Inhibition by succinyl concanavalin A of strong adjuvant activity of lipopolysaccharides which possess mannans as the O-specific polysaccharide chains

It has been reported that lipopolysaccharides (LPS) from Klebsiella O3 and O5 and Escherichia coli O8 and O9 exhibit extraordinarily strong adjuvant activity in augmenting antibody responses against protein antigens in mice as compared with other kinds of LPS. These four kinds of LPS all possess homopolysaccharides consisting of mannose (mannans) as the O-specific side chains. When these kinds of LPS were mixed in vitro with succinyl concanavalin A (Con A) which is known to bind specifically to alpha-mannoside and alpha-glucoside, their strong adjuvant activity was inhibited. Degree of the inhibition of the adjuvant activity of Klebsiella O3 LPS by succinyl Con A was dependent upon the dose of succinyl Con A. However, phytohemagglutinin, which is known to bind specifically to N-acetyl-D-galactosamine, did not inhibit the adjuvant activity of Klebsiella O3 LPS and O5 LPS. When Klebsiella O3 LPS was mixed with succinyl Con A in the presence of excess amounts of alpha-methyl mannoside or the polysaccharide fraction isolated from Klebsiella O3 LPS, the inhibitory effect of succinyl Con A on the adjuvant activity of Klebsiella O3 LPS was blocked. By contrast, the activity of Klebsiella O3 LPS as a polyclonal B-cell activator was not affected by treatment with succinyl Con A. From these results it is concluded that the mannans, as the O-specific polysaccharide chains of the LPS, significantly contribute to expression of their strong adjuvant activity.     

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.     

Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: common glycoproteins and glycoprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment

Whole cell extracts of 10 clones of bloodstream forms of African trypanosomes representing two strains of Trypanosoma brucei gambiense, one strain of T. b. rhodesiense and one strain of T. b. brucei were fractionated on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose paper, and probed with horseradish peroxidase conjugated lectins to detect glycoproteins. Variant specific glycoproteins of all 10 clones bound peroxidase labeled concanavalin A, but peroxidase labeled wheat germ agglutinin bound to the variant specific glycoproteins of only 3 of the 10 clones examined. In addition, 22 other glycoproteins expressed in common by all clones bound peroxidase labeled concanavalin A; 19 common glycoproteins bound peroxidase labeled wheat germ agglutinin. Lectin binding to transferred glycoproteins was specifically inhibited by appropriate monosaccharides, alpha-methyl mannoside for concanavalin A and N-acetyl glucosamine for wheat germ agglutinin. Prior incubation of blots in endo-beta-N-acetylglucosaminidase H eliminated binding of peroxidase-labeled concanavalin A to most of the 22 common glycoproteins. Two glycoproteins, designated Gp 81 and Gp 110, were the major Endoglycosidase H resistant components. Endoglycosidase H treatment also reduced binding of peroxidase labeled concanavalin A to the variant specific glycoproteins of 7 clones. The variant specific glycoproteins from the 3 clones that bound peroxidase labeled concanavalin A following enzyme treatment were those that bound peroxidase labeled wheat germ agglutinin. These results show that African trypanosomes express a greater number of glycoproteins than has been reported previously and that only a limited number of these glycoproteins bear Endoglycosidase H resistant oligosaccharides.     

Enhanced multiplication of intracellular (amastigote) stages of Trypanosoma cruzi in vitro

Amastigotes of different strains of Trypanosoma cruzi responded to stimulation with concanavalin A in an axenic medium by increased DNA synthesis and cell multiplication. These effects were inhibited by alpha-methyl mannoside. Other mitogens, i.e. phytohemagglutinin P, castor bean ricin Type II isolated from Ricinus communis, and a bacterial lipopolysaccharide, had no effect on amastigote growth. Amastigote stimulation by concanavalin A lends itself to studies on the biochemistry and cell cycle of this human pathogen.     

The 5-hydroxyl of myo-inositol is essential for uptake into HSDM1C1 mouse fibrosarcoma cells

We attempted to replace the myo-inositol in cellular inositol phosphatides with 5-deoxy-myo-inositol to evaluate the role of inositol 1,4,5-trisphosphate as a second messenger. This analog, lacking a 5-hydroxyl, might be incorporated into 5-deoxyphosphatidylinositol and converted to the corresponding phosphatidylcyclitol 4-phosphate but could not be converted to phosphatidylinositol 4,5-diphosphate, the precursor of the second messenger molecule inositol 1,4,5-trisphosphate. We synthesized 5-deoxy-myo-inositol and found that this analog does not replace myo-inositol as an essential growth factor for essential fatty acid deficient HSDM1C1 mouse fibrosarcoma cells. Furthermore, [5-3H]-5-deoxy-myo-inositol was neither incorporated into the phospholipids nor accumulated in the cytoplasm of these cells. It appears that this cell line has a specific myo-inositol uptake system that excludes a potentially harmful analog of inositol.     

Formation and activity of covalent conjugates of poliovirus and ligands binding to cell surface structures

Disulfide-linked conjugates of poliovirus with streptavidin or concanavalin A were formed and the binding of the conjugates to mouse L cells that lack natural poliovirus receptors was studied. The conjugate with streptavidin was specifically bound to biotinylated L cells, but not to unmodified L cells. The conjugate with conA was bound to L cells in the absence of, but not in the presence of alpha-methyl mannoside. Incubation of L cells with bound conjugates did not produce virus, although the conjugates were highly infectious in HeLa cells, containing natural poliovirus receptors. This suggests that the artificially bound virus was unable to penetrate the L cells and start replication. The possibility that binding of the virus to the natural receptor is required for efficient infection is discussed.     

Influence of lectins on the binding of 125I-labeled EGF to human fibroblasts.

Lectins that interact with mannose (concanavalin A), galactose (ricin, abrin), or N-acetylglucosamine (wheat germ agglutinin) block ¹²⁵I-labeled EGF binding to the surface of cultured human fibroblasts at 37° or 5°. Lectins specific for fucose or N-acetylgalactosamine, soybean agglutinin or gorse lectin, respectively, do not interfere with growth factor binding. The inhibition of ¹²⁵I-labeled EGF binding by concanavalin A at 37° or 5° could be reversed rapidly by the addition of α-methyl mannoside. The results suggest that the fibroblast membrane receptor for EGF is, or is closely associated with, a glycoprotein or glycolipid that contains mannose, galactose and N-acetylglucosamine residues.     

Synthesis of cluster mannosides via a Ugi four-component reaction and their inhibition against the binding of yeast mannan to concanavalin A

The Ugi four-component reaction (U-4CR) was utilized to prepare divalent and trivalent cluster mannosides with different scaffolds. The glycoclusters obtained were tested for their relative inhibitory potency against the binding of yeast mannan to concanavalin A by solid-phase enzyme-linked lectin assays (ELLA) using methyl alpha-D-mannopyranoside as a standard. Among them, a divalent mannoside containing aromatic groups showed the strongest binding affinity to concanavalin A.     

Interactions of plant lectins with glycolipids in liposomes

A panel of five plant lectins with different binding specificities was used to determine if plant lectins could bind specifically to membrane-associated glycolipids. $\text{Ricinis communis}$ and wheat germ agglutinins both bound specifically to mixed brain gangliosides and globoside I from human erythrocytes. Wheat germ agglutinin also bound to ganglioside G_M1 and human erythrocyte ceramide trihexoside, but not to ceramide dihexoside, mono-, or digalactosyl diglycerides. Concanavalin A bound to liposomes with or without glycolipid substituents, and this binding was partially inhibited by α-methyl mannoside. This study indicates that lectins can specifically recognize and bind to certain glycolipids in membranes.     

The interaction of N-acetylhexosaminidase with insolubilized concanavalin A.

The specific interaction between human N-acetylhexosaminidase and concanavalin A was evaluated with respect to temperature, time, pH and concentration of specific ligand in incubation mixtures containing the enzyme and insolubilized lectin. Elution of the enzyme from insolubilized concanavalin A is dependent on both temperature and concentration of alpha-methyl mannoside. Conditions for a high yield of the enzyme from chromatography on insolubilized concanavalin A are described.     

Lectin affinity chromatography of glycopeptides and oligosaccharides from normal and lectin-resistant Chinese-hamster ovary cells.

The [3H]mannose-labelled glycopeptides from two lectin-resistant lines of Chinese-hamster ovary cells were fractionated by chromatography on lentil lectin-Sepharose and concanavalin A-agarose columns and subsequently analysed by gel filtration in comparison with the glycopeptides of the parental cell line. Essentially all of the [3H]mannose-labelled asparaginyl-oligosaccharides from the 'single-mutant' cells selected for resistance to phytohaemagglutinin and the 'double-mutant' cells selected for additional resistance to concanavalin A were not bound to lentil lectin, whereas approximately one-sixth of the parental-cell glycopeptides were bound and specifically eluted with alpha-methyl mannoside. These bound and eluted glycopeptides represented a specific subset of the complex acidic-type asparaginyl-oligosaccharides. The percentage of radiolabelled glycopeptides and oligosaccharides from each cell line that were specifically bound to concanavalin A was consistent with the relative sensitivities of the three cell lines to this lectin. The major radiolabelled species in the endoglycosidase digest of the 'double-mutant'-cell glycopeptides (Man4GlcNAc1-size neutral oligosaccharides) were not bound to concanavalin A, whereas essentially all of the other neutral-type oligosaccharides were bound. In addition, the larger neutral-type oligosaccharides (Man8--9GlcNAc1) were more strongly bound to concanavalin A than were either the smaller neutral-type or the di-antennary acidic-type structures.     

Interaction of spermine with polyphosphoinositides containing liposomes and myo-inositol 1,4,5 triphosphate

The interaction of spermine with liposomes containing 2% phosphatidylinositol, phosphatidylinositol 4 phosphate and phosphatidylinositol 4,5 biphosphate was inferred from the ability of these liposomes to interfere with spermine binding to the resin heparin-Sepharose. The inositol phospholipids tested showed different affinities for spermine: the order of binding strength appear to be phosphatidylinositol phosphatidylinositol 4 phosphate phosphatidylinositol 4,5 biphosphate. The ability of vesicles containing 2% polyphosphoinositides to interact with spermine is comparable to that of either single stranded RNAs or highly negatively charged liposomes. Myo-inositol 1,4,5 triphosphate has a much lower ability to bind spermine.     

Inhibition of phosphatidylinositol synthase and other membrane-associated enzymes by stereoisomers of hexachlorocyclohexane

Hexachlorocyclohexanes (HCCH) are chlorinated analogs of inositol; the alpha, beta, gamma, and delta isomers of HCCH have the stereochemical configurations of (+/-)-, scyllo-, muco-, and myo-inositol, respectively. To assess their potential as specific tools for the study of agonist-stimulated phosphoinositide metabolism, we examined the effects of these four HCCH isomers on phosphatidylinositol (PI) synthase (CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase), PI:inositol exchange enzyme, and several membrane-associated enzymes unrelated to inositol metabolism. In pancreas microsomes, in the presence of saturating myo-inositol, the alpha, beta, gamma, and delta isomers (4 mM) inhibited PI synthase activity by 9, 4, 22, and 69%, respectively. Half-maximal inhibition by delta-HCCH occurred at 0.25 mM. A similar pattern of HCCH inhibition was obtained using n-octylglucopyranoside-solubilized and partially purified PI synthase preparations. The inhibition by delta-HCCH was noncompetitive versus myo-inositol. The PI:inositol exchange enzyme in mouse pancreas microsomes was inhibited 90% by 1 mM delta-HCCH in the presence of 0.25% Triton X-100, but not in its absence; half-maximal inhibition occurred with 0.5 mM delta-HCCH. delta-HCCH (4 mM) also inhibited to varying extents the following enzymes: pancreas CDP-choline:1,2-diacyl-sn-glycerol cholinephosphotransferase (75%), brain and erythrocyte (Na+,K+)-ATPase (87 and 70%), brain and erythrocyte Mg2+-ATPase (38 and -5%), brain 1,2-diacyl-sn-glycerol kinase (22%), and liver glucose 6-phosphatase (16%). gamma-HCCH (4 mM) inhibited these enzymes to a lesser extent, or not at all. The order of inhibition by HCCH stereoisomers was the same as the order of their saturation level in phospholipid vesicles (delta greater than gamma greater than alpha greater than beta). This suggests that the inhibitory action is due to insertion of the compounds either into hydrophobic domains of the enzymes or into annular lipid. The results indicate that the HCCHs are not selective inhibitors of inositol metabolism.     

The Influence of Divalent Cations and Substrate Concentration on the Incorporation of Myo-inositol into Phospholipids of Isolated Bovine Oligodendrocytes

The incorporation of myo-inositol into phosphatidylinositol by two routes (CTP-independent and CTP-independent) has been investigated in homogenates prepared from isolated bovine oligodendrocyte perikarya. The CTP-dependent route has the higher maximum velocity of inositol incorporation and can utilise either Mn2+ or Mg2+ as a divalent ion cofactor. This route of inositol incorporation is also strongly inhibited by Ca2+ ions at concentrations less than 1 mM. The primary site of the inhibitory action appears to be the enzyme CDP-diglyceride inositol phosphatidyl transferase (EC 2.7.8.11) though synthesis of CDP-diacylglycerol is also inhibited by endogenous Ca2+ present in the oligodendrocyte homogenate. In contrast, CTP-independent inositol incorporation into phosphatidylinositol is only stimulated by Mn2+ (Zn2+,Cu2+, Mg2+, Ca2+ and Co2+ are ineffective) and is not inhibited by Ca2+, at least up to a concentration of 1 mM.     

Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands.

1. The effect of Li+ on the agonist-dependent metabolism of [3H]inositol has been studied in rat brain, rat parotid and the insect salivary gland. 2. When brain or parotid slices were incubated in the presence of [3H]inositol, Li+ was found to amplify the ability of agonists such as carbachol, phenylephrine, histamine, 5-hydroxytryptamine and Substance P to elevate the amount of label appearing in the inositol phosphates. 3. A different approach was used with the insect salivary gland, which was prelabelled with [3H]inositol. After washing out the label, the subsequent release of [3H]inositol induced by 5-hydroxytryptamine was greatly decreased by Li+. During Li+ treatment there was a large accumulation of [3H]inositol 1-phosphate. 4. This ability of Li+ to greatly amplify the agonist-dependent accumulation of myo-inositol 1-phosphate offers a novel technique for identifying those receptors that function by hydrolysing phosphatidylinositol. 5. The therapeutic action of Li+ may be explained by this inhibition of myo-inositol 1-phosphatase, which lowers the level of myo-inositol and could lead to a decrease in the concentration of phosphatidylinositol, especially in those neurons that are being stimulated excessively. This alteration in phosphatidylinositol metabolism may serve to reset the sensitivity of those multifunctional receptors that generate second messengers such as Ca2+, cyclic GMP and the prostaglandins.     

A sensitive method to introduce membrane-bound proteins into recipient cells based on affinity enrichment of lipid vesicles to the recipient cell prior to fusion

A sensitive analytical procedure for studying membrane-bound structures has been developed. Membrane glycoproteins inserted into liposomes were transferred to recipient cells by use of a lectin, concanavalin A, bound to the cells as a bridge to generate proximity between the recipient cell and the glycoprotein-containing liposome, prior to exposure to the fusing agent, poly(ethylene glycol). Partially purified histocompatibility antigen from rats was introduced into the membrane of human lymphocytes. After treating the cells with poly(ethylene glycol) under fusion conditions, some of the antigen present in the preparation could not be eluted with alpha-methyl mannoside and EDTA, indicating that incorporation in the cell membrane had taken place. This antigen remained exposed on the lymphocyte surface for approximately 1 h as demonstrated by sensitivity of the lymphocytes to the lytic effect of an antiserum to the histocompatibility antigen in the presence of complement. Some of the lectin molecules seemed to be internalized in the cells but no induction of cell mitosis was observed. The described method gives an opportunity to work with small amounts of membrane proteins inserted into liposomes, introducing them into recipient cells for analysis of their biological activities.     

Neo-mannosylated liposomes: synthesis and interaction with mouse Kupffer cells and resident peritoneal macrophages

In order to target liposomes to cells expressing at their surface mannose receptors, e.g. mouse Kupffer cells and peritoneal macrophages, we have developed a new synthetic strategy which allows a chemically well defined preparation of neo-mannosylated vesicles. alpha-D-Thiomannopyranoside residues, substituted with a hydrophilic spacer arm and functionalized with a sulfhydryl group, were covalently coupled to preformed large unilamellar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. Liposomes, containing 15 mol% of mannosyl residues, were specifically aggregated with concanavalin A; this aggregation could be reversed by an excess of free methyl alpha-D-mannopyranoside indicating that the surface ligands were freely accessible to the lectin. The neo-mannosylated liposomes presented in vitro an increased binding to cells possessing alpha-D-mannose specific binding sites. At 37 degrees C a specific binding, up to 9-fold compared to control vesicles, was observed. These neo-mannosylated vesicles represent attractive tools for targeting bio-active molecules to macrophage-associated diseases.     

Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post-receptor mechanism

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.     

Receptor-mediated gonadotropin action in the ovary. Inhibitory actions of concanavalin A and wheat-germ agglutinin on gonadotropin-stimulated cyclic AMP and progesterone responses in ovarian cells.

Pretreatment of ovarian cells with concanavalin A and wheat-germ agglutinin blocked the gonadotropin-induced cyclic AMP and progesterone responses and this effect was time- and concentration-dependent. Basal production of either cyclic AMP or progesterone, however, was not affected by treatment of cells with lectin. The effect of concanavalin A on gonadotropin-mediated cyclic AMP and progesterone responses was blocked by alpha-methyl D-mannoside and alpha-methyl d-glucoside. Similarly the inhibitory effect of wheat-germ agglutinin was reversed by N-acetyl-D-glucosamine. Pretreatment of ovarian cells with concanavalin A or wheat-germ agglutinin had no effect on protein synthesis in the ovary as monitored by [3H]proline incorporation studies. Concanavalin A and wheat-germ agglutinin did not affect steroid production in response to dibutyryl cyclic AMP and 8-bromo cyclic AMP, indicating that the inhibitory action of lectin was occurring at a step before cyclic AMP formation. Lectins specific for L-fucose, D-galactose and N-acetyl-D-galactosamine, gorse seed agglutinin, peanut agglutinin and Dolichos biflorus agglutinin respectively, did not interfere with gonadotropin-induced cyclic AMP and progesterone responses. The present studies suggest that gonadotropin receptors may be glycoprotein in nature or closely associated with glycoprotein structures with the carbohydrate chain containing N-acetyl-D-glucosamine, mannose and possibly N-acetylneuraminic acid.     

Studies of the cell surface of Dictyostelium discoideum during differentiation. The binding of 125I-concanavalin A to the cell surface.

125I-concanavalin A (125I-Con A) was found to be equally effective as native Con A in binding to and agglutinating cells of Dictyostelium discoideum, suggesting that iodination of the molecule had no effect on the interaction of the protein with the cell surface. Almost all of the 125I-Con A binding to the cells was inhibited by alpha-methyl glucoside. The binding of 125I-Con A to the cells was extremely rapid, and once bound, the molecule was not readily displaced by prolonged incubation or by the addition of excess native concanavalin A (Con A). In contrast, the 125I-Con A was displaced rapidly from the cell surface by alpha-methyl glucoside. The binding of 125I-Con A to D. discoideum was identical at 22 degrees and 4 degrees, and was unaffected by metabolic inhibitors, suggesting that the protein was not subject to endocytosis. The cell surface Con A binding sites became saturated at high 125I-Con A concentrations. Scatchard plots of the data indicated that growing cells possessed 4 X 10(7) sites/cell, all of equal affinity. Similar plots for "aggregation phase" cells indicated at least two classes of binding sites. A small proportion of the sites had an affinity close to that for the sites on growing cells, but the majority of the sites had a markedly decreased affinity. The total number of binding sites increased only slightly during aggregation to 5.6 X 10(7) sites/cell.