Immunochemical studies on the combining site of the d-galactose/N-acetyl-d-galactosamine specific lectin from Erythrina cristagalli seeds

The combining site of the Erythrina cristagalli lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin precipitated best with two fractions of a precursor human ovarian cyst blood group substance with I and i activities. A₁, A₂, B, H, Le^a, and Le^b blood group substances precipitated poorly to moderately and substances of the same blood group activity precipitated to varying extents. These differences are attributable to heterogeneity resulting from incomplete biosynthesis of carbohydrate chains. Specific precipitates with the poorly reactive blood group substances were found to be more soluble than those reacting strongly. Precipitation was minimally affected by EDTA or divalent cations. Among the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl βdGal was most active and was 10 times more active than methyl βdGal, indicating involvement of hydrophobic contacts in site specificity. Methyl αdGalNAc, p-nitrophenyl αdGalNAc, methyl αdGal, N-acetyl-d-galactosamine, p-nitrophenyl αdGal, methyl βdGal, and p-nitrophenyl βdGalNAc were progressively less active than p-nitrophenyl βdGal. The best disaccharide inhibitor dGalβ1 → 4dGlcNAc was 7.5 times more potent than p-nitrophenyl βdGal. A tetraantennary and triantennary oligosaccharide containing four and three dGalβ1 → 4dGlcNAcβ1 → branches, respectively, were, because of cooperative binding effects, 1.6 and 2.5 times more active than the bi- and monoantennary oligosaccharides, respectively. dGalβ1 → 4dGlcNAcβ1 → 6dGal and dGalβ1 → 4dGlcNAcβ1 → 2dMan had the same activity, being 1.5 times more active than dGalβ1 → 4dGlcNAc, which was 2.6 and 8.5 times more active than dGalβ1 → 3dGlcNAc and dGalβ1 → 3dGlc, respectively. Substitutions by N-acetyl-d-galactos-amine or l-fucose on the d-galactose of inhibitory compounds blocked activity. These results suggest that a hydrophobic interaction with the subterminal sugar is important in the binding and that the specificity of the lectin combining site involves a terminal dGalβ1 → 4dGlcNAc and the β linkage of a third sugar.     

Immunochemical studies on the combining site of the d-galactopyranose and 2-acetamido-2-deoxy-d-galactopyranose specific lectin isolated from Bauhinia purpurea alba seeds

The combining site of the Bauhinia purpurea alba lectin was studied by quantitative precipitin and precipitin inhibition assays. Of 45 blood group substances, glycoproteins, and polysaccharides tested, 35 precipitated over 75% of the lectin. Precursor blood group substances with I activity (Cyst OG 10% from 20% and Cyst OG 20% from 10%), desialized fetuin, and desialized ovine salivary glycoprotein, in which more than 75% of the carbohydrate side chains have dGalN Ac linked through α1 → to the OH group of Ser or Thr of a protein core, completely precipitated the lectin. The poorly reactive blood group substances after mild acid hydrolysis or Smith degradation, as well as sialic acid-containing glycoproteins after removal of sialic acid, had substantially increased activity so that more than 80% of the lectin was precipitated. Precipitability with various blood group substances and glycoproteins is ascribable to the terminal nonreducing dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 3 or 4dGlcNAc, and dGalβ1 → 3 or 4dGlcNAcβ1 → 3dGal determinants on the carbohydrate moiety. Of the monosaccharides tested for inhibition of precipitation, dGalNAc and its p-nitrophenyl and methyl α-glycosides were best. These compounds were four to five times better than the corresponding dGal compounds but methyl βDGalNAcp was only about 40% more active than methyl βdGalp. The α-anomers of p-nitrophenyl DGalNAcp and dGalp, were twice as active as the corresponding β-anomers. Methyl αDGalNAcp was four times as active as the β-anomer but the inhibitory power of the methyl α- and β-anomers of dGal were about equal. Among the oligosaccharides tested, dGalβ1 → 3dGalNAc and its tosyl derivatives were most active, the tosyl glycosides being about twice as active as dGalβ1 → 3dGalNAc, which was somewhat more active than dGalNAcα1 → 6dGal and dGalNAc, and 2.5 and 5 times as active as dGalNAcα1 → 3dGalβ1 → 3dGlcNAc and dGalNAcαl → 3dGa1, respectively (blood group A specific). These findings suggest that a subterminal dGalNAc β-linked and substituted on carbon 3 plays an important role in binding. Consistent with this inference are the findings that dGalβ1 → 3dGlcNAc and dGalβ1 → 6dGal were poorer inhibitors although dGalβ1 → 3dGlcNAc was two to three times as active as glycosides of dGal. Oligosaccharides with terminal nonreducing dGal and subterminal α-linked dGal were as active or less active than dGal. dGalβ1 → 3dGlcNAcβ1 → 3dGalβ1 → 4dGlc (lacto-N-tetraose) and dGalβ1 → 3dGlcNAcβ1 → 3dGal-β1-O-(CH₂)₈COOCH₃ were equally active and 1.5 times as potent as dGalβ1 → 3dGlcNAc whereas dGalβ1 → 3dGlcNAcβ1 → 6dGal was only 40% as potent as dGalβ1 → 3dGlcNAc suggesting that a third sugar may be part of the determinant. Substitution of dGalβ1 → 3dGlcNAcβ1 → 3dGalβ1 → 4dGlc on the subterminal dGlcNAc by lFucα1 → 4 in lacto-N-fucopentaose II reduced activity fourfold; if the nonreducing dGal is substituted by lFucα1 → 3 as in lacto-N-fucopentaose I its activity is almost completely abolished. This suggests that a terminal nonreducing dGal as well as subterminal dGlcNAc are contributing to binding. The β → 3 linkage of the terminal dGal to the subterminal amino sugar is significant since dGalβ1 → 4dGlcNAc is a poorer inhibitor. Although the available data suggest that the combining site of the lectin Bauhinia purpurea alba may be most complementary to the structure dGalβ1 → 3dGalNAcβ1 → 3dGal, several other possibilities remain to be tested when suitable oligosaccharides become available.     

Properties of a lectin purified from the seeds of Cicer arietinum

A lectin was isolated from seed extracts of Cicer arietinum by (NH4)2SO4 precipitation and subsequent ion exchange chromatography and gel filtration. Affinity chromatography on desialylated human IgM coupled to AH-Sepharose was also performed, but the amount bound was very low. The lectin has a molecular mass of about 44000 Da, as determined by ultracentrifugation and gel filtration. Dodecyl sulphate polyacrylamide-gel electrophoresis showed one band corresponding to a molecular mass of 26000 Da. N-Terminal amino acid sequence analyses indicate only one type of chain, suggesting that the lectin is probably dimeric. The amino acid composition is given. Papainized human erythrocytes of the different ABO groups were agglutinated equally well by the Cicer lectin, whereas untreated cells reacted weakly and only in the presence of bovine serum albumin. Simple sugars did not inhibit the agglutination, but some glycoproteins did inhibit. The lectin is probably nonmitogenic against human lymphocytes. Antigenic analyses in an enzyme-linked immunosorbent assay (ELISA) showed only a weak cross-reaction between Cicer and the lectins in the Vicieae tribe. Thus, our physicochemical and antigenic studies of the Cicer lectin support the botanical reasons recently given for removing the genus Cicer from the Vicieae tribe.     

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.     

Carbohydrate binding studies on the lectin from Datura stramonium seeds

The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (M_r = 86,000) possesses two carbohydrate-binding sites for N,N′,N′',N?-tetraacetylchitotetritol/mol protein, with an apparent K_a = 8.7 × 10³M^?1 at 4 °C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N′- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N′,N?-triacetylchitotriose was over 6-fold more potent than the disaccharide (N′,N′-diacetylchitobiose) which, in turn, was 90 times more reactive than N-acetyl-d-glucosamine.N-Acetyllactosamine [β-d-Gal-(1 → 4)-d-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N′-diacetylchitobiose. The requirement for an N-acetyl-d-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.     

Developmental changes in the bark lectin of Sophora japonica L.

Lectin is the major protein in the phloem tissue of S. japonica. By immunohistochemistry using anti-seed lectin antibody it was demonstrated that the lectin was localized in the ray and the axial parenchyma. Neither lectin nor other cross-reactive materials were observed in the cambium, sieve tubes and companion cells. The distribution and localization changed in relation to tissue development. Lectin content in the bark changed during the year, the average in summer being about 50% of that in winter. The distribution of lectin in the bark in winter was similar from the innermost (youngest) to the outermost (oldest) region. In contrast, in summer the innermost region hardly contained any lectin, and the outermost region contained less lectin than the middle. Lectin localization in tissues and cells differed also depending on tissue age. In new tissue, produced in the current year, lectip was absent in summer, was located in the cytoplasmic layer between cell wall and vacuole in autumn, and sequestered in the vacuoles in winter. On the other hand, lectin in old tissue (formed in the previous year) was located throughout the year mainly within the vacuoles, with only very small contents in the cytoplasmic layer in autumn. Within the outermost (oldest) region, in which the lectin content was low in summer, the cells which bordered the outer bark never contained any lectin in summer. The intracellular localization in autumn in new tissue, determined by immunogold electron microscopy, was in the lumen of the endoplasmic reticulum and vesicles, with gold particles hardly present in the cytoplasm. From these findings we conclude that lectin is synthesized on the endoplasmic reticulum and most vigorously in the new tissue in autumn, and that it is mainly consumed in the outermost bark regions, where dilatation occurs and-or where cork cambium is differentiated.Abbreviations ELISA enzyme-linked immunosorbent assay - ER endoplasmic reticulum - kDa kilodalton Retired. Anatomical terms in this paper are used according to Multilingual glossary of terms used in wood anatomy edited by the Committee on Nomenclature, International Association of Wood Anatomists; reprints may be obtained from the Office of the Secretary-Treasurer, Universitätsstrasse 2, CH-8092 Zürich 6, Switzerland.     

Preliminary X-ray diffraction analysis of crystalline lectins from the seeds and leaves of Sophora japonica

The Japanese Pogada Tree (Sophora japonica) contains at least four distinct lectins distributed among seeds, bark, and leaf tissues of the plant. All of these lectins are N-acetylgalactosamine-specific, have molecular weights of about 130,000, are glycoproteins and possess substantial sequence homology. However, they are products of distinct genes. We have crystallized Sophora lectins from bark, seeds, and two different lectins from leaves. Multiple crystal forms of each variety have been obtained by vapor diffusion with polyethylene glycol 4,000 solutions, and five of the crystal forms have been characterized by X-ray diffraction. The data demonstrates that at least in the case of leaf lectin II, the tetrameric molecule contains at least one exact dyad axis. Several of the crystals appear suitable for high resolution structure analysis.     

Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia

The tissue distribution and the effects of starvation and streptozotocin-induced diabetes on insulin B chain-degrading neutral peptidase activity in the rat have been studied. The neutral peptidase activity in tissue extracts was determined by measuring the formation of trichloroacetic acid-soluble radioactivity from ¹²⁵I-labeled B chain of insulin in 0.1 m Tris buffer (pH 7.2). Inhibition by several different compounds (EDTA, dithiothreitol, and potassium phosphate) which are known to inhibit the purified enzyme and the effects of pH suggest that the B chain-degrading activity measured in each of 12 tissue extracts may be similar to the neutral peptidase recently purified from rat kidney (P. T. Varandani and L. A. Shroyer, 1977, Arch. Biochem. Biophys., 181, 82–93). Neutral peptidase activity was observed in all tissues examined and varied in the order kidney ? intestine > pancreas, testis > liver > thymus > heart, skeletal muscle, diaphragm > lung, spleen > fat. Neutral peptidase activity in kidney, liver, fat, and skeletal muscle from diabetic animals was significantly depressed when compared with the levels in these tissues from normal animals. Insulin treatment of diabetic animals raised the neutral peptidase activity in kidney, liver, and fat to levels equivalent to or even exceeding normal levels; however, activity in skeletal muscle persisted at depressed levels. Heart muscle neutral peptidase activity was not significantly affected in either diabetes or starvation. In the liver, starvation reduced the level of neutral peptidase activity while subsequent refeeding raised the activity to a level exceeding the control. Opposite effects were observed in kidney: starvation increased neutral peptidase activity while refeeding brought the activity back to normal levels. Only small decreases in neutral peptidase activity were observed in fat and skeletal muscle after 24 h starvation, but were not evident after 64 h starvation. The changes in neutral peptidase activity correlated well with the changes in glutathione-insulin transhydrogenase activity previously reported in liver and kidney.     

Isolation and characterization of a novel lectin with antifungal and antiproliferative activities from Sophora alopecuroides seeds

Sophora alopecuroides lectin (SAL), a novel lectin from the seeds of Sophora alopecuroides, was purified by ion-exchange chromatography on diethylaminoethyl (DEAE)- and carboxymethyl (CM)-Sepharose columns, followed by gel filtration on a Sephadex 75 10/300 GL column. SAL was found to be a monomer of 39916.3 Da, as determined by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC). The N-terminal 10-amino acid sequence of SAL, KPWALSFSFG, resembles those of other legume lectins. SAL exhibits hemagglutinating activity against rabbit erythrocytes at 11.9 μg/ml. Its hemagglutinating activity is stable in the pH range 7-11 and in the temperature range 30-90°C, and is stimulated by Mn(2+). The hemagglutinating activity of SAL is most potently inhibited by 50-mM d-galactose. SAL suppresses mycelial growth in Penicillium digitatum and Alternaria alternata; the IC(50) of the antifungal activity toward P. digitatum and A. alternata were found to be 3.125 and 3.338 μM, respectively. SAL suppresses the proliferation of human cervical cancer cells (HeLa) at an IC(50) of 6.25 μM (P< 0.05). But it has no inhibiting effect on bacteria. This is the first report of a lectin from seeds of S. alopecuroides.     

A novel mannose-specific and sugar specifically aggregatable lectin from the bark of the Japanese pagoda tree (Sophora japonica)

A new D-mannose/D-glucose-specific lectin (B-SJA-II) was isolated from the bark of the Japanese pagoda tree, Sophora japonica. B-SJA-II was separated from a well known D-galactose/N-acetyl-D-galactosamine-specific lectin (B-SJA-I) by affinity chromatography on lactamyl-Sepharose, then purified by affinity chromatography on maltamyl-Sepharose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, B-SJA-II gave four bands: subunit a-1 (Mr = 19,400), a-2 (Mr = 18,200), b-1 (Mr = 15,000), and b-2 (Mr = 13,200). Carbohydrate analysis and binding study with horseradish peroxidase-labeled lectins on the bands electroblotted onto polyvinylidene difluoride membrane showed that the three subunits other than b-2 have N-linked oligosaccharides typical of plant glycoproteins. The binding assay with horseradish peroxidase-glycoproteins revealed that all the subunits can bind sugar specifically with fetuin and asialofetuin. Furthermore, B-SJA-II aggregated to form precipitates in the absence of a specific sugar and became soluble upon addition of the specific sugar. The results indicate that each subunit has a sugar-binding site for the mannosyl core of N-linked oligosaccharide chains and recognizes each other sugar specifically to form aggregates. According to the N-terminal amino acid sequences obtained, the subunits are classified into two groups. The first group (a-1 and a-2) has an N-terminal sequence 50% identical with that of other S. japonica lectins (Hankins, C. N., Kindinger, J. I., and Shannon, L. M. (1988) Plant Physiol. 86, 67-70) and the amino acid sequence initiating at position 123 of concanavalin A (Cunningham, B. (1975) J. Biol. Chem. 250, 1503-1512), while the N-terminal sequence of the second group (b-1 and b-2) is homologous to that of concanavalin A, but completely different from that of the first group.     

Circular dichroism studies on the alpha-D-galactopyranosyl binding lectin isolated from the seeds of Bandeiraea simplicifolia.

The conformation of the alpha-D-galactopyranosyl binding lectin isolated from Bandeiraea simplicifolia seeds has been investigated over a broad range of pH in the presence of various solvents by circular dichroism (CD) spectroscopy in the region 200-300 nm. Analyses of the spectra obtained on the native protein show the lectin to contain a considerable proportion of beta structure (30-40%). The native conformation was found to be largely insensitive to changes in pH, but was influenced by sodium dodecyl sulfate or trifluoroethanol. Alterations in conformation in the presence of these agents were reflected in the CD spectra and show the presence of alpha helix under these conditions. These changes in conformation are accompanied by a loss in polysaccharide-precipitating activity. The protein is irreversibly denatured in 8 M urea. Neither removal of the intrinsic calcium ions from the protein nor addition of methyl alpha-D-galactopyranoside induces any appreciable change in the CD spectra of the protein although the former treatment abolishes the polysaccharide-precipitating capacity of the lectin. The conformational data obtained in the present study are compared with data available from conformational studies of other lectins and leads to the hypothesis that most lectins probably contain beta structure as the predominant conformational feature.     

国槐叶黄酮的抗氧化活性研究

本文采用ABTS清除体系、DPPH清除体系、H2O2/Fe2+/水杨酸检测体系、亚硝基清除体系、普鲁士蓝法、邻苯三酚自氧化法研究了国槐叶黄酮的抗氧化活性,并同芦丁、Vc进行了比较。结果表明,国槐叶黄酮对几种自由基均有不同的清除效果,在质量浓度为50μg/mL时,国槐叶黄酮对羟基自由基(·OH)、ABTS、DPPH、亚硝基自由基(NO2-)和超氧阴离子自由基(O2-·)的清除率为分别62.09%、47.70%、43.28%、22.11%、21.83%;与芦丁、Vc相比较,国槐叶黄酮清除ABTS、DPPH自由基的能力低于芦丁和Vc,而清除NO2-、.OH的能力高于芦丁和Vc,对O2-·的清除能力低于Vc而高于芦丁,其次国槐叶黄酮具有很好的还原力。由此说明,国槐叶提取物可以作为天然抗氧化剂,可以应用在医药食品领域。     

Purification and characterization of lectin II from Ulex europaeus seeds and an immunochemical study of its combining site.

The lectin II from Ulex europaeus seeds was purified by adsorption on insoluble polyleucyl hog A + H blood group substance and elution with 35% ethylene glycol, and by chromatography on ?-aminocaproyl-fucosyl-amine-agarose. In immunodiffusion against rabbit antiserum to the crude extract, the isolated lectin formed one line which fused with one of the five formed by crude extract. The purified lectin showed two bands on acrylamide electrophoresis under alkaline or acid conditions but only one band of molecular weight 23,000 if the electrophoresis was in the presence of 0.1% sodium dodecyl sulfate at pH 8.8. The agglutinating and precipitating abilities are abolished by EDTA and can be restored by bivalent cations. The purified lectin precipitated to different extents with blood group A₁, A₂, B, HLe^b, Le^a, and I precursor substances and with acid- or Smith-degraded substances. Inhibition of precipitation indicated that the lectin site was unusual in that it interacted most strongly with the h-specific oligosaccharide

and with 2′-fucosyllactose, followed by β1 → 4 linked oligomers of dGlcNAc. Molecular models showed that all these inhibitors have a similarity in three-dimensional structures that could account for their activities.     

苦豆子豆籽油化学成分研究

采用气相色谱/质谱联用,首次对苦豆子豆籽油化学成分进行了研究,检出了43种化学成分鉴定了25种成分。苦豆子豆籽油的主要成分为生物碱、有机酸及甾体类化合物。     

Immunochemical studies on the role of the Golgi complex in protein-body formation in rice seeds

Antibodies raised against purified glutelins and prolamines were employed as probes to study the cellular routes by which these proteins are deposited into protein bodies of rice (Oryza sativa L.) endosperm. Three morphologically distinct protein bodies, large spherical, small spherical, and irregularly-shaped, were observed, in agreement with existing reports. Immunocytochemical studies showed the presence of glutelins in the irregularly-shaped protein bodies while the prolamines were found in both the large and small spherical protein bodies. Both the large and small spherical protein bodies, distinguishable by electron density and gold-labeling patterns, appear to be formed by direct deposition of the newly formed proteins into the lumen of the rough endoplasmic reticulum (ER). In contrast, glutelin protein bodies are formed via the Golgi apparatus. Small electron-lucent vesicles are often found at one side of the Golgi. Electron-dense vesicles, whose contents are labeled by glutelin antibody-gold particles, are commonly observed at the distal side of the Golgi apparatus and fuse to form the irregularly shaped protein bodies in endosperm cells. These observations indicate that the transport of rice glutelins from their site of synthesis, the ER, to the site of deposition, the protein bodies, is mediated by the Golgi apparatus.Abbreviations BSA bovine serum albumin - Da dalton - DAF days after flowering - ER endoplasmic reticulum - GL irregularly shaped - L large spherical - S small spherical (protein bodies) - PBS phosphate-buffered saline - PTA phosphotungstic acid