,702 Da), amino acid composition, p I, carbohydrate content, deglycosylated form MW, thermal stability and Ca 2+ and Mn 2+ requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin’s specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.

Carbohydrate specificity of an insecticidal lectin isolated from the leaves of Glechoma hederacea (ground ivy) towards mammalian glycoconjugates

Preliminary studies indicated that the potent insecticidal lectin, Gleheda, from the leaves of Glechoma hederacea (ground ivy) preferentially agglutinates human erythrocytes carrying the Tn (GalNAcalpha1-Ser/Thr) antigen. However, no details have been reported yet with respect to the fine specificity of the lectin. To corroborate the molecular basis of the insecticidal activity and physiological function of Gleheda, it is necessary to identify the recognition factors that are involved in the Gleheda-glycotope interaction. In the present study, the requirement of high-density multivalent carbohydrate structural units for Gleheda binding and a fine-affinity profile were evaluated using ELLSA (enzyme-linked lectinosorbent assay) with our extended glycan/ligand collections, a glycan array and molecular modelling. From the results, we concluded that a high-density of exposed multivalent Tn-containing glycoproteins (natural armadillo and asialo ovine salivary glycoproteins) were the most potent factors for Gleheda binding. They were, on a nanogram basis, 6.5x10(5), 1.5x10(4) and 3.1x10(3) times more active than univalent Gal (galactose), GalNAc (N-acetylgalactosamine) and Tn respectively. Among mono- and oligo-saccharides examined, simple clustered Tn (molecular mass <3000 Da) from ovine salivary glycoprotein was the best, being 37.5 and 1.7x10(3) times better than GalNAc and Gal respectively. GalNAc glycosides were significantly more active than Gal glycosides, indicating that the N-acetamido group at C-2 plays an important role in Gleheda binding. The results of glycan array support the conclusions drawn with respect to the specificity of Gleheda based on the ELLSA assays. These findings combined with the results of the molecular modelling and docking indicate the occurrence of a primary GalNAcalpha1-binding site in the Gleheda monomer. However, the extraordinary binding feature of Gleheda for glycoproteins demonstrates the importance of affinity enhancement by high-density multivalent glycotopes in the ligand-lectin interactions in biological processes.

Polyvalent GalNAcα1→Ser/Thr (Tn) and Galβ1→3GalNAcα1→Ser/Thr (T α) as the most potent recognition factors involved in Maclura pomifera agglutinin–glycan interactions

The agglutinin isolated from the seeds of Maclura pomifera (MPA) recognizes a mucin-type disaccharide sequence, Galbeta1-->3GalNAc (T) on a human erythrocyte membrane. We have utilized the enzyme-linked lectinosorbent assay (ELLSA) and inhibition assay to more systematically analyze the carbohydrate specificity of MPA with glyco-recognition factors and mammalian Gal/GalNAc structural units in lectin-glycoform interactions. From the results, it is concluded that the high densities of polyvalent GalNAcalpha1-->Ser/Thr (Tn) and Galbeta1-->3GalNAcalpha1-->Ser/Thr (T(alpha)) glycotopes in macromolecules are the most critical factors for MPA binding, being on a nanogram basis 2.0 x 10(5), 4.6 x 10(4) and 3.9 x 10(4) more active than monovalent Gal, monomeric T and Tn glycotope, respectively. Other carbohydrate structural units in mammalian glycoconjugates, such as human blood group Sd (a+) related disaccharide (GalNAcbeta1-->4Gal) and Pk/P1 active disaccharide (Galalpha1-->4Gal) were inactive. These results demonstrate that the configurations of carbon-4 and carbon-2 are essential for MPA binding and establish the importance of affinity enhancement by high-density polyvalencies of Tn/T glycotopes in MPA-glycan interactions. The overall binding profile of MPA can be defined in decreasing order as high density of polyvalent Tn/T(alpha) (M.W. > 4.0 x 10(4)) >> Tn-containing glycopeptides (M.W. < 3.0 x 10(3)) > monomeric T/Tn and P (GalNAcbeta1-->3Gal) > GalNAc > Gal >> Man, L: ARA: , D: Fuc and Glc (inactive). Our findings should aid in the selection of this lectin for elucidating functions of carbohydrate chains in life processes and for applications in the biomedical sciences.

A Novel Lectin (Morniga M) from Mulberry (Morus nigra) Bark Recognizes Oligomannosyl Residues in N-Glycans

Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra). In order to understand the function and application of this novel lectin, the binding property of Morniga M was studied in detail using an enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay with extended glycan/ligand collection. From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans such as those of the bovine α₁-acid glycoprotein (gp) and lactoferrin were the most active gps, but not the O-glycans or polysaccharides including mannan from yeast. The binding affinity of Morniga M for ligands can be ranked in decreasing order as follows: gps carrying multiple N-glycans with oligomannosyl residues >> N-glycopeptide with a single trimannosyl core > Tri-Man oligomer [Manα1→6(Man α1→3) Man], Penta-Man oligomer [Manα1→6(Manα1→3)Manα1→6(Manα1→3) Man] ≧ Man α1→2, 3 or 6 Man > Man > GlcNAc, Glc >> L-Fuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans.

A novel lectin (morniga M) from mulberry (Morus nigra) bark recognizes oligomannosyl residues in N-Glycans

Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra). In order to understand the function and application of this novel lectin, the binding property of Morniga M was studied in detail using an enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay with extended glycan/ligand collection. From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans such as those of the bovine alpha1-acid glycoprotein (gp) and lactoferrin were the most active gps, but not the O-glycans or polysaccharides including mannan from yeast. The binding affinity of Morniga M for ligands can be ranked in decreasing order as follows: gps carrying multiple N-glycans with oligomannosyl residues >> N-glycopeptide with a single trimannosyl core > Tri-Man oligomer [Man alpha1-->6(Man alpha1-->3) Man], Penta-Man oligomer [Man alpha1-->6(Man alpha1-->3)Man alpha1-->6(Man alpha1-->3) Man] > or = Man alpha1-->2, 3 or 6 Man > Man > GlcNAc, Glc >> L-Fuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans.

Lectinochemical studies on the affinity of Anguilla anguilla agglutinin for mammalian glycotopes

Anguilla anguilla agglutinin (AAA) is a fucose-specific lectin found in the serum of the fresh water eel. It is suggested to be associated with innate immunity by recognizing disease-associated cell surface glycans, and has been widely used as a reagent in hematology and glycobiology. In order to gain a better understanding of AAA for further applications, it is necessary to elucidate its binding profile with mammalian glycotopes. We, therefore, analyzed the detailed carbohydrate specificity of AAA by enzyme-linked lectinosorbent assay (ELLSA) with our extended glycan/ligand collection and lectin-glycan inhibition assay. Among the glycans tested, AAA reacted well with nearly all human blood group A h (GalNAcα1→3[ lFucα1→2]Gal), B h (Galα1→3[ lFucα1→2]Gal) , H lFucα1→2Gal) and Le b (Fucα1→2Galβ1→3[Fucα1→4]GlcNAc) active glycoproteins (gps), but not with blood group Le a (Galβ1→3[Fucα1→4]GlcNAc) substances, suggesting that residues and optimal density of α1-2 linked lFuc to Gal at the non-reducing end of glycoprotein ligands are essential for lectin-carbohydrate interactions. Blood group precursors, Galβ1-3GalNAc ( T), GalNAcα1-Ser/Thr ( Tn) containing glycoproteins and N-linked plasma gps, gave only negligible affinity. Among the mammalian glycotopes tested, A h, B h and H determinants were the best, being about 5 to 6.7 times more active than lFuc, but were weaker than p-nitrophenylαFuc indicating that hydrophobic environment surrounding the lFuc moiety enhance the reactivity. The hierarchy of potency of oligo- and monosaccharides can be ranked as follows: p-nitrophenyl-αFuc > A h, B h and H > lFuc > lFucα1→2Galβ1→4Glc (2′-FL) and Galβ1→4[ lFucα1→3]Glc (3′-FL), while LNDFH I ( Le b hexa-), Le a, Le x (Galβ1→4[Fucα1→3]GlcNAc), and LDFT (gluco-analogue of Le y ) were inactive. From the present observations, it can be concluded that the combining site of AAA should be a small cavity-type capable of recognizing mainly H/crypto H and of binding to specific polyvalent ABH and Le b glycotopes.

Effect of polyvalencies of glycotopes on the binding of a lectin from the edible mushroom, Agaricus bisporus.

Agaricus bisporus agglutinin (ABA) isolated from edible mushroom has a potent anti-proliferative effect on malignant colon cells with considerable therapeutic potential as an anti-neoplastic agent. Since previous studies on the structural requirement for binding were limited to molecular or submolecular levels of Galbeta1-3GalNAc (T; Thomsen-Friedenreich disaccharide glycotope; where Gal represents D-galactopyranose and GalNAc represents 2-acetamido-2-deoxy-D-galactopyranose) and its derivatives, the binding properties of ABA were further investigated using our collection of glycans by enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay. The results indicate that polyvalent Galbeta1-related glycotopes, GalNAcalpha1-Ser/Thr (Tn), and their cryptoforms, are the most potent factor for ABA binding. They were up to 5.5x10(5) and 4.7x10(6) times more active than monomeric T and GalNAc respectively. The affinity of ABA for ligands can be ranked as: multivalent T (alpha) (Galbeta1-3GalNAcalpha1-), Tn and I / II (Galbeta1-3GlcNac/Galbeta1-4GlcNAc, where GlcNAc represents 2-acetamido-2-deoxy-D-glucopyranose)>>>>monomeric T (alpha) and Tn > I >>GalNAc>>> II, L (Galbeta1-4Glc, where Glc represents D-glucopyranose) and Gal (inactive). These specific binding features of ABA establish the importance of affinity enhancement by high-density polyvalent (versus multiantennary I / II) glycotopes and facilitate our understanding of the lectin receptor recognition events relevant to its biological activities.

Binding profile of Artocarpus integrifolia agglutinin (Jacalin)

Artocarpus integrifolia agglutinin (Jacalin) from the seeds of jack fruits has attracted considerable attention for its diverse biological activities and has been recognized as a Galβ1→3GalNAc ( T) specific lectin. In previous studies, the information of its binding was limited to the inhibition results of monosaccharides and several T related disaccharides, but its interaction with other carbohydrate structural units occurring in natural glycans has not been characterized. For this reason, the binding profile of this lectin was studied by enzyme linked lectinosorbent assay (ELLSA) with our glycan/ligand collection. Among glycoproteins (gps) tested for binding, high density of multi-Galβ1→3GalNAcα1→ (m T α) and GalNAcα1→Ser/Thr (m Tn) containing gps reacted most avidly with Jacalin. As inhibitors expressed as nanograms yielding 50% inhibition, these m T α and m Tn containing glycans were about 7.1 × 10 3, 4.0 × 10 5, and 7.8 × 10 5 times more potent than monomeric T α, GalNAc, and Gal. Of the sugars tested and expressed as nanomoles for 50% inhibition, Tn containing peptides, T α, and the human P blood group active disaccharide ( P α, GalNAcβ1→3Galα1→) were the best and about 283 times more active than Gal. We conclude that the most potent ligands for this lectin are m Tn, m T, and possibly P α glycotopes, while GalNAcβ1→4Galβ1→, GalNAcα1→3Gal, GalNAcα1→3GalNAc, and Galα1→3Gal determinants were poor inhibitors. Thus, the overall binding profile of Jacalin can be defined in decreasing order as high density of m Tn, and m T α ⋙ simple Tn cluster > monomeric T α > monomeric P α > monomeric Tn > monomeric T > GalNAc > Gal > Methylα1→Man ≫ Man and Glc (inactive). Our finding should aid in the selection of this lectin for biological applications.

Carbohydrate specificity of a toxic lectin, abrin A, from the seeds of Abrus precatorius (jequirity bean)

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Galβ1→4GlcNAc ( II), Galα1→4Gal ( E), and Galβ1→3GalNAc ( T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the D Fuc pyranose ring to penta-saccharides and probably internal Galα,β→; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Galα1→3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I >> Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.

Carbohydrate specificity of a lectin isolated from the fungus Sclerotium rolfsii

In order to investigate the functional roles of a phytopathogenic fungal lectin (SRL) isolated from the bodies of Sclerotium rolfsii, the binding properties of SRL were studied by enzyme linked lectinosorbent assay and by inhibition of SRL-glycan interaction. Among glycoproteins(gp) tested for binding, SRL reacted strongly with GalNAcα1→Ser/Thr ( Tn) and /or Galβ1→3GalNAcα1→( T α) containing gps: human T α and Tn glycophorin, asialo salivary gps, and asialofetuin, but its reactivity toward sialylated glycoproteins was reduced significantly. Of the sugar ligands tested for inhibition of SRL-asialofetuin binding, Thomsen-Friedenreich residue ( T α) was the best, being 22.4 and 2.24×10 3 more active than GalNAc and Galβ1→ residues, respectively. Other ligands tested were inactive. When the glycans used as inhibitors, T α, and/or Tn containing gps, especially asialo PSM, asialo BSM, asialo OSM, active antifreeze gp, asialo glycophorin and Tn-glycophorin were very active, and 1.0×10 4 times more potent than GalNAc. From these results, it is clear that the combining site of SRL should be of a cavity type and recognizes only Tn and T α residues of glycans; it is suggested that T α and Tn glycotopes, which are present only in abnormal carbohydrate sequences of higher orders of mammal, are the most likely sites for phytopathogenic fungal attachment as an initial step of infection. The affinity of SRL for ligands can be ranked in decreasing order as follows: multivalent T α and Tn >> monomeric T α and Tn > GalNAc >>> II (Galβ1→4GlcNAc), L (Galβ1→4Glc), and Gal.

Carbohydrate specificity of an agglutinin isolated from the root of Richosanthes kirilowii

The root of Trichosanthes kirilowii, which has been used as Chinese folk medicine for more than two thousand years, contains a Gal specific lectin (TKA). In order to elucidate its binding roles, the carbohydrate specificities of TKA were studied by enzyme linked lectinosorbent assay (ELLSA) and by inhibition of lectin-glycoform binding. Among glycoproteins (gp) tested, TKA reacted strongly with complex carbohydrates with Galβ1 → 4GlcNAc clusters as internal or core structures (human blood group ABH active glycoproteins from human ovarian cyst fluids, hog gastric mucin, and fetuin), porcine salivary glycoprotein and its asialo product, but it was inactive with heparin and mannan (negative control). Of the sugar inhibitors tested for inhibition of binding, Neu5Acα2 → 3/6Galβ1 → 4Glc was the best and about 4, 14.6 and 27.7 times more active than Galβ1 → 4GlcNAc(II), Galβ1 → 3GalNAc(T) and Gal, respectively. From these results, it is suggested that this agglutinin is specific for terminal or internal polyvalent Galβ1 → 4GlcNAcβ1→, terminal Neu5Acα2 → 3/6Galβ1 → 4Glc and cluster forms of Galβ1 → 3GalNAcα residues. The unusual affinity of TKA for terminal and internal Galβ1 → glycotopes may be used to explain the possible attachment roles of this agglutinin in this folk medicine to target cells.