Confocal microscopy confirms the presumed cytoplasmic/nuclear location of plant, fish and fungal orthologs of the Galanthus nivalis agglutinin.
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Animals↗
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[Isolation of new flavonol oligosaccharie from the petals of Leucojum vernum L. and Galanthus nivais L].
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Flavonoids↗
[Galanthus Krasnovii A. Chochr.--a source for obtaining galanthamine].
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Alkaloids↗
Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities.
The thermodynamics of binding of a system of plant lectins specific for the oligosaccharide methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside have been studied calorimetrically. This system of lectins consists of concanavalin A, the lectin isolated from Dioclea grandiflora, and the lectin from Galanthus nivalis. The group thus contains lectins with similar structures and similar binding properties as well as lectins with different structures but similar binding properties. Concanavalin A and the lectin from Dioclea are highly homologous, while the lectin from Galanthus nivalis shares no sequence homology with either of the legume lectins, although it also binds the mannose trisaccharide tightly. Calorimetric data for oligosaccharide binding to both of the legume lectins suggests that the total binding site comprises a single high-affinity site and an additional extended site. The pattern of binding for the lectin from Galanthus is significantly different. Binding studies with the same saccharides indicate that the lectin has binding sites designed specifically for the 1-->3 and 1-->6 arms of the mannose trisaccharide that are unable to accommodate other saccharides. Enthalpy--entropy compensation was observed for several saccharides as a function of lectin structure. Contributions of solvation effects to the enthalpy of binding and the configurational entropies were determined experimentally. For those systems studied here, solute-solute attractive interactions and configurational entropies were the greatest contributors to enthalpy-entropy compensation. Our studies clearly demonstrate that, despite their common affinity for the mannose trisaccharide, the three lectins bind oligosaccharides very differently.
Calorimetry↗
Comparison of serological tests for the diagnosis of feline immunodeficiency virus infection of cats.
A new enzyme-linked-immunosorbent assay (ELISA) for the detection of antibodies to feline immunodeficiency virus was compared with previously described ELISAs. Serum samples from 184 infected or uninfected cats were tested using a whole virus lysate kit and ELISAs based on recognition of one of two synthetic peptides (P237 and P253) localized in the transmembrane domain of the viral envelope. The whole virus lysate commercial kit led to the detection of 6% false positive and 4.3% false negative sera. The ELISA based on peptide P253 gave no false positive result and failed to detect only one serum that was subsequently shown to be positive by radio-immunoprecipitation assay. A sandwich-ELISA test using Galanthus nivalis agglutinin, a lectin that specifically binds terminal mannose groups of the envelope proteins was used as a confirmatory test for equivocal results with peptide ELISA and gave similar results. This study indicates that recognition of P253 could serve as a sensitive and specific test for the diagnosis of seropositivity to feline immunodeficiency virus, and moreover that the Galanthus nivalis ELISA could be useful in equivocal cases as a confirmatory test.
Amino Acid Sequence↗
Phagocytosis stimulates alternative glycosylation of macrosialin (mouse CD68), a macrophage-specific endosomal protein.
Macrosialin (mouse CD68), a macrophage-specific member of the lysosomal-associated membrane protein family, displays N-linked glycosylation and a heavily sialylated, mucin-like domain. We show that phagocytosis of zymosan by inflammatory peritoneal macrophages potently alters glycan processing of macrosialin in vitro. The phagocytic glycoform is not induced by other forms of endocytosis and depends on particle internalization. Zymosan uptake does not influence macrosialin protein synthesis, but increases the specific incorporation of D-[2-3H]mannose, D-[6-3H]galactose, N-acetyl-D-[1-3H]glucosamine and L-[5,6-3H]fucose by 2-15-fold. The phagocytic glycoform displays increased binding of agglutinins from peanut, Amaranthus caudatus and Galanthus nivalis, whereas binding of the sialic-acid-specific Maakia amurensis agglutinin is slightly reduced. Digestion by N-Glycanase abolishes the incorporation of [3H]mannose label and Galanthus nivalis agglutinin binding activity, but preserves the incorporation of galactose and N-acetylglucosamine and specific lectin binding. We also show that phagocytosis increases the complexity and length of O-linked chains. The data presented highlight the importance of differential glycosylation in the biology of macrosialin, phagosomes and macrophages in general.
Animals↗
CBP70, a glycosylated nuclear lectin.
Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by beta-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-l agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal beta Gal residues, Gal beta 1-3 GalNAc, Man alpha 1-3 Man, sialic acid alpha 2-6 linked to Gal or GalNAc; and sialic acid alpha 2-3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and alpha-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting.
Agglutinins↗
Characterization of endopolygalacturonase (EC 3.2.1.15) from Aspergillus niger as glycoprotein by electrophoretic methods and lectin affino-blotting.
Chromatographically purified endopolygalacturonase (PG) from Aspergillus niger was deglycosylated with N-glycosidase F (PNGase F) and characterized by means of sodium dodecyl sulfate (SDS)-electrophoresis, polyacrylamide gel electrophoresis (PAGE) without denaturing agents, isoelectric focusing (IEF) and lectin affino-blotting. The results show that PG, which is apparently homogeneous in SDS-PAGE but heterogeneous in IEF and PAGE, consists of at least two polypeptide chains with different glycosylation patterns. The component with the higher electrophoretic mobility is deglycosylated with PNGase F and reacts with concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA), indicating a "high mannose" or "hybrid"-type of glycoprotein (GP). The other component may contain O-glycosidically linked mannose, N-acetylglucosamine or glucose.
Aspergillus niger↗
Studies on the carbohydrate moieties of the timothy grass pollen allergen Phl p I.
Timothy grass pollen was investigated in order to determine the carbohydrate moieties of its major grass group I (Phl p I) and to study its impact on allergenicity. Based on computer calculations one N-glycosylation site was deduced from the cDNA data of Phl p I. After two-dimensional polyacrylamide gel electrophoresis, followed by blotting of pollen extract and by use of the monoclonal antibody IG 12 we identified at least six isoallergens of Phl p I with the main spots at a molecular mass of 35-37 kDa and a pI range of 6.5-7.3. Deglycosylation by trifluoromethanesulfonic acid resulted in a decrease of about 2 kDa. Treatment with N-glycosidase A resulted in a partial deglycosylation, while N-glycosidase F and O-glycosidase had no effect. Ten lectins were investigated for their binding to Phl p I components: Aleuria aurantia agglutinin showed strong reactivity (indicating fucose residues), while Galanthus nivalis agglutinin (indicating mannose residues) and concanavalin A (indicating mannose, glucose or N-acetylglucosamine residues) showed weak binding. By neutral sugar analysis we determined similar contents of the monosaccharides in the isoallergens. In order to study the influence of the carbohydrate structures of Phl p I on IgE reactivity we tested some patient sera for their reactivity with intact and deglycosylated Phl p I. Even though most of the IgE antibodies bind at the protein core, we detected one serum that recognized carbohydrate moieties on the Phl p I.
Allergens↗
Glycosylation of beta-1 integrins in B16-F10 mouse melanoma cells as determinant of differential binding and acquisition of biological activity.
Studying B16-F10 cells we could identify beta-1 integrins as laminin, fibronectin and collagen receptors. Gradient ionic strength elution analysis of affinity chromatography showed differential interactions between laminin-binding beta-1 integrins (two beta-1 polypeptides of 105 and 120 kDa) and fibronectin and collagen-binding beta-1 integrins (elution of one major beta-1 polypeptide of 120 kDa) and their respective ligands. To evaluate this diversity we submitted B16-F10 extracts to IEF and SDS-PAGE and found that one beta-1 integrin formed acidic and larger isoforms, while another formed basic and smaller isoforms. To study this difference we also submitted material eluted from WGA-Sepharose columns to IEF but now only the acidic beta-1 isoform was found. Extracts of B16-F10 treated with neuraminidase showed only the basic beta-1 isoform, suggesting that terminal sialic acid residues may be responsible for this acidic pattern, an interpretation supported by the fact that MAA (Maackia ammurensis agglutinin) reacts only with the acidic isoform. Differential glycosylation of beta-1 integrin isoforms in B16-F10 was also demonstrated since the smaller laminin-binding beta-1 integrin isoform reacted only with GNA (Galanthus nivalis agglutinin), whereas the mature larger form reacted with DSA (Datura stramonium agglutinin) and MAA; thus this heterogeneity of beta-1 chains is essentially due to variable glycosylation. Autoradiography and immunoblotting analysis of material separated by 2-dimensional electrophoresis show that only the processed forms of beta-1 integrins are expressed at the cell surface.
Animals↗
Vitelline coat of Unio elongatulus: III. Glycan chain analysis of the 220- and 180-kD components by means of lectins.
Lectins of different binding specificity were used to analyze the oligosaccharide chains of the 220- and 180-kD proteins of the Unio elongatulus egg vitelline coat (vc). The lectins ConA and RCA1 reacted with both glycoproteins, and four other lectins reacted with one or other vc components. The lectin from Galanthus nivalis, which recognizes terminal mannose residues of N-linked high mannose type oligosaccharide chains, bound specifically to the 180-kD protein. Binding sites for this lectin were found throughout the vc of the differentiating oocyte and the mature egg. Lectins specific for the O-linked oligosaccharide chains, such as AIA and PNA, reacted only with the 220-kD protein species. Binding sites for these lectins were found only in the crater region. The presence of fucosyl residues on the glycan chains was investigated with lectins from Lotus tetragonolobus and Aleuria aurantia. The latter was positive on both glycoproteins, whereas LTA was only positive to the 220-kD species. The binding sites of both these lectins were in the same areas as those of PNA and AIA. These results suggest that while the 180-kD protein is part of the entire vc structure, the 220-kD protein is prevalently accumulated in the crater region. Since this is where sperm recognition and interaction take place, it has been suggested the 220-kD protein acts as a ligand molecule in the sperm-egg interaction.
Animals↗
Mannose-specific lectins bind alpha-2-macroglobulin and an unknown protein from human plasma.
GNA, the mannose-specific lectin from Galanthus nivalis was confirmed to bind alpha-2-macroglobulin (A2M) but another protein was copurified with A2M from total human plasma. A total of 23 other lectins with diverse specificities were tested for reaction with human A2M and with three other members of the A2M family. NPA, a mannose-specific lectin isolated from Narcissus pseudonarcissus bulbs, and RSA, the Rhizoctonia solani agglutinin, were selected for further testing. For isolation of A2M, immobilized NPA was superior to GNA because its binding capacity was an order of magnitude higher. The specificity of these lectins must be very similar however, because the same unknown plasma protein was also bound by NPA. A2M and the unknown protein must share a unique mannose carbohydrate structure not present in any other human plasma protein. The copurified protein subunit size of 185 kDa is very similar to that of A2M, but the native molecular mass of 350 kDa indicated a noncovalent homodimer structure. Together with the acid isoelectric point this is not typical for any known plasma protein nor for any unidentified spot on the two-dimensional map of human plasma proteins. No immunological reaction with available antisera was evident. A specific antiserum raised to the unknown protein demonstrated its presence in all human plasma samples examined. The N-terminal residue was blocked, whereas internal protein sequences obtained after CNBr fragmentation and proteolysis were not homologous to any known protein sequence. These data demonstrate that this protein is unknown and not a proteinase inhibitor of the A2M family.
Amino Acid Sequence↗
Secretion of two beta-fructofuranosidases by Aspergillus niger growing in sucrose.
Aspergillus niger is induced to secrete two invertases, named SUC 1 and SUC 2, when grown on a minimal medium containing sucrose. Although, both have been classified as beta-D-fructofuranoside fructohydrolases, SUC 2 also possesses inulin hydrolytic activity (sucrose/inulin activity ratio of 4). These activities have been separated from each other and almost completely purified by anion-exchange, lectin affinity chromatography, and chromatofocusing. SUC 1 appeared as a single glycoprotein band on PAGE and SDS-PAGE corresponding in size to 250 and 125 kDa, respectively, compared with a much broader band (suggesting greater glycan heterogeneity) of 210-240 and 90-120 kDa for SUC 2. Therefore, both may be dimers, in their natural conformation. The glycan part of both contained the same monosaccharides: mannose, glucose, galactose, and N-acetylglucosamine; however, SUC 1 had approximately 10-fold more mannose and this was utilized to separate it from SUC 2 by Galanthus nivalis lectin affinity. Both the apparent Km values and the pH activity curves were different; SUC 1 did not show normal Michaelis-Menten kinetics to sucrose and apparent Michaelis constants of 30 and 160 mM were obtained. Activity was observed over a large range of pH 4.5-9 with a maximum at pH 6. In contrast, SUC 2 exhibited a Km of 40 and 1.7 mM to sucrose and inulin, respectively, with a pH optimum of 5.0 for both. Treatment with endo-beta-N-acetylglucosaminidase suggests that in both SUC 1 and SUC 2 some of the glycan present was N-linked glycan but that the differences in enzyme activities were not due to the N-linked moiety.
Aspergillus niger↗
Maackia amurensis agglutinin discriminates between normal and chronic leukemic human lymphocytes.
Altered glycosylations of cell surface glycoproteins often accompany malignant transformation and lectins are useful for probing these alterations. Lymphocytes exhibit characteristic surface glycoproteins which serve as markers of cell status and development. The present work was undertaken to compare, on blots, the binding characteristics of membranes isolated from normal peripheral blood lymphocytes and chronic lymphatic leukemia cells to five different lectins, from Datura stramonium, Maackia amurensis, Sambucus nigra, Galanthus nivalis and Peanut. The Maackia amurensis lectin interacted with the normal lymphocytes but showed no binding to malignant cells. Hence, we suggest the Maackia lectin may be used to differentiate normal from leukemic cells.
Cell Membrane↗
T cell-B cell interaction: autoreactive T cells recognize B cells through a terminal mannose-containing superantigen-like glycoprotein.
Rat autoreactive T cells (ATs) recognize a membrane component(s) on syngeneic B cells in association with class II MHC antigens resulting in proliferation of ATs as well activation and differentiation of B cells. Results presented herein indicate that ATs recognize a stimulating antigen(s) SA, in association with class II MHC antigens, on the B cell surface. Our studies using inhibitors of carbohydrate and protein synthesis suggest that SA is a glycoprotein(s) with a high turnover rate but is not an immunoglobulin. Treatment of B cells with mannosidase abrogates their ability to stimulate AT proliferation. Furthermore, pretreatment of B cells with GNA (a lectin from Galanthus nivalis that reacts with free terminal-mannose residues on glycoconjugates) also inhibits their ability to stimulate ATs. However, these treatments do not affect the competence of B cells to stimulate an allogeneic MLR or present a conventional antigen to T cells. The frequency of CD4+ T cells proliferating in response to syngeneic B cells is very high (0.2-0.5%) and is in line with frequencies seen in "superantigen"-type responses. Moreover, T cell receptors expressed on ATs use mainly V beta 6, V beta 11, and V beta 8 regions. Based on these data, SA appears to be a fast turnover, terminal mannose-containing, superantigen-like glycoprotein on the B cell surface.
Animals↗
Specific glycoconjugates are present at the oolemma of the fertilization site in the egg of Discoglossus pictus (Anurans) and bind spermatozoa in an in vitro assay.
In the egg of the anuran Discoglossus pictus, the site of fertilization is restricted to the central portion of an animal hemisphere indentation (the dimple). Previous studies showed that the acrosome reaction of D. pictus sperm is triggered in the jelly, and yet sperm arrive at the dimple surface with the plasma membrane at an early stage of vesiculation. Reactivity of the dimple surface with specific lectins suggests that fucose might be utilized as a marker of glycoproteins located at the dimple surface. In this paper, proteins of the egg surface were labeled with the membrane impermeable sulfo-NHS-biotin. Four main bands of 200, 230, 260, and 270 kDa labeled only at the dimple surface, although they were detected in the cortex of the whole egg. The 270-kDa band reacted with Galanthus nivalis agglutinin only in the cortex of the dimple, suggesting that this band is differently glycosylated according to its localization. The alpha-l-fucose-specific lectin Ulex europaeus agglutinin I was utilized both in lectin blotting and in affinity chromatography and cross-reacted with the 200- and 270/260-kDa bands. Furthermore, two polypeptides were obtained by exposure of intact eggs to lysylendoproteinase C. They were also reactive to Ulex europaeus agglutinin I. The 200- and 270/260-kDa bands were eluted from the acrylamide gels and adsorbed to polystyrene beads. An assay for sperm binding to 200-kDa glycoprotein-bound beads was developed. Sperm stuck to the beads before but not after Ca-ionophore treatment. When the beads were coated with the 270/260-kDa glycoproteins, binding occurred after ionophore treatment. In these assays, the 200- and 270/260-kDa glycoproteins competitively inhibited sperm binding to the beads coated with the corresponding glycoprotein. These results indicate that the assayed glycoproteins, located either in the glycocalyx or in the plasma membrane of the fertilization site, are involved in sperm binding.
Animals↗
Insights into carbohydrate recognition by Narcissus pseudonarcissus lectin: the crystal structure at 2 A resolution in complex with alpha1-3 mannobiose.
Carbohydrate recognition by monocot mannose-binding lectins was studied via the crystal structure determination of daffodil (Narcissus pseudonarcissus) lectin. The lectin was extracted from daffodil bulbs, and crystallised in the presence of alpha-1,3 mannobiose. Molecular replacement methods were used to solve the structure using the partially refined model of Hippeastrum hybrid agglutinin as a search model. The structure was refined at 2.0 A resolution to a final R -factor of 18.7 %, and Rfreeof 26.7 %. The main feature of the daffodil lectin structure is the presence of three fully occupied binding pockets per monomer, arranged around the faces of a triangular beta-prism motif. The pockets have identical topology, and can bind mono-, di- or oligosaccharides. Strand exchange forms tightly bound dimers, and higher aggregation states are achieved through hydrophobic patches on the surface, completing a tetramer with internal 222-symmetry. There are therefore 12 fully occupied binding pockets per tetrameric cluster. The tetramer persists in solution, as shown with small-angle X-ray solution scattering. Extensive sideways and out-of-plane interactions between tetramers, some mediated via the ligand, make up the bulk of the lattice contacts.A fourth binding site was also observed. This is unique and has not been observed in similar structures. The site is only partially occupied by a ligand molecule due to the much lower binding affinity. A comparison with the Galanthus nivalis agglutinin/mannopentaose complex suggests an involvement of this site in the recognition mechanism for naturally occurring glycans.
Amino Acid Sequence↗
A corm-specific gene encodes tarin, a major globulin of taro (Colocasia esculenta L. Schott).
A gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (G1, ca. 28 kDa) was isolated from a lambda Charon 35 library, using a cDNA derived from a highly abundant corm-specific mRNA, as probe. The gene, named tar1, and the corresponding cDNA were characterized and compared. No introns were found. The major transcription start site was determined by primer extension analysis. The gene has an open reading frame (ORF) of 765 bp, and the deduced amino acid sequence indicated a precursor polypeptide of 255 residues that is post-translationally processed into two subunits of about 12.5 kDa each. The deduced protein is 45% homologous to curculin, a sweet-tasting protein found in the fruit pulp of Curculigo latifolia and 40% homologous to a mannose-binding lectin from Galanthus nivalis. Significant similarity was also found at the nucleic acid sequence level with genes encoding lectins from plant species of the Amaryllidaceae and Lilliaceae families.
Amino Acid Sequence↗