Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

Recognition of foreignness by blood cells of the freshwater snail Lymnaea stagnalis, with special reference to the role and structure of the cell coat

The structure and function of the cell coat of the blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were studied with ultrahistochemical tests, including concanavalin A (Con A) labeling, and with in vitro phagocytosis experiments. The cell coat is intensely stained by ruthenium red and tannic acid. The cells possess binding sites for Con A. Proteolytic enzymes destroy the receptors for Con A and totally inhibit the phagocytic activity of amoebocytes. Incubation experiments with proteases, carbohydrases, and inhibition sugars revealed that (1) the Con A binding sites are anchored in the plasma membrane by proteins, and (2) glucose, fructose, mannose, and to a lesser extent N-acetylglucosamine and N-acetylgalactosamine, inhibit the binding of Con A to amoebocytes, suggesting that these carbohydrates might form part of these binding sites.     

Concanavalin a perturbation of membrane enzymes of mammary gland

The plant lectin concanavalin A (Con A) specifically inactivates the 5′ -nucleotidase of a plasma membrane-enriched fraction from lactating mammary gland. The lectin also causes an activation of the membrane Mg⁺⁺ -ATPase, but does not affect galactosyltransferase or alkaline phosphatase. The enzyme perturbations are prevented by α-methylmannoside, an inhibitor of Con A binding, indicating that specific binding to carbohydrate structures rather than nonspecific protein-protein interaction is involved. Solubilization of the 5′ -nucleotidase in detergents (0.2% Triton X-100 or 1% deoxycholate) does not prevent Con A inactivation, indicating that incorporation into the membrane structure is not a requirement for the Con A effect. The results suggest that Con A inactivates the 5′ -nucleotidase by a direct interaction with the enzyme and that this enzyme is a Con A receptor site on the surface of mammary cells.     

Characterization and quantitation of concanavalin a binding by plasma membrane enriched fractions from soybean root

The binding of concanavalin A (Con A) to soybean root membranes in plasma membrane enriched fractions (recovered from the 34/45% interface of simplified discontinuous sucrose density gradients) was studied using a radiochemical assay employing tritiated (³H)-Con A. The effect of lectin concentration, time, and membrane protein concentration on the specific binding of ³H-Con A by the membranes was evaluated. Kinetic analyses showed that Con A will react with membranes in that fraction in a characteristic and predictable manner. The parameters for an optimal and standard binding assay were established. Maximal binding occurred with Con A concentrations in the range of 8 to 16% of the total membrane protein with incubation times greater than 40 min at 22 C. Approximately 10¹⁵ molecules of ³H-Con A were bound per microgram of membrane protein at saturation. Binding was reversible. Greater than 92% of the total Con A bound at saturation was released by addition of α-methyl mannoside.     

Variations in distribution of con A receptor sites and anionic groups during red blood cell differentiation in the rat

The distribution of receptors for concanavalin A (Con A) and anionic groups on plasma membranes of developing blood cells was investigated in the rat. Glutaraldehyde-fixed bone marrow and circulating blood cells were exposed to ferritin-conjugated Con A or positively chaged ferric oxide (CI) and processed for electro n microscopy. The frequency of Con A and CI binding sites varied during different erythroid developmental stages and amont different leukoid cell types. There was a constant inverse relationship between Con A and CI binding sites. Among leukoid cells, Con A binding was high on plasma cells and macrophages, lower on neutrophils and lymphocytes, and still lower on eosinophils and basophils; CI binding was highest in the latter and lowest in plasma cells and macrophages. Among erythroid cells, there was a progressive increase in Con A and a decrease in CI binding after successive divisions of erythroblasts, and a progressive decrease in Con A and an increase in CI binding upon maturation of the orthochromatic erythroblast to the reticulocyte. The most pronounced modification in distribution of these sites occurred during nuclear expulsion: that protion of the plasma membrane surrounding the extruded nucleus was heavily labeled by Con A (up to four times that of the orthochromatic erythroblast) whereas the reticulocyte had considerably fewer sites. The situation was reversed with CI. The results suggest that the concentration of nonsialated glycoproteins (to which Con A binds) varies inversely to that of sialoproteins (to which CI binds) in the membrane of the differentiating erythroid cell. The remarkable changes observed at the time of nuclear extrusion suggest that there is either local modification of glycosylated groups with removal of sialyl residues from the membrane surrounding the extruded nucleus of selective segregation of membrane glycoproteins leading to a high concentration of sialoproteins (glycophroin) in the membrane of the mature erythrocyte.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

A confocal microscopic evaluation of the effects of insulin imprinting on the binding of Concanavalin A by Tetrahymena pyriformis.

With confocal microscopy it is possible to study the Concanavalin A (Con A) binding characteristics of the surface and interior of a single cell by viewing optical sections. It was observed in Tetrahymena pyriformis that Con A bound both to the plasma membrane and to intracellular structures. Incubation of cells with a competing sugar a-methylmannopyranoside, decreased binding. Hormonal imprinting with insulin resulted in an increase in binding of Con A to the cell surface and a decrease in intracellular binding. It is possible that the intracellular binding sites may migrate to the plasma membrane.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Cytotoxicity of lymphoid cells induced by Maclura pomifera (MP) lectin.

The cytotoxic activity of lymphoid cells stimulated with Maclura pomifera (MP) lectin was investigated. Spleen cells of Lewis (LEW) or Brown Norway (BN) rats induced a cell-dependent release of ⁵¹Cr from syngeneic, allogeneic, and xenogeneic erythrocytes when incubated with MP for 4–16 hr. The activity of MP differed from that of concanavalin A (Con A). MP exhibited a greater activity with spleen cells while Con A was more active when bone marrow cells were tested. Activity induced by MP required the presence of the lectin for at least 4 hr and was inhibited by melibiose, an inhibitor of MP binding. MP also stimulated phagocytosis by peritoneal macrophages of LEW rats, but phagocytosis was not responsible for the cytotoxic effect measured by ⁵¹Cr release. The ability of aggressor cells to bind MP did not correlate with their cytotoxic activity. The cytotoxic activity of spleen cells from athymic nude mice was equivalent to that of cells from euthymic littermates when stimulated with MP.     

A comparative evaluation of the level of concanavalin a binding by enriched plasma membrane fractions from developing soybean roots

The Concanavalin A (Con A) binding capacity of plasma membranes isolated from meristematic and mature regions of four-day-old soybean (Glycine max L. Merr. cv. Wells) roots was compared. Con A binding was studied using a radiochemical assay with tritiated (³H)-Con A and by an electron microscope technique using Con A-ferritin (Con A-F). In both cases, plasma membranes isolated from meristematic tissue bound significantly more Con A than did corresponding membranes from mature tissue. The relative difference in reactivity, as determined by the two procedures, was approximately 49% (³H-Con A) and 46% (Con A-F). In contrast, K_m values, determined from ³H-Con A binding curves, were approximately the same, indicating that receptor sites on plasma membranes from both sources were qualitatively similar.     

TCDD causes stimulation of c-ras expression in the hepatic plasma membranes in vivo and in vitro

A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21^ras protein, or indirectly measured by the specific binding of ³H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P < 0.05, Student t test) levels of hepatic p21^ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the differencebecame statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21^ras protein which did not become statistically significant even at 24 hr post-dosing. TCDD evoked increases in hepatic p21^ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a ³H-GTP binding protein with MW of approximately 21 Kd. ³H-GTP binding in total hepatic membranes was also elevated (P<0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of ≈ 21 Kd.     

Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton.

Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes.     

Progesterone-induced down-regulation of an electrogenic Na+, K+-ATPase during the first meiotic division in amphibian oocytes

Summary Progesterone initiates the resumption of the meiotic divisions in the amphibian oocyte. Depolarization of theRana pipiens oocyte plasma membrane begins 6–10 hr after exposure to progesterone (1–2 hr before nuclear breakdown). The oocyte cytoplasm becomes essentially isopotential with the medium by the end of the first meiotic division (20–22 hr). Voltage-clamp studies indicate that the depolarization coincides with the disappearance of an electrogenic Na⁺, K⁺-pump, and other electrophysiological studies indicate a decrease in both K⁺ and Cl^– conductances of the oocyte plasma membrane. Measurement of [³H]-ouabain binding to the plasma-vitelline membrane complex indicates that there are high-affinity (K _d-4.2×10^–8 m), K⁺-sensitive ouabain-binding sites on the unstimulated (prophase-arrest) oocyte and that ouabain binding virtually disappears during membrane depolarization. [³H]-Leucine incorporation into the plasma-vitelline membrane complex increased ninefold during depolarization with no significant change in uptake or incorporation into cytoplasmic proteins or acid soluble pool(s). This together with previous findings suggests that progesterone acts at a translational level to produce a cytoplasmic factor(s) that down-regulates the membrane Na⁺, K⁺-ATPase and alters the ion permeability and transport properties of both nuclear and plasma membranes.     

Heterogeneity of the population of lymphoma NK/Ly and leukemia L-1210 cells according to the carbohydrate structure of cell surfaces: Immunocytochemical analysis of lectin binding

This paper investigates the heterogeneity of surface carbohydrate determinants in a population of ascites cells of lymphoma NK/Ly and leukemia L-1210 according to the binding of lectins in lentil, wheat germs, peanut, and concavalin A. Bound lectins were detected by an indirect immunochemical method using obtained by the authors colloidal gold-labeled polyclonal antilectin antibodies with further development with silver acetate. Significant differences were detected in the binding of concavalin A between NK/Ly (67% Con A⁺) and L-1210 (7.2% Con A⁺) cells, while the difference in the binding of other lectins with both types of tumor cells were not significant. A relatively high percentage of PNA⁺ cells was recorded; it can indicate on a high degree of desialization of membrane glycoproteins.     

S100 and annexin proteins identify cell membrane damage as the Achilles heel of metastatic cancer cells

Mechanical activity of cells and the stress imposed on them by extracellular environment is a constant source of injury to the plasma membrane (PM). In invasive tumor cells, increased motility together with the harsh environment of the tumor stroma further increases the risk of PM injury. The impact of these stresses on tumor cell plasma membrane and mechanism by which tumor cells repair the PM damage are poorly understood. Ca²⁺ entry through the injured PM initiates repair of the PM. Depending on the cell type, different organelles and proteins respond to this Ca²⁺ entry and facilitate repair of the damaged plasma membrane. We recently identified that proteins expressed in various metastatic cancers including Ca²⁺-binding EF hand protein S100A11 and its binding partner annexin A2 are used by tumor cells for plasma membrane repair (PMR). Here we will discuss the involvement of S100, annexin proteins and their regulation of actin cytoskeleton, leading to PMR. Additionally, we will show that another S100 member – S100A4 accumulates at the injured PM. These findings reveal a new role for the S100 and annexin protein up regulation in metastatic cancers and identify these proteins and PMR as targets for treating metastatic cancers.     

Detection and immunolocalization of glycoproteins of the plasma membrane of maize sperm cells

Summary After purifying plasma membranes from isolated maize sperm cells by aqueous polymer two-phase partition, peripheral and integral proteins were solubilized from the plasma membrane with Triton X-114 and separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining revealed 10 bands (19–68 kDa) of peripheral membrane proteins and about 40 bands (12–120 kDa) of integral proteins. Peroxidase-conjugated Con A was used to detect the surface glycopeptides. It was found that Con A particularly stained 8 peripheral polypeptide bands, including 68, 66, 55, 51,48, 44, 36, and 32 kDa, and 6 integral polypeptide bands, 68, 51, 48, 44, 38, and 34 kDa. These bands differed from those of somatic samples. Staining specificity was demonstrated by the control in the presence of competing inhibitory sugar. The above result indicates the existence of mannosyl and glucosyl residues in the surface glycoproteins of maize sperm cells. The prominent peripheral 68 kDa polypeptide was further separated into 4 spots by isoelectric focusing and sodium dodecyl sulfate two-dimensional (IEF-SDS 2-D) electrophoresis, showing pI values from 5.5 to 5.8. Three prominent glycopeptides (68, 48, and 32 kDa) were localized on the plasma membrane of maize sperm cells via the fluorescein isothiocyanate (FITC) technique. About 25% of sperm cells showed an intense positive reaction in each immunological labelling. The results agree with our previous labelling of the surface of isolated viable maize sperm cells with Con A-FITC.Abbreviations FITC fluorescein isothiocyanate - Con A Canavalia ensiformis agglutinin - HRP horseradish peroxidase - RCA Ricinus communis agglutinin - WGA Triticum vulgaris agglutinin     

Use of lectins as probes for analyzing embryonic induction

Summary Lectins were used as probes to investigate the mechanism of embryonic induction. Concanavalin (Con A) and gorse agglutinin out of 7 species of lectins tested were found to have strong neural-inducing effect on the presumptive ectoderm of newt gastrulae. Their effects were abolished by the addition of -methyl-D-mannoside and -L-fucose, respectively. Succinyl-Con A had a weak inducing activity in comparison to Con A. Autoradiography of³H-Con A-treated explants revealed that Con A bound to the inner surface, but not to the outer surface of ectoderm and was successively incorporated into cytoplasm.³H-Thymidine incorporation was lower in the first half and higher in the second half of the 60 h cultivation period in Con A-treated explants as compared to controls.Con A-Sepharose had a strong inductive effect. This suggests that neural induction is caused through Con A binding to the plasma membrane, but not through incorporation into the cytoplasm of the ectoderm cells.     

Isolation of concanavalin a caps during various stages of formation and their association with actin and myosin

Regions of plasma membrane of dictyostelium discoideum amoebae that contain concanavalin A (Con A)-receptor complexes are more resistant to disruption by Triton X-100. This resistance makes possible the isolation of Con A-associated membrane fragments in sufficient quantity and homogeneity to permit the direct biochemical and ultrastructural study of receptor-cytoskeletal interactions across the cell membrane. After specific binding of Con A to the cell surface, a large amount of the cell’s actin and myosin copurifies with the plasma membrane fragments. Myosin is more loosely bound to the isolated membranes that actin and is efficiently removed by treating membranes with ATP and low ionic strength. If cells are not lysed immediately after lectin binding, all of the Con A that is bound to the cell surface is swept into a cap in a process requiring metabolic energy. When cells are lysed at different stages of cap formation, the amount of actin and myosin that copurifies with the isolated membranes remains the same. Thick and thin filaments that are attached to the protoplasmic surface of the isolated membranes underlie lectin-receptor complexes during all stages of cap formation. Once the cap is complete, the amount of actin and myosin that tightly bound to the plasma membrane is concentrated into the cap along with the Con A-receptor complexes. These results suggest that the ATP-dependent sliding of membrane-associated actin and myosin filaments is responsible for the accumulation of Con A-receptor complexes into a cap on the cell surface.     

Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.     

The role of the membrane skeleton in concanavalin A-mediated agglutination of human erythrocytes

In the erythrocyte membrane, the mobility of band 3 protein, the receptor for concanavalin A (Con A), is drastically reduced by the membrane skeleton. Yet, the vesicles free of membrane skeletal proteins, isolated from the highly agglutinable proteinase-treated cells, are found to be devoid of Con A agglutinability. The vesicles bind Con A in normal amounts, and remain agglutinable with the wheat germ and Ricinus agglutinins. Intracellular entrapment of monospecific antibodies to spectrin and 4.1 protein (two of the major skeletal components of the membrane) is also found to inhibit agglutination by 30-50%. Thus the membrane skeleton appears to play a positive role in the agglutination of the cells with Con A. The anti-ankyrin antibodies are found to be without any effect. The anti-band 3 (cytoplasmic domain) antibodies are also inhibitory to agglutination. Since Con A binding to cells alters the shape responses and deformability of the cells, and the cells resist fragmentation at 49 degrees C, the properties of the whole skeleton, especially spectrin, appear to be changed. The Con A-bound membranes also do not release the complex of spectrin-band 4.1-actin when extracted with a hypotonic medium. It appears that Con A binding leads to interaction of the cytoplasmic domain of the receptor with a skeletal component, possibly spectrin. Subsequent to this, the receptor molecules and the skeletal proteins undergo aggregation in the membrane, which is detected by their crosslinking by an 8.6-A span bifunctional reagent. The contractility believed to be associated with the membrane skeleton may be responsible for the aggregation.