Sophora Japonica Lectin Research Articles

Bacterial adherence in otitis media: Determination of N-acetylgalactosamine (GalNAc) residues in the submucosal glands and surface epithelium of the normal and diseased Eustachian tube

Background Acute otitis media (AOM) is the most common childhood infection caused by bacteria. The pathogenesis of AOM implicates initial adherence of a pathogen to the nasopharyngeal epithelium, which is followed by bacterial colonization of the middle ear cavity through the Eustachian tube. N-acetylgalactosamine (GalNAc) is an important constituent of mucins and GalNAc containing sugar residues seem to be essential for initial adherence of respiratory bacteria to the surface of epithelial cells. Objective To explore the localization of GalNAc residues, we incubated Eustachian tube sections from Streptococcus pneumoniae infected and normal control rats with seven biotinylated, GalNAc recognizing lectins: Bauhinia purpurea lectin (BPA), Psophocarpus tetragonolobus lectin (PTA), Helix aspersa lectin (HAA), Helix pomatia lectin (HPA), Phaseolus lunatus lectin (PLA), Sophora japonica lectin (SJA) and Vicia Villosa isolectin B4 (VVA-B4). Results The mucin producing epithelium and submucosal glands of the normal Eustachian tube contained GalNAc residues, as evidenced by binding of several of the lectins. Lectin binding specificity and intensity changed following acute middle ear infection. BPA was the only lectin that exclusively stained the surface epithelium and the serous acini of the submucosal glands in the infected animals, whereas no binding was detected in the normal controls. HPA, HAA, PTA and VVA-B4 binding to surface epithelial cells increased after infection, indicating an active secretion of GalNAc containing glycans. Quantitative analysis of submucosal gland staining intensity showed significantly more GalNAc residues in the normal Eustachian tube, compared to infected animals. Conclusion We conclude that the mucous producing elements of the normal rat Eustachian tube contain GalNAc residues essential for respiratory pathogen adherence. In addition, the GalNAc residue specificity and reacting intensity change in relation to acute infection, which may be important in relation to subsequent development of secretory otitis media or formation of a bacterial biofilm in the middle ear. The results show that GalNAc residues increased in both the submucosal serous glands and in the surface epithelium of the Eustachian tube after middle ear infection with S. pneumoniae

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts.

Carbohydrates have been suggested to account for some IgE cross-reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti-horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.

Amino acid sequence, glycan structure, and proteolytic processing of the lectin of Vatairea macrocarpa seeds

VML is a galactose-binding lectin isolated from Vatairea macrocarpa seeds. By SDS-polyacrylamide gel electrophoresis, VML is a glycoprotein composed of a major 32–34 kDa double band (alpha-chain) and minor 22 kDa and 13 kDa bands. N-terminal sequencing of electroblotted samples showed that the 22 and 13 kDa bands corresponded to C-(beta) and N-(gamma) terminal fragments of the alpha-chain, respectively. The primary structure of VML displays similarity with other leguminous lectins, particularly with Erythrina variegata, Robinia pseudoacacia and Sophora japonica lectins. VML is N-glycosylated at asparagine residues at positions 111 and 183 with one major glycan structure. Tandem mass spectrometry and methylation analysis indicated the presence of Manα1-6[(Manα1-3)(Xylβ1-2)]Manβ1-4-GlcNAcβ1-4(Fucα1-3)GlcNAc, a typical plant N-glycan. Equilibrium sedimentation analysis by analytical centrifugation showed that VML had a mass of 122–130 kDa, which did not change within the pH range 2.5–8.5. These data indicated that VML is a pH-independent homotetrameric protein and that a small proportion of the alpha-subunits is cleaved into noncovalently associated N- and C-terminal fragments. Mass spectrometric analysis suggested a mechanism for the proteolytic processing of VML. V. macrocarpa lectin contains a mixture of doubly (28 525 Da) and singly (27 354 Da) glycosylated alpha-chains. Deglycosylation of Asn-111 correlates with proteolytic cleavage of the Asn-114-Lys-115 bond yielding glycosylated gamma (residues 1–114, 12 304 Da) and nonglycosylated beta- (residues 115–239, 14 957 Da) chains. Some beta-chain molecules are further deglycosylated and N-terminally processed yielding products of molecular masses of 13 783 Da and 13 670 Da.

Primary structure of an N-glycosidic carbohydrate unit derived from Sophora japonica lectin.

The lectin isolated from Sophora japonica seeds is a glycoprotein which binds oligosaccharides with non-reducing terminal Gal beta(1----3/4)GlcNac beta 1----units. The carbohydrate moiety of the lectin is composed of fucose, xylose, mannose and N-acetylglucosamine. The major glycopeptide of the lectin, prepared by pronase digestion, was derivatized with fluorescein isothiocyanate, purified by PAGE and examined by exoglycosidase digestion as well as purified by gel filtration through Bio-Gel P6-DG and investigated by methylation analysis and 400-MHz 1H-NMR spectroscopy. The primary structure of the glycopeptide was established to be as follows. (Formula: see text). Structures similar to this containing a (beta 1-2)xylosyl substituent on the core beta-mannosyl residue and an inner core (alpha 1-3)fucosyl substituent seem to occur frequently in plant glycoproteins.

Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes.

Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various 125I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeus and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.

Identification of liver cell membrane galactoglycoproteins involved in the process of insulin binding

The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [ 125I]insulin binding to soluble receptors from mouse liver was inhibited by digestion with β-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[ 3H]hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10 −7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified within two galactoglycoproteins of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with β-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10 −7 M proinsulin slightly hindered labeling while pretreatment with 10 −7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate the glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties.

Immunochemical studies on the carbohydrate specificity of Maclura pomifera lectin

Maclura pomifera lectin was purified from crude extracts of seeds by adsorption onto soluble polyleucyl hog A + H blood group substance and elution with either melibiose or N-actyl- d-galactosamine. The purified lectin formed a single band in immunodiffusion and immunoelectrophoresis against rabbit antiserum to the crude extract. It migrated as a broad band of five very closely packed fine bands on polyacrylamide electrophoresis at both acidic and alkaline pH. A broad band was seen on isoelectrofocusing of the purified lectin in acrylamide gels. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, three bands with molecular weights of 14,500, 13,500, and 12,000 were seen. The carbohydrate specificity of the M. pomifera lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin precipitated well with blood group substances which had been subjected to mild acid hydrolysis or to Smith degradation and with blood group precursor substances. The active and inactive antifreeze glycoproteins, composed of repreating units of dGalβ1 → 3 dGalNAcαl → OThrAlaAla, were the best, precipitating 90% of the lectin with less than 3.0 μg of glycoprotein being required for precipitation of 50% of 6.0 μg lectin N. Precipitability of this lectin with various native and blood group substances and glycoproteins is attributable to the terminal nonreducing DGalNAc and dGalβ1 → 3 dGalNAc on the carbohydrate moiety of those compounds. Among the monosaccharides and glycosides tested for inhibition of precipitation, the nitrophenyl α-glycosides of dGalNAc and dGal were most active and were more than 130 times as active as dGalNAc and dGal, respectively. dGalNAc was 3.3 times as active as dGal. Nitrophenyl α- and methyl α-glycosides of dGalNAc and dGal were much more active than the corresponding β-anomers. Nitrophenyl α-glycosides were substantially better than the corresponding methyl α-glycosides, indicating a hydrophobic contribution to the site subterminal to the sugar moiety as has been found for Wistaria floribunda and Sophora japonica lectins. dGalβ1 → 3 dGalNAc and its β-tosyl derivative were the best of the oligosaccharides tested. They were as active as methyl α dGalNAc, about 5.6 times less active than nitrophenyl α-glycosides of DGalNAc, and 5 and 18.5 times as active as dGalNAcα1 → 6 dGal and melibiose, respectively. dGalβ1 → 3 dGlcNAc, lactose, lacto- N-tetraose, N-acetyllactosamine as well as blood-group-specific oligosaccharides gave no inhibition in amounts comparable to those used with dGalβ → 3 dGalNAc. From these results, it can be concluded that hydrophobic interaction is important for binding and that the combining size of the lectin is at least as large as a disaccharide, and of the compounds studied is most complementary to a mucintype disaccharide on a human erythrocyte membrane, DGalβ1 → 3 dGalNAc.

Immunochemical studies on the reactivities and combining sites of the d-galactopyranose- and 2-acetamido-2-deoxy- d-galactopyranose-specific lectin purified from Sophora japonica seeds

Sophora japonica lectin agglutinates human B erythrocytes strongly and A 1 erythrocytes weakly. Bivalent metal ions such as Ca 2+, Mn 2+, or Mg 2+ were shown to be essential for hemagglutinating and precipitating activities. At optimal concentrations of bivalent metal ions, hemagglutinating activity was highest between pH 8.5 and 9.0 and decreased sharply below pH 8.5, whereas precipitating capacity was maximal between pH 6.7 and 9.5. The combining site of the S. japonica lectin was explored by quantitative precipitin and precipitin inhibition assays. This lectin showed substantial differences in precipitation with several blood group B substances ascribable to heterogeneity resulting from incomplete biosynthesis of their carbohydrate side chains. The lectin precipitated moderately well with A 1 substance and precursor blood group I fractions (OG). It precipitated weakly or not at all with A 2, H, or Le a substances. In inhibition assays, glycosides of dGalNAc were about five to six times better than those of dGal; dGalNAc itself was about six times better than dGal. Nitrophenyl glycosides were all substantially better than the methyl glycosides, indicating a hydrophobic contribution to the site subterminal to the nonreducing moiety. Although nitrophenyl β-glycosides were much better than the corresponding α-glycosides, the methyl α-and βDGalNAc p were equal in activity as were methyl α- and βDGal p. Among the oligosaccharides tested, the β-linked N-tosyl- l-serine glycoside of dGalβ1 → 3 dGalNAc was best and was as active as p-nitrophenyl βDGalNAc p, whereas dGalβ1 → 3 dGalNAc α- N-tosyl serine and the nitrophenyl and phenyl α-glycosides of dGalβ1 → 3 dGalNAc were much less active, suggesting that the hydrophobic moiety and/or a subterminal dGalNAc β-linked and substituted on carbon 3 play an important role in binding and that a β-linked glycoside of dGalβ1 → 3 dGalNAc may be an essential requirement for binding. The results of inhibition studies with other oligosaccharides indicate that a subterminal dGlcNAc substituted on carbon 3 or 4 by dGalβ may contribute somewhat to binding and that whether the dGlcNAc is linked β1 → 3 or β1 → 6 to a third sugar does not contribute to or interfere with binding. The β1 → 3 linkage of the terminal dGal to the subterminal amino sugar is significant since dGalβ1 → 4 dGlcNAc was one-half as active as the corresponding β1 → 3-linked compound and the subterminal sugar must be unsubstituted for optimal binding. N-Acetyllactosamine was 50% more active than lactose, indicating that the subterminal N-acetamido group was also contributing significantly to binding. A variety of other sugars, glycosides, and oligosaccharides showed little or not activity. From the oligosaccharides available, the combining size of this lectin would appear to be least as large a β-linked disaccharide and most complementary to dGalβ1 → 3 dGalNAc β-linked to tosyl- l-serine the most active compound tested.

Circular dichroism study on structural reorganization of lectins by sodium dodecyl sulfate.

The circular dichroism (CD) spectra of lectins from Dolichos biflorus, Helix pomatia, Lotus tetragonolobus, Phaseolus vulgaris (erythroagglutinin), Pisum sativum, Sophora japonica, and Ulex europaeus I were analyzed, before and after the conformational transitions which were effected by sodium dodecyl sulfate (SDS) and change of pH. It was found that the pleated sheet conformation dominated in these lectins and that the best estimates of the amounts of the sheets can be made from the positive CD bands centered at 195 . 197 nm. The mean residue ellipticities of these bands in native lectins varied between 1.0 . 10(4) and 1.7 . 10(4) deg . cm2 . dmol-1. The conformation of the lectins was perturbed by SDS, which caused the CD bands related to the tyrosine and tryptophan chromophores to diminish and which also caused the reorganization of the main chains into new forms having approximately 15--40% of alpha-helix. Acid facilitated these transitions. The secondary structure of Ulex europaeus I lectin was resistant to SDS in neutral solutions but reorganization occurred in acid solutions. In Helix pomatia lectin the helices were formed chiefly from the aperiodic segments of the main chain. As structural perturbant, SDS was most effective at relatively high concentrations of 5 . 10(-3) to 5--10(-2) M, i.e. above the critical micelle concentration. Experiments with Pisum sativum and Sophora japonica lectins indicated that SDS below critical micelle concentration did not effect the tertiary structure of the lectins.

Properties and subunit characterization of affinity purified Sophora japonica lectin.

The affinity purified Sophora japonica lectin exhibits an anomalous behavior on polyacrylamide gel electrophoresis (PAGE). Electrophoresis at pH 8.9 produces three protein staining bands. Extraction and re-electrophoresis of the fastest and slowest migrating components demonstrates that the lectin solution is an equilibrium mixture of interconvertible forms. Addition of a bindable saccharide, D-galactose, during PAGE causes the equilibrium to be shifted toward a single form. As indicated by analytical gel filtration, sedimentation velocity ultracentrifugation and ion-exchange chromatography experiments, the equilibrium mixture consists of charge and not molecular weight variants of the native molecule of 132,800 g/m. Results from end-group and cysteine analyses and PAGE in sodium dodecyl sulfate indicate that the native lectin is composed of the non-covalent association of two dissimilar subunits. One subunit consists of two identical polypeptide chains attached by two disulfide bonds and the other subunit of two identical polypeptide chains stabilized by a single cysteine bridge.

Hapten inhibition of adsorption: Specificity of the Sophora japonica lectin

Since hapten inhibition of precipitation is relatively time consuming, we have developed a hapten inhibition of adsorption assay to explore the sugar binding specificity of the Sophora japonica lectin. Adsorbents were prepared with Sephadex and p-aminiphenyl β-D-galactopyranoside using the CNBr procedure. The ability of simple saccharides to inhibit the binding of lectin to the adsorbent was performed employing a fixed amount of adsorbent and lectin and varying quantities of inhibitor. Plots of the percent inhibition of adsorption with respect to the logarithm of micromoles of inhibitor yielded sigmoid shaped curves. The quantities of various saccharides required to cause 50% inhibition of the lectin—adsorbent interaction relative to D-galactose were within ±5% of the relative inhibiting potencies of the sugars in the lectin—blood group substance precipitation reactions.

Alteration of the Specificity of Lectin by Chemical Modification

Summary Acylation of lima bean and Sophora japonica lectins with p- phenylazobenzoyl chloride greatly increased the anti-A agglutinating activity and, in the case of S. japonica, diminished the anti-B and anti-O activity. Amino acid analyses of purified peptides isolated from the modified lectins suggested that the change in specificity might be due to combination of the introduced residues with the ε-amino groups of lysine.

A Specific Anti-B Lectin for Routine Diagnostic Purposes

Summary Privine hydrochloride, an inhibitor of anti-A hemagglutinins, can be used as an inhibitor of the anti-A of the Sophora japonica lectin, which then becomes a specific anti-B reagent suitable for routine diagnostic purposes.