Lectin-mediated attachment and ingestion of yeast cells and erythrocytes by hamster fibroblasts

Yeast cell attachment to Concanavalin A (ConA)-coated fibroblasts depends on the degree of saturation of ConA-binding sites on the fibroblast. Under comparable conditions, fresh mouse erythrocytes fail to establish stable contacts with ConA-coated fibroblasts. The interaction of ConA-coated erythrocytes with fibroblasts and of non-coated erythrocytes with wheat germ agglutinin (WGA)-coated fibroblasts is remarkably less efficient than that of yeast cells interacting with ConA-coated fibroblasts. Ingestion of attached cells was not observed in any of the above lectin-mediated cell-cell interactions. Yeast cells coated with ConA show a high extent of attachment to fibroblasts (three-fold that of yeast cell attachment to ConA-coated fibroblasts). The attachment is highly temperature sensitive, being 3 times more at 37 °C than at 14 °C. A significant fraction of attached yeast cells (˜46%) is ingested by the fibroblasts during the 60 min incubation at 37 °C. The ingestion exhibits a strong temperature dependence, being nil at 14 °C and amounting to 150 and 600 ingested yeast cells per 100 fibroblasts at 24 °C and 37 °C, respectively. Transmission and scanning electron microscopy of ConA-mediated yeast cell-fibroblast interaction indicates a tighter interaction when the yeast cells are coated with ConA than when the fibroblasts are coated with ConA. Thus spreading of the plasma membrane around the attached yeast cell as well as transduction of attachment to ingestion could be triggered only under conditions of a very extensive multibridge interaction between the two apposing surfaces. Such an interaction is not achieved when the mobility of ConA-receptors within the fibroblast membrane plane is restricted as a result of crosslinking with ConA.     

Concanavalin A-mediated attachment and ingestion of yeast cells by macrophages

The ConA-mediated interaction of yeast cells with macrophages was brought about in two steps. The first step involved the interaction of either macrophages or yeast cells with ConA, MConA or YConA in brief, respectively. The second step consisted of interacting the ConA-coated cells with their non-coated counterpart, yielding MConA-Y or M-YConA. The extent of yeast cell attachment to macrophages depends on the degree of saturation of ConA binding sites on the cell coated with ConA in the first step and on the temperature at which the two cell types interact. The temperature dependence in the range of 10–25 °C implies that cell-cell attachment is sensitive to the physical state of membrane lipids as reflected in increased lateral mobility of ConA receptors in the membrane plane. The extent of ConA-mediated cell association is not influenced significantly by colchicine, cytochalasin B (CB) or hydrocortisone. A mild treatment of macrophages with glutaraldehyde reduces, however, the association of yeast cells, further indicating a need for lateral mobility of ConA receptors. ConA-mediated yeast cell attachment could be totally reversed by α-methyl mannoside in the case of MConA-Y and only partially in the case of M-YConA. Yeast cell ingestion is highly temperature-dependent; in MConA-Y a 50% interiorization of the associated yeast cells is reached at 32 °C and detectable interiorization starts only above 19 °C, while in M-YConA a 50% value of interiorization is reached at 18 °C and about 15% of yeast cells are interiorized already at 5 °C. Interiorization of attached yeast cells is not affected by colchicine. Cytochalasin B (CB) (10 μg/ml) inhibits 82% of yeast interiorization in MConA-Y and only 12% in M-YConA. Hydrocortisone has a similar differential effect of inhibition of ingestion; at 25 °C inhibition in MConA-Y amounts to 78% and in M-YConA to 22%. Sodium azide inhibits 90% of interiorization of yeast cells in both MConA-Y and M-YConA. The following working hypothesis was proposed to explain both the characteristics of attachment and the remarkable difference in ingestion pattern of yeast cells in MConA-Y and M-YConA. ConA-mediated yeast cell attachment to macrophages involves multipoint interaction between the two cells achieved by a certain clustering of ConA receptors in the membrane plane. To achieve interiorization a higher extent of bridge formation between the cells is required, and a higher number of ConA-membrane receptors have to be recruited to the area of apposition of the two membranes. This requires lateral mobility of either ConA receptors conjugates (in the case of MConA) or of mobile non-crosslinked ConA receptors in macrophages interacting with YConA). Mobility of ConA receptor conjugates is more sensitive to membrane fluidity than that of non-crosslinked receptors and hence the differential temperature-dependence of ingestion. The effect of CB suggests an involvement of the cytoskeleton in the reorganization of ConA receptors at the membrane level.     

Concanavalin A mediated attachment and ingestion of red blood cells by macrophages.

The interaction of red blood cells and macrophages mediated by Concanavalin A (ConA) was studied using mouse peritoneal macrophages and fresh, homologous red cells. Erythrocytes exposed to ConA at 0.5 μg/ml, a condition that leads to a saturation of 3% of the ConA sites, were bound by macrophages at 22 °C. The ConA inhibitor, α-methylmannoside, prevented this attachment of red cells and largely reversed it when added to preformed macrophage-red cell rosettes up to 90 min. However, red cell attachment was essentially irreversible by 3 h. Electron microscopy showed a progressive increase in the degree of contiguity between red cells and macrophages with time, some macrophage projections distorting and partially encircling red cells at 3 h. Macrophages pretreated with high concentrations of ConA (25 μg/ml) also bound red cells. However, phagocytosis of adherent red cells did not occur at either 22 or 37 °C, even when both red cells and macrophages were pretreated with ConA. In contrast, phagocytosis of attached red cells was observed when preformed rosettes were exposed to ConA at a concentration of 5 μg/ml, and it was complete with ConA at a concentration of 25 μg/ml. These studies demonstrate that ConA in low concentration on red cells is detected by macrophages which form a progressively tighter bond with the red cell surface. However, it appears that phagocytosis can occur only under conditions in which a high density of ConA is established on the surface of the red cell.     

Erythrocytes attached to a wheat germ agglutinin coated surface display an altered phospholipid metabolism

Erythrocytes were bound to a lectin-coated surface; the multivalent attachment to this surface resulted in a severe deformation of the cells and an alteration in the cellular phospholipid metabolism. Human erythrocytes were allowed to bind for 20 min at 20 degrees C to polystyrene beads coated with wheat germ agglutinin (WGA beads). The bound erythrocytes were then lysed to produce stroma bound to WGA beads. Control stroma and stroma-WGA beads were incubated at 37 degrees C with gamma-32P-ATP to examine the phospholipid labeling patterns. The control stroma incorporated 32P-label into phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, in agreement with earlier studies. However, the stroma-WGA beads showed incorporation of 32P-label into phosphatidic acid in addition to that in the phosphoinositides. The quantity of 32P-phosphatidic acid produced during the 20-min assay was 3.23 +/- 0.84 (n = 7) picomoles/micrograms stromal cholesterol; the amount synthesized, however, was dependent on the procedure used to prepare the stroma-WGA beads. If the erythrocytes were bound to the WGA beads at 0 degrees C instead of 20 degrees C, the quantity of 32P-phosphatidic acid produced during the subsequent 37 degrees C assay with gamma-32P-ATP was decreased 4.2 fold; the phosphoinositide labeling pattern was unchanged. In addition, when the time for binding of intact erythrocytes to the WGA beads was varied from 1 to 20 minutes, there was a time-dependent increase in the amount of 32P-phosphatidic acid produced. This induction of phosphatidic acid synthesis could not be duplicated with fluid phase WGA. Therefore, the multivalent binding of intact erythrocytes to WGA beads causes an alteration in phospholipid metabolism.     

Binding of I-Concanavalin a and agglutination of embryonic neural retina cells : Age-dependent and experimental changes

Embryonic chick neural retina cells dissociated from retina tissue by treatment with EGTA (a calcium chelator) show an age-dependent decline in ability to agglutinate with concanavalin A (ConA). This developmental change in cell surface properties is not due to loss of ConA-binding sites, since mature retina cells can be rendered agglutinable by mild trypsinization. It is also not due to masking of ConA receptors, or to a decrease in their amount, since retina cells from late embryos (19 days) bind four times as much ¹²⁵I-ConA as cells from early embryos (8 days). Our findings lead us to suggest that, as the retina differentiates the lateral mobility of ConA receptors in the cell membrane decreases resulting in a reduction of cell agglutinability; trypsinization of late embryo retina cells increases the mobility of the receptors and thereby facilitates their clustering by the lectin into a configuration conducive to cell agglutination.The ability of late embryo (19 day) retina cells dispersed with EGTA to agglutinate with ConA could be increased by still other treatments: by pre-incubation of the cell suspension in Tyrode's balanced salt solution (1 h, 37 °C); and by brief pre-exposure to glutaraldehyde. These two treatments did not enhance cell agglutination with wheat germ agglutinin (WGA). Glutaraldehyde treatment of trypsinized cells made them agglutinable with ConA also at 4 °C; cells treated otherwise agglutinated only at higher temperature. Surface-saturation of monodispersed retina cells with ConA at 37 °C—but not at 4 °C—prevented their agglutination with this lectin, but not with WGA; this inhibition was reversible by methyl a-D-glucopyranoside (αMG).     

Threshold effects on the concanavalin A-mediated agglutination of modified erythrocytes.

Bovine erythrocytes, which are not concanavalin A (ConA)-agglutinable, can be rendered so by attaching alpha-D-mannose residues to their outer membrane. The sugars are incorporated by mildly oxidizing the cells with periodate followed by coupling the liberated aldehyde groups with an alpha-thiomannosyl containing hydrazide (I). The rate and extent of ConA-mediated aggregation of the modified cells are not linearly dependent on the amount of sugar incorporated. For example, treatment of the erythrocytes with 0.075 mM periodate for 5 min followed by I led to the introduction of 1.05 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA demonstrated the presence of 66,525 ConA receptors/cell with an average KA = 4.9 X 10(6) M-1 yet the cells failed to aggregate with ConA at concentrations up to 500 microgram ml-1. Treating the cells with 0.1 mM periodate followed by I led to the introduction of 1.42 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA indicated the presence of 78,780 binding sites/cell (KA = 5.9 X 10(6) M-1). These cells were readily aggregated by ConA at concentrations greater than or equal to 64 microgram ml-1. We show here that the sugar incorporation technique is random and that no functional differences were detected in the receptors introduced at the different periodate concentrations. Therefore, the ConA-mediated aggregation of these modified erythrocytes is exquisitely sensitive to small changes in functionally identical receptor densities.     

Macrophage recognition of periodate-treated erythrocytes: involvement of disulfide formation of the erythrocyte membrane proteins

Upon exposure to 2 mM periodate at 0 degrees C for 15 min, mouse erythrocytes underwent membrane lipid oxidation, oxidation of cell surface sialyl residues into aldehyde-bearing derivatives, and oxidation of SH groups of the membrane proteins into disulfides. The periodate-treated erythrocytes exhibited a remarkable increase in rosette attachment to resident mouse peritoneal macrophages in the absence of serum. The relationship between the oxidation of the membrane constituents and the macrophage recognition of these cells was investigated. Periodate treatment of erythrocytes in the presence of butylated hydroxytoluene, an inhibitor of lipid oxidation, did not affect the subsequent attachment of the erythrocytes to the macrophages. Reduction of the periodate-treated erythrocytes with borohydride or cyanoborohydride did not affect the erythrocyte attachment. Neuraminidase treatment of erythrocytes before periodate did not affect the attachment either. On reduction of the disulfides of the membrane proteins with dithiothreitol, the periodate-treated erythrocytes lost their ability to attach to the macrophages. Erythrocytes treated with an SH-oxidizing agent, diamide, were then examined for the macrophage recognition. The diamide-treated cells also showed rosette attachment to the macrophages in the absence of serum, but did not when reduced with dithiothreitol. These results indicate that oxidation of the SH groups of the membrane proteins to disulfides causes reversible membrane changes that macrophages recognize, and it is this mechanism that is responsible for the macrophage recognition of the periodate-treated erythrocytes.     

Temperature-sensitive hamster cell line with altered membrane properties

The altered properties of a concanavalin A-resistant Chinese hamster ovary cell line with obvious temperature-sensitive growth properties is described. The variant cell line, C^R-7, was shown to have a higher efficiency of colony formation than the parental wild-type population after treatment with various concentrations of concanavalin A (ConA). The variant cells had the properties of a temperature-sensitive cell line as judged by growth studies performed on solid surfaces or in suspension culture at the permissive (34 °C) and non-permissive (39 °C) temperatures; by colony efficiency determinations performed at 34 °C and 39 °C; and by the altered ability to incorporate DNA, RNA, and protein precursors into acid-precipitable material at the non-permissive temperature. Evidence for changes in the membrane properties of C^R-7 cells included: a reduced agglutinability in the presence of ConA, an altered cellular morphology on solid surfaces, an enhanced sensitivity to the toxic effects of membrane-active agents, altered and temperature-sensitive adhesiveness properties, and a reduced ability to bind labelled ConA.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus) : II. Effects of pretreatment with a small number of HVJ

Ehrlich ascites tumor cell membranes were completely modified after incubation at 37 °C for 30 min with a small dose of HVJ (about 0.7% of the maximum number of the virus particles that could be adsorbed onto the cells). After this treatment, the cells could adsorb further added HVJ onto their surfaces at 0 °C. But the cell agglutination which was induced by viral adsorption at 0 °C was very weak, and the interaction of the adsorbed virus with the lipid layer of the cell membrane at 37 °C preceding fusion or lysis of the cells was not strong. A discrepancy was observed between acquisition of the modification and liberation of sialic acid (destruction of viral receptors) by viral neuraminidase. The modification proceeded well on incubation at 37 °C but not at lower temperatures. The possibility that the modification is induced by fusion of viral envelopes with cell membranes is discussed.     

Thermostabilization of protective antigen—the binding component of anthrax lethal toxin

Protective antigen (PA) is the binding component of anthrax lethal toxin produced by Bacillus anthracis, and constitutes a major ingredient of the vaccine against anthrax. PA and lethal factor when added together are cytolytic to mouse macrophages and J774G8 macrophage cell line. This in vitro lethal toxicity assay is very useful in understanding the molecular mechanism of action of lethal toxin. Effective utilization of PA is, however, hampered due to its thermolability. On prolonged storage at 37 ° C, PA was found to lose its activity almost completely. The effect of solvent additives like trehalose, sorbitol, xylitol, sodium citrate and magnesium sulphate on the thermal stabilization of PA was examined. The results indicated an increase in the stability of PA when the incubation at 37 ° C was carried out in the presence of solvent additives used in the 1–3 M range. Magnesium sulphate helped retain the activity up to 82.7% against the control in which no additive was used, as judged by cytolytic assay using J774G8 macrophage cell line. Trehalose or sodium citrate also showed an appreciable protection of PA activity, while sorbitol or xylitol were not very effective. Competitive binding assay using radiolabeled PA showed that PA had lost capacity of binding to macrophage cells on prolonged incubation at 37 ° C. Circular dichroism results at 4, 18 and 37 ° C indicated an increase in secondary structure at 37 ° C relative to that at 4 or 18 ° C, supporting the activity data.     

Relationships between cell-substratum interactions and the distribution of concanavalin A receptors of mouse embryo fibroblasts

The mobility of concanavalin A (ConA) receptors on the surfaces of mouse embryo cells are affected by cell-substratum interactions. When the cells are grown on a substratum from which they are easily detached by EDTA, their receptors have an increased mobility and are redistributed into patches after incubation with ConA at 37 °C.     

Effect of ConA—receptor interaction on the structure of cell membrane

Recently,the effect of ligand receptor interaction on the membrane structure of liposomes has been studied extensively,However,little is known about how it exists on biological membranes,In this paper,the effect of Concanavalin A(ConA) receptorinteratcion on the structure of cell membranes was studied by Circular DIchrosim(CD) and 31P Nuclear Magnetic Resonance(NMR).CD results of both the purified macrophage membranes and human erythrocyte hgosts(EG) showed that the conformation of membrane proteins changed after ConA binding.For further research,31P-NMR was used to detect the orgainzation of phosp[holipid molecules on macrophage membranes.After ConA binding,the tendercy to form non bilayer structure increased with the amount of ConA.The changes of 31P-NMR spectra of living macrophages might be partly due to the above stated reason too.In addition,ConA-receptor interaction also induced similar results of 31P-NMR spectra in EG.In contrast,wheat germ agglutinin (WGA),another kind of lectin,rarely showed the same influence.     

Effects of low temperature storage upon subsequent energy metabolism of rabbit embryos

The oxidation of pyruvate by two-cell rabbit embryos occurred at a very low rate at 10 °C, but increased 10–15 times upon rewarming to 37 °C. Pyruvate utilization of these embryos increased with continued development parallel to embryos not cooled to 10 °C, but incubated directly at 37 °C. Storage at 10 °C did not affect the amount of glucose subsequently oxidized at 37 °C, nor the shift from the pentose shunt to glycolysis and the Krebs cycle that normally occurs between the morula and blastocyst stage. Thus, the temporary inhibition of carbohydrate metabolism induced through temperature reduction did not impair the subsequent ability of the embryos to metabolize pyruvate and glucose normally.     

Thermodynamic and kinetic analysis of the interaction between hepatitis B surface antibody and antigen on a gold electrode modified with cysteamine and colloidal gold via electrochemistry

Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 °C were easily distinguished and the kinetic rate constants (k_ass and k_diss) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, ΔG, ΔH and ΔS, of the interaction at 37 °C were calculated, which were −56.65, −64.54 and −25.45 kJ mol⁻¹, respectively.     

Regulation of colominic acid biosynthesis by temperature: role of cytidine 5''-monophosphate N-acetylneuraminic acid synthetase

Synthesis of colominic acid in Escherichia coli K-235 is strictly regulated by temperature. Evidence for the role of cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37°C. No activity was found in E. coli grown at 20°C. CMP-Neu5Ac started to be quickly synthesized when bacteria grown at 20°C were transferred to 37°C and was halted when cells grown at 37°C were transferred to 20°C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP-Neu5Ac necessary for the biosynthesis of colominic acid.     

Rearrangement of concanavalin A receptor sites on cells tagged with dinitrofluorobenzene: Evidence for the chemical induction of a change usually associated with malignant transformation

Normal human peripheral blood granulocytes which are tagged with 1-fluoro-2,4-dinitrobenzene (DNFB) are agglutinated by concanavalin A (ConA) in a way which resembles the pattern of reactivity displayed by leukemic cells. The present study further defines this reaction. The binding of ConA to untagged and DNP-tagged granulocytes, treated with DNFB at a ratio of 10¹¹ molecules/cell, was quantified by isotopic dilution experiments employing [³H]ConA. Similar amounts of the lectin were bound to untagged and DNP-tagged cells following incubation for 5 min at 4 °C or 30 min at 24 °C: 1.1 × 10⁵ molecules/cell, 4.6 × 10²/μ² of surface area, and 1.6 × 10³/μg of protein. The binding of [³H]ConA to both untagged and DNP-tagged cells was inhibited to the same degree by α-methylglucopyranoside (α-MG). Fixation with either glutaraldehyde or formaldehyde, which immobilizes ConA receptor sites, completely inhibited the agglutination of both untagged and DNP-tagged cells although lectin binding was unchanged. This suggests that the inhibition of agglutination was not due to the blocking of ConA-binding sites by aldehyde groups but rather to the immobilization of lectin receptors. We conclude that dinitrophenylation of normal granulocytes facilitates the rearrangement of lectin receptors in a way which resembles the ConA-induced clustering of sites which have been observed with malignant and transformed cells.     

Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at −5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme.     

Changes in oligosaccharide expression on plasma membrane of the mouse oocyte during fertilisation and early cleavage

It has been reported that various structural and functional changes occur on the surface of the plasma membrane of the ovum and embryo during fertilisation and cleavage in preparation for implantation. Glycoproteins are thought to be one of the factors in cell attachment. Thus, we investigated the changes in glycoprotein expression on the cell surface membrane of the mouse embryo by using lectins. Among seven types of lectin (ConA, WGA, UEA-I, MPA, LCA, DBA and PNA), the fluorescent intensities of ConA and WGA markedly increased from unfertilised ova to blastocysts. By quantitative analysis using immuno-scanning electron microscopy, the numbers of ConA-gold particles were small until 4-cell cleavage, but increased significantly at the blastocyst stage. In contrast, an increased number of WGA-gold particles was detected even at the 4-cell stage, and this increase continued to the blastocyst stage. From the above observations, we conclude that the numbers of sugar chains bound to both ConA andWGA increases with blastocyst formation and earlier expression is observed with WGA. The present study dearly shows that glycoproteins on the cell membrane surface of the mouse embryo quantitatively increase at the time of implantation, and the possibility has been indicated that glycoproteins are involved in intercellular recognition and adhesion between the embryo and endometrial epithelium.     

Localization of receptors and early events of phagocytosis in the macrophage

We studied the attachment of MRBC, 1.1 μm latex (foreign surface receptor), hemocyanin-DNP antiDNP Ab and sensitized SRBC (Fc receptor) to mouse peritoneal macrophages in culture, and the phagocytosis of 1.1 μm latex, AbAg complex, and sensitized SRBC by macrophages. Both types of receptors appeared over the whole cell surface. The particles attached mostly in the central area of the cell, but peripheral attachment was also observed. Internalization of 1.1 μm latex beads appeared to be mostly perinuclear, by sinking in of the cytoplasm with the attached particles, rather than a flow of membrane process over the beads. Also in the case of the AbAg complex attached to the Fc receptor internalization seemed mostly perinuclear and similar in mode to the latex ingestion. When attachment occurred in the periphery, active ruffling was observed. Sensitized SRBC internalization was seen mostly in the extreme periphery of the cells after the attached SRBC were moved from the perinuclear area where many of them first attached.     

Modulation of the locomotion of polymorphonuclear leukocytes by a synthetic amphiphile, poly(ethyleneglycol) palmitate

To mimic in vitro the effect of biologically active amphipathic molecules on the locomotion of polymorphonuclear leukocytes (PMNL), the interaction and the consequences of the interaction between poly(ethyleneglycol) (PEG) esterified with palmitic acid (P-PEG) and PMNL, was studied. It was shown that P-PEG was more liable to hydrophobic interaction than PEG, and that P-PEG associated to a greater extent than PEG with PMNL. This association was inhibited by decreasing the interaction temperature from 37 to 4 °C, but it was insensitive to inhibitors of glycolysis. P-PEG decreased colchicine-facilitated capping by concanavalin A (ConA). P-PEG also decreased random locomotion of individual as well as population of PMNL. Neither cell viability nor the ability to respond by stimulated locomotion to an attractant was affected. The results indicate that P-PEG modulates PMNL locomotion by altering the membrane structure, e.g. by decreasing the lateral mobility of membrane constituents.