The effect of temperature on the agglutinability by lectins of liposomes prepared from total lipids of erythrocytes

Multilamellar liposomes prepared from total lipids of red blood cells are agglutinable by the addition of soybean lectin. At 5 °C the rate of agglutination is significantly slower than at 37 °C, in contrast to erythrocyte ghosts and ghosts sonicated to 1 μ vesicles. The slower lateral mobilities of the lectin glycolipid receptor in the lipid liposomes due to increased microviscosity of the bilayer at the lower temperature, might be one explanation of our agglutination results. However, the opposite temperature dependence seen with ghosts argues for a possible protein modulation of the agglutination reaction.     

Temperature-sensitive loss of sexual agglutinability in Saccharomyces cerevisiae

Temperature dependency of sexual agglutination in Saccharomyces cerevisiae was found. Of 31 strains tested, which showed normal agglutination when cultured at 25°C, 29 strains lost their sexual agglutinability when they were grown at 37°C.     

Study of membrane glycoproteins of human erythrocytes using lectins

A method to study the glycoprotein composition of cell membranes, in particular of human red blood cells, has been developed. It includes the separation of membrane components by the SDS-polyacrylamide gradient slab gel electrophoresis, electroblotting of the phoretograms onto the nitrocellulose sheets and detection of glycoprotein fractions with FITC and peroxidase labeled lectins. PNA detected asialoglycoproteins with O-linked oligosaccharide chains, corresponding to all the PAS-positive bands of the phoretogram. SBA interacted more selectively and revealed only certain PAS-positive bands. Glycoproteins with N-linked carbohydrate chains were PAS-negative and can be identified only by the interaction with WGA, LCL, RCA. Group-specific agglutinins have shown that the ABO antigenic determinants are located in N-linked carbohydrate chains of membrane glycoproteins.     

Distribution of ferritin-conjugated lectins on sialidase-treated membranes of human erythrocytes

The labeling of sialidase-treated, human erythrocyte membranes with ferritin-conjugates of four plant lectinss, concanavalin A, Ricinus communis hemagglutinin, Bauhinia purpurea hemagglutinin and Arachis hypogoea hemagglutinin, is reported. Among these ferritin-conjugated lectins, ferritin-conjugated concanavalin A and ferritin-conjugated R. communis hemagglutinin were found in clusters on the sialidase-treated membranes, whereas ferritin-conjugated B. pupurea hemagglutinin and ferritin-conjugated A. hypogoea hemagglutinin were found in a random distribution on the membranes. Furthermore, when the membranes were labeled with a mixture of concanavalin A and ferritin-conjugated B. purpurea hemagglutinin, ferritin particles were found in clusters, indicating that the membrane receptors for B. purpurea hemagglutinin were forced to move together with those for concanavalin A. A method for thentitative estimation of the clustering of ferritin particles on the membranes was also devised and applied to the labeling of sialidase-treated, human erythrocyte membranes with the above four ferritin-conjugated lectins.     

Distribution of ferritin-conjugated lectins on sialidase-treated membranes of human erythrocytes.

The labeling of sialidase-treated, human erythrocyte membranes with ferritin-conjugates of four plant lectins, concanavalin A, Ricinus communis hemagglutinin, Bauhinia purpurea hemagglutinin and Arachis hypogoea hemagglutinin, is reported. Among these ferritin-conjugated lectins, ferritin-conjugated concanavalin A and ferritin-conjugated R. communis hemagglutinin were found in clusters on the sialidase-treated membranes, whereas ferritin-conjugated B. purpurea hemagglutinin and ferritin-conjugated A. hypogoea hemagglutinin were found in a random distribution on the membranes. Furthermore, when the membranes were labeled with a mixutre of concanavalin A and ferritin-conjugated B. purpurea hemagglutinin, ferritin particles were found in clusters, indicating that the membrane receptors for B. purpurea hemagglutinin were forced to more together with those for concanavalin A. A method for the quantitative estimation of the clustering of ferritin particles on the membranes was also devised and applied to the labeling of sialidase-treated, human erythrocyte membranes with the above four ferritin-conjugated lectins.     

Binding of lectins to "young" and "old" human erythrocytes

"Old" human erythrocytes showed a 21.2% decrease in cell surface area and a 2% decrease in the number of WGA receptor sites, but a 27% increase in the distribution density of the WGA (lectin) receptor site, when compared with "young" human erythrocytes. For a list of lectin abbreviations, see Materials and methods). Both "young" and "old" erythrocytes exhibited very weak binding activity for 125I-labeled PNA, but there was no difference in binding activity for PNA between "young" erythrocytes and "old" ones. Compared with "young" erythrocytes, decreases in the number and distribution density of receptor sites for five lectins including LPA, Con A, RCA-II, SBA and BPA on the cell surface were observed in aged erythrocytes. "Old" erythrocytes also showed a decrease in the number of PHA-E receptor sites, while the distribution density of the same receptor site remained unchanged. In view of these and other observations, it is thought that human erythrocyte aging is accompanied by elimination of some glycoconjugates which have affinity for six lectins, LPA, Con A, RCA-II, PHA-E, SBA and BPA, whereas no WGA receptor-containing glycoconjugates are released from erythrocyte membranes. Elimination of the glycoconjugates results in shrinkage of erythrocytes to reduce their cell surface areas.     

Reaction of lectins with human erythrocytes : II. Mapping of con A receptors by freeze-etching electron microscopy

Native concanavalin A (con A) molecules bound to human erythrocytes were visualized directly by freeze-etching. This technique revealed 400–700 randomly distributed con A molecules/μm² at saturation (or approx. 100 000 per cell, based on a surface of 145 μm²) on both untreated and neuraminidase treated cells. Temperature-dependent mobility of lectin receptors was demonstrated by a redistribution of con A following reactions with anti-con A antibodies. Since the extent of cell agglutination was temperature-independent, clustering or mobility of the receptor sites cannot be an important factor in erythrocyte agglutination. Comparison of the distribution of con A molecules on surfaces of cells with that of the intramembranous particles suggests that there is no direct relationship between these entities.     

Reaction of lectins with human erythrocytes : III. Surface charge density and agglutination

A number of commercially available enzymes were used to modify the cell surface of human erythrocytes to varying degrees. In protease-treated erythrocytes the decrease in surface charge (determined by cell electrophoresis or analysis of sialic acid content) correlates with an increase in agglutinability with concanavalin A (ConA) and wheat germ agglutinin (WGA). On the other hand, treatment with neuraminidase leads to very large decrease in surface charge with only an intermediate increase in agglutinability with both lectins. Subsequent protease treatment of these cells enhances their agglutinability appreciably without further altering their surface charge. It is concluded that the increased agglutinability following protease treatment is due both to a decrease in the net negative charge and a removal of peptides and glycopeptides from the cell surface that may sterically hinder the agglutination reaction.     

Studies on the relationship between lectins from Axinella polypoides agglutinating bacteria and human erythrocytes

A new lectin, called Axinella IV, with bacteria agglutinating activity was detected in the crude extract of the sponge Axinella polypoides. Immunofluorescence studies and immunoelectrophoresis revealed no cross-reactivities with the already known lectins Axinella I. II, and III. d-Galactose and oligosaccharides with d-Galactose in terminal nonreducing position are inactive in agglutination inhibition tests demonstrating also different specificity of Axinella IV lectin when compared with the three aforementioned lectins.     

Isolation of lectins from hemolymph of decapod crustaceans by adsorption on formalinized erythrocytes

Hemolymph of decapod crustaceans contains lectins of important specificity. An isolation procedure, based on adsorption of hemolymph lectins on red blood cells (RBC) fixed with formaldehyde, is described. Hemolymph is let to clot for 3 h at 22-28 degrees C (RT) and for 24 h at 5 degrees C; centrifuged at 13000 g for 30 min; filtered through 5-microm filters; diluted with an equal volume of 50 mM NaCl, 100 mM CaCl(2); supplemented with protease as well as phenoloxidase inhibitors; centrifuged at 13000 g for 20 min. Formalinized RBC (FRBC) are mixed with diluted hemolymph to a suspension of about 20% v/v FRBC. After incubation for 30 min at RT, FRBC are washed five times with 150 mM NaCl, 10 mM CaCl(2). The lectins adsorbed on FRBC are desorbed using either 100-500 mM of carbohydrate solutions in 0.9% NaCl or 50 mM Tris-HCl buffer, pH 8.0 containing 100 mM NaCl and 20 mM entylenediaminetetraacetate (EDTA). The procedure is efficient in isolating the hemolymph lectins of the decapods Liocarcinus depurator and Potamon potamios.     

Recognition of microbial glycans by soluble human lectins

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Comparative studies of the haemagglutination of adult and umbilical cord erythrocytes by animal lectins

1. The sugar specificities of four lactose-binding lectins were studied through the agglutination of adult and/or umbilical cord human erythrocytes (AHRBC and/or CHRBC). 2. Rana catesbeiana egg lectin specifically agglutinated both intact blood group A-AHRBC and intact blood group A-CHRBC. 3. Rana catesbeiana liver lectin agglutinated intact A-AHRBC much more strongly than intact A-CHRBC. 4. Xenopus laevis skin lectin nonspecifically agglutinated AHRBC and CHRBC. 5. Plecoglossus altivelis egg lectin specifically agglutinated intact B-AHRBC, but weakly agglutinated intact B-CHRBC. 6. Comparative studies of lectin-induced AHRBC or CHRBC agglutination clarified the sugar-binding specificities of these lectins.     

Temperature-sensitive mutants of human cytomegalovirus.

Eight temperature-sensitive mutants of human cytomegalovirus have been isolated after mutagenesis with nitrosoguanidine. Three of these mutants have been classified into three separate complementation groups and are capable of synthesizing virus DNA at the nonpermissive temperature (39.5 degrees C). Two others appear unable to synthesize virus DNA at the elevated temperature.