Lectin Ligands Research Articles

Sweet and shapely: Ligands for animal lectins

Sweet and shapely: Ligands for animal lectins

Fragmentation and dispersal of Golgi proteins and redistribution of glycoproteins and glycolipids processed through the Golgi apparatus after infection with herpes simplex virus 1.

In Vero monkey cells and HEp-2 human epidermoid carcinoma cells infected with herpes simplex virus 1 the proteins beta-COP, galactosyltransferase, and alpha-mannosidase II associated with the Golgi apparatus appear to be associated with numerous smaller structures dispersed throughout the cytoplasm. Concomitantly, the intracytoplasmic ligands of lectins normally associated wholly (Helix pomatia or Ricinus communis agglutinin) or in part (wheat germ agglutinin) with the Golgi apparatus increased in amount and became dispersed. This phenomenon was seen in some of the baby hamster kidney cells analyzed but not in others and not in the human 143TK- cells. The fragmentation and dispersal of the Golgi apparatus was a late event in the reproductive cycle coinciding with virion assembly, processing of viral glycoproteins, and exocytosis from infected cells. The fragmentation of the Golgi apparatus is morphologically different from that seen with brefeldin A and may reflect disequilibration between the anterograde and retrograde Golgi transport caused by the huge influx of viral glycoproteins contained in virions and membranes flowing through the exocytic pathway.

Lesion-induced re-expression of neonatal recognition molecules in adult rat cerebellum

It has been previously shown that sectioning of parallel fibers in the cerebellar molecular layer of adult rats gave rise to rapid reinnervation of the target cells i.e., Purkinje cells. This paper reports that such a reinnervation is accompanied by reexpression (partial and total) of two developmentally regulated complementary molecules. These are an endogenous mannose-binding lectin, called R1, which reappears at the surface of the dendrites of Purkinje cells, and an endogenous glycoprotein ligand of R1, the 31 kDa glycoprotein, which seems to be neosynthetized and transported to the surface of parallel fibers. In this system, embryonic N-CAM is not reexpressed in neurons but reappears in reactive astrocytes in the vicinity of the lesion. The reexpression of recognition molecules (lectin and glycoprotein ligand) involved in normal synaptogenesis, may constitute the molecular basis for repair of nervous circuits in the adult as well.

Human brain lectin: a soluble lectin that binds actin.

A biotinylated probe was used for detection of endogenous ligands of a human brain lectin on blotted human brain soluble proteins. Of the various proteins from brain extract resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five reacted with the biotinylated probe. After elimination of saccharidic moieties by periodic treatment of the same extract, a single band with Mr approximately 43,000 was recognized by the lectin. This band was identified as actin using an anti-actin antibody. These results were confirmed by binding of biotinylated lectin to purified actin.

Regulation of a beta-galactoside-binding lectin and potential ligands during postnatal maturation of rat brain.

Variations in expression and activity of a beta-galactoside-specific lectin were quantified during postnatal maturation of the rat brain. Lectin expression, estimated from enzymatic immunoassay data, and lectin activity, evaluated by rabbit erythrocyte agglutination, are higher in the brains of young animals (5 days after birth) than in older ones (2 months of postnatal life). Concurrently, modifications in glycosylation during cellular differentiation were detected by affinoblotting using a biotinylated derivative of a beta-galactoside-specific brain lectin. This study shows that membrane and cytosolic glycoconjugates of the rat brain bear appropriate beta-galactoside moieties which may be recognized by an endogenous lectin expressed in the tissue itself in relation to brain maturation. The observed variations in the expression of lectin and complementary glycoconjugates can be correlated with significant events of rat brain development.

Hepatocyte adhesion to carbohydrate-derivatized surfaces. II. Regulation of cytoskeletal organization and cell morphology.

Rat hepatic lectins mediate adhesion of isolated rat hepatocytes to synthetic surfaces derivatized with galactosides. Initial weak adhesion is followed by rapid adhesion strengthening. After hepatocytes contact galactose-derivatized gels, the hepatic lectins move rapidly into an inaccessible patch at the adhesive surface (Weisz, O. A., and R. L. Schnaar. 1991. J. Cell Biol. 115:485-493). Hepatic lectin patching, which occurs both at 37 degrees C and 4 degrees C, is not responsible for adhesion strengthening, which does not occur at 4 degrees C. Of various cytoskeletal and metabolic perturbants tested, only a combination of hyperosmotic medium, colchicine, and cytochalasin caused a marked (72%) reduction of adhesion strengthening (without reducing weak cell adhesion). Clathrin and actin were readily detected in the adhesive patch by immunofluorescence microscopy. Rat hepatocytes also adhered avidly to surfaces derivatized with asialofetuin, a high-affinity ligand for the rat hepatic lectins. However, hepatic lectin molecules did not migrate into a patch on the asialofetuin-derivatized surface, suggesting that hepatic lectin-asialofetuin binding may have resulted in the rapid formation of a ring of essentially irreversibly adherent receptors that prevented diffusion of additional lectin molecules into the contact site. The cells were unable to increase their adhesive contact area by flattening onto the derivatized surface. Treatment of cells with cytochalasin, however, did result in an increase in the size of the contact area. Cells adhering to surfaces derivatized with an adhesion-promoting peptide (containing an arg-gly-asp sequence) had larger contact areas than those adhering to galactoside-derivatized surfaces. A model is proposed in which carbohydrate-mediated adhesion causes specific reorganization of cytoskeletal components, leading to strengthened adhesion and a characteristic spherical cell morphology.

Mac-2-binding glycoproteins. Putative ligands for a cytosolic beta-galactoside lectin.

Mac-2, a galactose-binding lectin secretion by activated macrophages, is the major non-integrin laminin-binding protein in these cells. Mac-2 is also expressed by epithelial cells in the intestine and kidney. We wished to identify intestinal glycoproteins other than laminin that have a high affinity for Mac-2 and that could be considered as candidate ligands or partners for this lectin in intestinal epithelium. Certain lines of human colon adenocarcinoma cells produce two Mac-2-binding glycoproteins (M2BP-1 and M2BP-2) that were identified by their avid association with Mac-2 following detergent lysis and immunoprecipitation. These glycoproteins do not share a common epitope with Mac-2, and the interaction between Mac-2 and these proteins is mediated through the carbohydrate-binding domain of Mac-2 and sugar moieties on M2BP-1 and M2BP-2. M2BP-1 (98 kDa) and M2BP-2 (70 kDa) were purified by immunoaffinity chromatography and were specifically eluted with either galactose or lactose. Peptide maps revealed that M2BP-1 and M2BP-2 are structurally related. M2BP-1 is secreted and could conceivably associate with Mac-2 extracellularly. N-terminal sequence analysis of M2BP-2 suggests that these glycoproteins represent a unique subset of candidate ligands for this mammalian beta-galactoside lectin.

Malignant transformation in hepatocytes is associated with the general increase of glycoprotein ligands specifically binding to the endogenous lectin CSL

Several hepatoma cell lines and hepatic ascite tumour cells were studied for the presence of glycoprotein ligands of an endogenous lectin, the “Cerebellar Soluble Lectin” (CSL). This lectin is also present in hepatocytes in vivo and in vitro and can be detected biochemically and immunologically. In transformed cells, the level of CSL glycoprotein ligands is increased 50-fold as compared to the control cells. Such an increase is not observed for the ligands of the plant lectin, concanavalin A, which is, as CSL, a d-mannose-binding lectin. These results indicated that the changes in glycans during malignant transformation, in these cells, is specifically important for minor glycans binding to CSL.

Tumor cell surface beta 1-4-linked galactose binds to lectin(s) on microvascular endothelial cells and contributes to organ colonization.

Cell surface carbohydrate structures acting as ligands for tissue specific mammalian lectins have been implicated in cell-cell interactions during embryogenesis, lymphocyte homing, and tumor cell metastasis. In this report, we provide evidence that beta 1-4 linked galactose (Gal) residues in N-linked oligosaccharides on the surface of blood born tumor cells serve as a ligand for binding to microvascular endothelial cells. D36W25, a class 1 glycosylation mutant of the MDAY-D2 lymphoreticular tumor cell line, lacks sialic acid and Gal in cellular glycans due to a defect in the Golgi UDP-Gal transporter. Using UDP-Gal and bovine galactosyltransferase in vitro, beta 1-4 Gal was restored to the surface of the cells and 70% of the galactosylated glycans persisted for 8 h in vitro at 37 degrees C. Compared to mock-treated D36W25 cells, galactosylated D36W25 cells showed an 80% increase in binding to microvascular endothelial cell monolayers in vitro. The enhanced binding of galactosylated D36W25 cells to endothelial cell was inhibited by the addition of lactosamine-conjugated albumin to the assay. Consistent with these observations, swainsonine and castinospermine, two inhibitors of N-linked processing that result in loss of lactosamine antennae inhibited the binding of wild-type MDAY-D2 cells to endothelial cells in vitro. Injection of radiolabeled tumor cells into the circulation of syngeneic mice, showed that galactosylation of D36W25 cells resulted in 2-3 more tumor cells retained in the lungs and livers. In addition, galactosylation of D36W25 cells increased by 30-fold the number of visible liver metastases on inspection 4 wk after tumor cell injection. These results suggest that beta 1-4Gal-binding lectins on microvascular endothelial cells can contribute to retention and secondary tumor formation of blood born tumor cells. With the increasing availability of purified glycosyltransferases, reconstruction of a variety of carbohydrate sequences on the surface of class 1 mutants provides a controlled means of studying carbohydrate-lectin interactions on viable cells.

Spatial differences of endogenous lectin expression within the cellular organization of the human heart: A glycohistochemical, immunohistochemical, and glycobiochemical study

Protein-carbohydrate recognition may be involved in an array of molecular interactions on the cellular and subcellular levels. To gain insight into the role of proteins in this type of interaction, surgically removed specimens of human endomyocardial tissue were processed for histochemical and biochemical analysis. The inherent capacity of these sections to bind individual sugar moieties, which are constituents of the carbohydrate part of cellular glycoconjugates, was assessed using a panel of biotinylated neoglycoproteins according to a standardized procedure. Together with appropriate controls, it primarily allowed localization of endogenous lectins. Differences in lectin expression were observed between layers of endocardial tissue, myocardial cell constituents, connective-tissue elements, and vascular structures. The endocardium proved to be positive with beta-galactoside-bearing probes; with neoglycoproteins carrying beta-xylosides, alpha-fucosides, and galactose-6-phosphate moieties; and with probes containing a carboxyl group within the carbohydrate structure, namely sialic acid and glucuronic acid. In contrast, only fucose-and maltose-specific receptors were apparent in the elastic layers of the endocardium. Aside from ascertaining the specificity of the protein-carbohydrate interaction by controls, i.e., lack of binding of the probe in the presence of the unlabelled neoglycoprotein and lack of binding of the labelled sugar-free carrier protein, respective sugar receptors were isolated from heart extracts by using histochemically effective carbohydrates as immobilized affinity ligand. Moreover, affinity chromatography using immobilized lactose as affinity ligand as well as the use of polyclonal antibodies against the predominant beta-galactoside-specific lectin of heart demonstrated that the lactose-specific neoglycoprotein binding was due to this lectin. Remarkably, the labelled endogenous lectin, preferred to plant lectins for detecting ligands of the endogenous lectin, localized ligands in tissue parts where the lectin itself was detected glycohistochemically as well as immunohistologically. This demonstration of receptor-ligand presence in the same system is a further step toward functional assignment of the recorded protein-carbohydrate interaction. Overall, the observed patterns of lectin expression may serve as a guideline to elucidate the precise physiological relevance of lectins and to analyze pathological conditions comparatively.

Hodgkin's cell lectin, a lymphocyte adhesion molecule and mitogen.

We have previously shown a novel galactose/N-acetylgalactosamine specific lectin activity (Hodgkin's disease (HD) lectin) on the surface of cultured HD cells (lines L428, its variants, and line L540) to mediate lymphocyte adhesion. We here demonstrate that both surface membrane-bound and secreted HD lectin activities participate in the activation of agglutinated lymphocytes. Among known adhesion molecules expressed by the HD cells, only the intercellular adhesion molecule-1 (ICAM-1) contributed to this activation as an alternative PBL binding site. As yet we have not identified the cellular ligand(s) for the HD lectin on the lymphocyte surface. Pretreatment of lymphocytes with mAb to the accessory molecules CD2, CD3, CD4, CD8, CD11b, or CD11c did not interfere with their response to HD cells. mAb to CD11a (LFA-1), the alleged ligand of ICAM-1, inhibited the ICAM-1 but not the HD lectin-mediated lymphocyte stimulation. Although lymphocyte binding could proceed via either pathway, lymphocyte activation always depended upon factors secreted by the HD cells, one of which we identified as a soluble form of the HD lectin based on its shared properties with the membrane-bound form including immunologic cross-recognition and carbohydrate-binding specificity. Although HD cell-conditioned medium alone stimulated lymphocytes, HD cell plasma membranes could compensate for low concentrations of this medium. In addition, resting lymphocytes, normally unresponsive, were triggered into DNA synthesis by growth medium when cocultured with HD cell membranes. The unique functions of the surface-expressed HD lectin and its soluble counterpart as lymphocyte adhesion molecule and mitogen might be physiologically relevant to the severe immunodeficiencies occurring in patients with HD.

ChemInform Abstract: Tumor Lectinology: At the Intersection of Carbohydrate Chemistry, Biochemistry, Cell Biology, and Oncology

The scientific description of lectins, a class of sugar-binding proteins distinct from both sugar-specific enzymes and antibodies, began nearly 100 years ago. The natural ligands of lectins include the sugar moieties of cell glycoproteins, glycolipids, and proteoglycans. Lectins can selectively recognize these potential information-storing or -transferring structural elements and so contribute to regulating biological processes. Plant lectins are now a well-established means of characterizing and localizing glycoconjugate-bound sugar determinants. However, work on endogenous lectins is still confronted by many open questions, whose answers require detailed experiments using a suitable model system. Tumors offer an obvious model, particularly since they allow the possible diagnostic and therapeutic uses of lectins to be explored directly. Tumor lectinology employs a broad range of methods, from the chemical synthesis of the sugar components of neoglycoproteins, and their use to seek out cellular sugar receptors with histochemical and cell-biological techniques, to the biochemical and immunological characterization of the lectins expressed by tumor cells and the use of homology analysis to find further functional domains. The outlook for clinical applications depends on the harmonious integration of these disparate disciplines in tumor lectinology.

Tumor Lectinology: At the Intersection of Carbohydrate Chemistry, Biochemistry, Cell Biology, and Oncology

AbstractThe scientific description of lectins, a class of sugar‐binding proteins distinct from both sugar‐specific enzymes and antibodies, began nearly 100 years ago. The natural ligands of lectins include the sugar moieties of cell glycoproteins, glycolipids, and proteoglycans. Lectins can selectively recognize these potential information‐storing or ‐transferring structural elements and so contribute to regulating biological processes. Plant lectins are now a well‐established means of characterizing and localizing glycoconjugate‐bound sugar determinants. However, work on endogenous lectins is still confronted by many open questions, whose answers require detailed experiments using a suitable model system. Tumors offer an obvious model, particularly since they allow the possible diagnostic and therapeutic uses of lectins to be explored directly. Tumor lectinology employs a broad range of methods, from the chemical synthesis of the sugar components of neoglycoproteins, and their use to seek out cellular sugar receptors with histochemical and cell‐biological techniques, to the biochemical and immunological characterization of the lectins expressed by tumor cells and the use of homology analysis to find further functional domains. The outlook for clinical applications depends on the harmonious integration of these disparate disciplines in tumor lectinology.

Isolation and characterization of endogenous ligands for liver mannan-binding protein

Endogenous ligands for the hepatic lectin which is specific for mannose and N-acetylglucosamine (mannan-binding protein, MBP) were isolated from rat liver rough microsomes and primary cultured hepatocytes by affinity chromatography on an immobilized MBP column. Western blotting using specific antisera revealed that serum glycoproteins, α 1-macroglobulin, α 1-antitrypsin, and α 1-acid glycoprotein, and a lysosomal enzyme, β-glucuronidase were the major constituents of the endogenous ligands. These endogenous ligands consisted of high mannose-type oligosaccharides of Man 9GlcNAc 2 and Man 8GlcNAc 2, and had rapid turnover rates with an average half-life of 45 min, indicating that they were mainly composed of biosynthetic intermediates of glycoproteins. In view of the identification of the endogenous ligands as the biosynthetic intermediates of glycoproteins, the possible functions of the intracellular lectin are discussed in relation to the intracellular transport of glycoproteins.

Effect of lectins on auxin‐induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

Auxin‐induced elongation of coleoptile segments of oat (Avena sativa L. cv. Victory) was suppressed by agglutinin from Canavalia ensiformis DC. (concanavalin A), Triticum vulgaris Vill. germ, Dolichos biflorus L., Glycine max Merr. and Limulus polyphemus L. at 0.5 mg ml−1, while that of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by agglutinin from Canavalia ensiformis DC., Tetragonolobus purpureus Moench and Ulex europaeus L. I when segments were incubated at pH 6.0. These lectins suppressed elongation without any prominent lag period. This suppression of elongation by the lectins was not apparent at pH 4.0. When a ligand for lectins, for instance methyl‐α‐D‐mannopyranoside, was added simultaneously with the lectin, elongation of segments was completely or partly restored. The lectins that suppressed elongation were bound to the cell walls of the segments and inhibited the auxin‐induced cell wall loosening (decrease in the minimum stress‐relaxation time of the cell wall). Thus, the lectins may, at least partly, suppress elongation by inhibiting cell wall loosening. Our results demonstrate that the wall components containing α‐D‐mannose (α‐D‐glucose), N‐acetyl‐D‐glucosamine, N‐acetyl‐D‐galactosamine or D‐glucuronic acid play an important role in wall loosening of oat coleoptiles, while the wall components containing α‐D‐mannose (α‐D‐glucose) or L‐fucose are important for that of azuki epicotyls.

Muscarinic receptor-detergent complexes with different biochemical properties: Selective solubilization, lectin affinity chromatography and ligand binding studies

Muscarinic receptors were solubilized by nonionic, zwitterionic and ionic detergents from porcine striatum. A mixture of digitonin and gitonin (3:2) was found to be most suitable in respect to receptor yield and stability. The solubilization of muscarinic receptors by this detergent appears to be dependent on the existence of free detergent micelles. Consequently, the receptor solubilization was studied at different protein-to-detergent ratios. Based on these experiments, a double extraction procedure was developed in which the receptor is solubilized subsequent to the solubilization of other membrane proteins. After elimination of the detergent excess, the binding of the receptor-detergent complex to six immobilized lectins was studied. In accordance with previous reports, we found a considerable portion of the digitonin/gitonin solubilized receptors (one step extraction procedure) specifically bound to wheat germ agglutinin via sialic acid residues. Muscarinic receptors solubilized by a double extraction procedure (either from porcine striatum or rat brain) did not bind to the lectin. This is not owing to selective extraction or partial denaturation, and indicates that considerable portions of the glycan residues are not covalently bound to the receptor polypeptide. A GTP-insensitive heterogenous agonist binding was found only at the non-wheat germ agglutinin binding receptors. The data analysis was performed by the affinity spectra method.

34] Cluster glycosides

Many animal lectins bind clustered glycosides much more tightly than can be accounted for by the arithmetic sum of the glycosyl residues in the ligand. For example, neoglycoproteins derived from bovine serum albumin show logarithmic increase in binding affinity when the sugar density on the protein is increased linearly. The chapter reviews the “cluster effect”. Cluster glycosides of much simple design can be readily prepared, and some of them are found to be quite potent ligands for animal lectins. Cluster glycosides can be in the form of branched oligosaccharides frequently encountered in natural glycoconjugates. This chapter discusses preparations of various glycosides and the materials required. Tris(hydroxymethyl)aminomethane is the easiest to prepare cluster glycoside.

A flow cytometric method for intracellular labeling and purification of rare neuronal populations: Isolation of fixed neurophysin neurons

A method is described for flow cytometric analysis and fluorescence-activated cell sorting of small populations of neurons following dissociation of fixed brain tissue and immunofluorescent labeling of intracellular antigens. This method has been successfully applied to neurophysin-containing magnocellular neurons of the rat supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. These neurons constitute a rare population in the context of flow cytometry, comprising less than 2% of all cells present in dissociated tissue punches of SON and PVN. Following labeling with anti-neurophysins sera and fluorescein-conjugated second antibody, a highly enriched population containing 80–85% neurophysin-positive neurons was isolated by fluorescence-activated cell sorting. Recovery of 29% of all neurophysin-containing neurons in the SON/PVN was achieved. Perikarya were recovered largely intact, frequently with attached proximal dendritic processes. Applications of this method include purification of specific neuronal types for use as immunogens in production of monoclonal antibodies to cell-type-specific antigens, and rapid surveys of fluorescent lectin or other ligand binding to cell populations identified by the presence of particular intracellular antigens.

Fluorescence quenching of tryptophan by trifluoroacetamide

Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1·mol −1 and 1.9·10 9 1·mol −1·s −1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555–567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1·mol −1 and 092·10 9 1·mol −1· −1, respectively. In the presence of a specific lectin ligand, the methyldi- N, N′-trifluoroacetyl- β- chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.

Discoidin-binding polysaccharide from Dictyostelium discoideum.

Extracts of Dictyostelium discoideum grown axenically in a chemically defined medium were evaluated for binding to discoidin I and discoidin II, endogenous lectins of this slime mold. Binding activity was measured by competitive inhibition of 125I-lactosyl-bovine serum albumin binding to the immobilized lectins. With the solubilization procedure used extracts of vegetative cells and of early aggregates had no significant inhibitory activity, but an abundant discoidin-binding substance was detected in late aggregates and fruiting bodies. This material was purified by ethanol and acid precipitation followed by precipitation with discoidin. It is a polysaccharide composed of 77% galactose, 15% N-acetylgalactosamine, 5% glucose, and 3% N-acetylglucosamine and may be a biologically functional ligand for the slime mold lectins, in particular discoidin II. Use of axenic cells was critical in these experiments, since extracts of Escherichia coli and Klebsiella aerogenes, commonly used as food for D. discoideum, were found to contain substances that react with discoidin. This would complicate isolation of endogenous discoidin ligands from cells raised on bacteria.