Occurrence of novel Cu2+-dependent sialic acid-specific lectin,on the outer surface of mature caprine spermatozoa

Effects of several bivalent metal ions on the autoagglutination event in mature caprine epididymal sperm cells have been investigated using a chemically defined medium. This study demonstrates for the first time that Copper (Cu²⁺) ion (300 μM) has high specificity for autoagglutination of mature cauda-epididymal sperm. Head-to-head interaction of the male gametes is responsible for this event. Studies on the effect of various sugars reveal that the autoagglutinated cells can be dissociated specifically with neutralized sialic acid (50 mM), which also inhibits the sperm cell autoagglutination phenomenon. Blood serum protein fetuin, that contains terminal sialic acid residue, showed high efficacy for inhibiting this autoagglutination event at 4 μM concentration. However, asialofetuin is not capable of inhibiting this Cu²⁺-dependent cellular event. Mature sperm cells bound with caprine erythrocytes at their head region in presence of Cu²⁺ ion. The purified sperm membrane fraction isolated by aqueous two phase polymer method showed high efficacy to agglutinate erythrocytes. These sperm-erythrocyte interactions as well as sperm membrane induced haemagglutination were strongly blocked by neutralized sialic acid (50 mM). The results confirm the occurrence of unique Cu²⁺ dependent, sialic acid-specific lectin on the outer surface of a mammalian cell using caprine sperm as the model. The observed Cu²⁺-mediated cellular autoagglutination is caused by the interaction of the cell surface lectin with the lectin receptor on the surface of the neighboring homologous cell.     

Synchronous modulation of cell surface lectin and its receptor in a homologous cell population: a novel mechanism of cellular regulation

Testicular immotile sperm undergo maturation during epididymal transit when these cells pass through caput, corpus, and cauda-epididymal regions. Maturing goat spermatozoa specifically at the distal corpus epididymal stage show head-to-head autoagglutination when incubated in vitro in a modified Ringer's solution. Here, we show the biochemical mechanism of autoagglutination event and its functional significance. A lectin-like molecule located on sperm surface specifically interacts with its receptor of the neighboring homologous cells to cause autoagglutination. Lectin is a Ca++-dependent galactose-specific protein. Failure of the pre- and post-distal corpus sperm to show autoagglutination is due to lack of lectin-like molecule and its receptors, respectively. Maturing sperm at distal corpus stage acquire lectin-like molecule followed by sharp disappearance of its receptor, and this event is synchronously associated with the initiation of sperm forward motility that is essential for fertilization in vivo. Lectin and its receptor isolated from sperm plasma membrane showed high efficacy for blocking autoagglutination phenomenon. The data are consistent with the view that synchronous modulation of homologous cell surface lectin and their receptors constitutes a novel mechanism for cellular regulation by generating waves of signals by manipulating lectin-sugar-dependent "self-talk" and cell-cell "cross-talk".     

Cell surface carbohydrates in adhesion and migration

Cell surface sugar chains extend away from the cell membraneand offer a first line of contact with approaching cells andsubstrates. These sugars are candidates for mediating cell-celladhesion and migration. Here, I review experiments that implicatecarbohydrate-containing molecules in cell-cell adhesion of ascitestumor and embryonic cells and that correlate the mobility ofcarbohydrate-containing receptor sites in the membrane withcellular migratory activity. The experiments show that L-glutamineis required to form complex carbohydrates implicated in mediatingintercellular adhesion and that a controlling factor in determiningcellular adhesiveness may be the specific activity of intracellularglutamme synthetase. Molecules that promote ascites tumor celladhesion have been isolated. These molecules are large, appearto contain terminal D-galactose residues that bind to cell surfacereceptor sites and consist of more than one component on DEAEcellulose. Studies with sea urchin embryonic cells, utilizingplant lectins that bind to surface carbohydrates, indicate thatcell surface sugar-containing receptor sites change during development.In addition only the micromeres, that become actively migratory,possess mobile cell surface lectin receptor sites. Other seaurchin embryonic cell populations (mesonieres and macromeres)do not exhibit lectin receptor site mobility. Cell surface sugar-containingreceptor sites potentially mediate adhesion and migration inembryos and tumors.     

Involvement of blood-group-B-active trisaccharides in Ca2+-dependent cell-cell adhesion in the Xenopus blastula

 Despite their wide distribution in various organisms, no physiological roles have been proposed for the human blood-group-ABO (ABH)-active trisaccharides. Here we show that monoclonal antibodies against human blood-group-B-active trisaccharides (B-substance) completely block the Ca²⁺-dependent cell-cell adhesion system of frog (Xenopus laevis) embryonic cells. Synthetic B-substance or B-active glycopeptides also disrupt the Ca²⁺ -dependent cell-cell adhesion. These results suggest that blood-group-B-active substances play a role in cell-cell adhesion. Blood-group-B-active substances were found as glycoproteins and as glycosphingolipids. In order to identify B-active glycoproteins active in cell-cell adhesion, we purified B-active membrane glycoproteins by two-dimensional electrophoresis and found that they are 45- to 58-kDa proteins with pI(s) ranging from 4.0 to 5.3. They are glycosylphosphatidyl inositol (GPI) anchored. Amino acid sequence analysis showed that the purified B-active GPI-anchored proteins are homologues of soluble Xenopus cortical granule lectins (CGL). The results suggest that the B-active membrane glycoproteins are GPI-anchored forms of the lectin and are directly involved in frog Ca ²⁺-dependent cell-cell adhesion. Received: 16 September 1997 / Accepted 19 November 1997     

Inositol- and Galactose-Binding Lectins of Chicken Brain Fractions Enriched with Glial Cells

Galactose- and inositol-binding proteins with lectin activity (GL-GAL and GL-I, respectively) were isolated from membranes enriched with cells of chicken brain fractions. Both lectins are glycoproteins of molecular mass 13.5 and 11.5 kDa, respectively; they show a high affinity to EDTA (GL-I) and EGTA (GL-GAL, GL-I), which indicates an important role of Ca⁺² in molecular organization of these lectins. In brain glial cells of chick embryos, unlike adult chickens, a soluble form of lectins has been revealed; it is easily extracted with 2 mM EDTA and shows sensitivity to L-lactose, D-galactose, and N-acetyl-D-galactosamine. It is suggested that in the course of embryonal and postembryonal development of the chicken brain, a transformation and qualitative changes of the lectin spectrum occur due to a change of function of glial cells.     

Isolation and cloning of a C-type lectin from the hexactinellid sponge Aphrocallistes vastus: a putative aggregation factor

Among the sponges (Porifera), the oldest group of metazoans in phylogenetic terms, the Hexactinellida is considered to have diverged earliest from the two other sponge classes, the Demospongiae and Calcarea. The Hexactinellida are unusual among all Metazoa in possessing mostly syncytial rather than cellular tissues. Here we describe the purification of a cell adhesion molecule with a size of 34 kDa (in its native form; 24 kDa after deglycosylation) from the hexactinellid sponge Aphrocallistes vastus. This adhesion molecule was previously found to agglutinate preserved cells and membranes in a non-species-specific manner (Müller, W. E. G., Zahn, R. K, Conrad, J., Kurelec, B., and Uhlenbruck, G. [1984] Cell adhesion molecules in the haxactinellid Aphrocallistes vastus: species-unspecific aggregationfactor. Differentiation, 26, 30--35). The fact that the aggregation process required Ca(2+) and was inhibited by bird's nest glycoprotein and D-galactose but not by D-mannose or N-acetyl-D-galactosamine suggests that this cell adhesion molecule is a C-type lectin. To test this assumption, two highly similar C-type lectins were cloned from A.vastus. The deduced polypeptides of the two cDNA species isolated classified these molecules as C-type lectins. The calculated M(r) of the 191 aa long sequences were 22,022 and 22,064, respectively. The C-type lectins showed highest similarity to C-type lectins (type-II membrane proteins) from higher metazoan phyla; these molecules are absent in non-Metazoa. The two sponge C-type lectins contain the conserved domains known from other C-type lectins (e.g., disulfide bonds, the amino acids known to be involved in Ca(2+)-binding, as well as the amino acids involved in the specificity of binding to D-galactose) and a hydrophobic N-terminal region. The N-terminal part of the purified C-type lectin was identical with the corresponding region of the deduced polypeptide from the cDNA. It is proposed that the A.vastus lectins might bind to the cell membrane by their hydrophobic segment and might interact with carbohydrate units on the surface of the other cells/syncytia.     

Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.     

Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.     

Studies on cellular adhesion of Xenopus laevis melanophores: modulation of cell-cell and cell-substratum adhesion in vitro by endogenous Xenopus galactoside-binding lectin

We have investigated cell-cell and cell-substratum adhesion of Xenopus laevis neural crest cells at various stages of melanophore differentiation. Single-cell suspensions were obtained by trypsinization and aggregated in a cell-cell adhesion assay. Unpigmented cells did not adhere while the rate of adhesion of melanophores correlated with the degree of melanization. Melanophore cell-cell adhesion decreased significantly in the presence of beta-galactosidase, which suggests that cell-surface galactose is involved. Beta-galactoside-binding lectin has been isolated and purified from embryos at the stage of neural crest migration. When added to aggregating cells smaller, looser clusters formed compared to controls. When lectin was added to cells in stationary culture to test cell-substratum adhesion, melanophores spread more smoothly and formed more regular spacing patterns. These results suggest that this lectin can modulate receptors used in cell-cell and cell-substratum adhesion of melanophores.     

Calcium-dependent adhesion of Drosophila embryonic cells

Summary By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca²⁺-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca²⁺ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca²⁺. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca²⁺ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins.     

Endogenous lectins as mediators of tumor cell adhesion

Endogenous carbohydrate-binding proteins have been found in various normal tissues and cells. Although lectins with different sugar-binding specificities have been described, the most prevalent ones are those that bind beta-galactosides. The ability of some normal and malignant cells to bind exogenous carbohydrate-containing ligands suggested that lectinlike activity is associated with the cell surface and that carbohydrate-binding proteins might mediate intercellular recognition and adhesion. We found that extracts of various cultured murine and human tumor cells exhibit a galactoside-inhibitable hemagglutinating activity. This activity was associated with two proteins of molecular weights of 34,000 and 14,500 daltons, which were purified by affinity chromatography by using immobilized asialofetuin. That these lectins are present on the cell surface was indicated by the binding of monoclonal antilectin antibodies to the surface of various tumor cells and by the immunoprecipitation of 125I-labeled lectins from solubilized cell-surface iodinated cells by polyclonal antilectin antibodies. That these cell surface lectins are functional was demonstrated by the ability of the galactose-terminating asialofetuin to enhance cell aggregation and of asialofetuin glycopeptides to block this homotypic aggregation as well as to suppress cell attachment to substratum, and by the inhibition of both asialofetuin-induced cell aggregation and cell attachment to substratum by the binding of monoclonal antilectin antibodies to the cell surface. These findings implicate cell surface lectins as mediators of cell-cell and cell-substratum adhesion. Some of these cellular interactions might be important determinants of tumor cell growth and metastasis.     

Complex carbohydrate-lectin interaction at the interface: a model for cellular adhesion. II. Reactivity of both the oligosaccharide chain and sugar-binding domain of a glycoprotein lectin.

We describe studies of a new model cell adhesion system involving liposomes bearing lectins and the glycosphingolipid, asialomonosialoganglioside (asialoGM1). The model provides a simple analysis of experimental data to elucidate the mechanism of heterophilic cell-cell adhesion mediated by multiple protein-carbohydrate interactions. Phospholipid vesicles bearing the fatty acid conjugate of a glycoprotein lectin from Ricinus communis (RCAI vesicle) are shown to react with vesicles bearing the fatty acid conjugate of Concanavalin A (Con A) and asialoGM1 (Con A vesicle). The kinetics of aggregation and monosaccharide-induced disaggregation of the two types of vesicles were followed by monitoring the time-dependent change in turbidity. Depending on the surface density of the asialoGM1, 40-60% of the resulting precipitin complex was dissociable only in the presence of both RCAI-specific galactose and Con A-specific alpha-methyl-D-mannoside. Results indicate simultaneous participation of both the saccharide-binding domain and carbohydrate sequence of RCAI, a model cell adhesion molecule, to stabilize the encounter complex by two types of interactions. These findings support the possibility of stable cell-cell adhesion in vivo occurring via interactions between cell adhesion molecules on apposing cell-surface membranes.     

Changes in the cell surface coat during the development ofXenopus laevis embryos,detected by lectins

Summary The composition of the surface coat in embryonic cells ofXenopus laevis was examined by agglutination and fluorescent staining with lectins.Cells of early and mid gastrula stages were agglutinated by lectins specific for D-mannose, D-galactose, L-fucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. No differences in agglutinability among ectoderm, mesoderm and endoderm cells were observed with lectins specific for D-mannose, D-galactose and N-acetyl-D-galactosamine, though agglutination of gastrula cells with fluorescent lectins revealed considerable differences in the intensity of lectin binding among cells within an aggregate. These differences in amount of lectin bound were not related to cell size or morphology. Patches of fluorescent material formed on the cells, suggesting that lectin receptors are mobile in the plane of the plasma membrane.In the early cleavage stages intensive lectin binding occurs only at the boundary between preexisting and nascent plasma membranes. The external surface of the embryo has few lectin receptors up to the late gastrula stage. The unpigmented nascent plasma membranes, when exposed to fluorescent lectins, do not assume any fluorescence distinguishable from the background autofluorescence of yolk, in stages up to the mid-blastula. From this stage onwards lectin binding was observed on the membranes of the reverse side of surface layer cells and on the membranes of deep layer cells. During gastrulation there is an accumulation of lectin-binding material on surfaces involved in intercellular contacts.The significance of lectin binding material for morphogenesis is discussed.     

Intracellular Ice Formation Is Affected by Cell Interactions

Cell-to-cell and cell-to-surface interactions are important to the structure and function of tissues. These interactions are also important determinants of low-temperature responses in tissues. Four in vitro models using hamster fibroblast cells in tissue culture were used to investigate the influence of cell-cell and cell-surface interactions on intracellular ice formation in these systems. The four models were: (a) single cells in suspension; (b) cells individually attached to glass with only cell-to-surface adhesion; (c) colonies of cells attached to glass with both cell-cell and cell-surface interactions; and (d) multicellular spheroids with extensive cell-cell contacts. Cryomicroscopy was used to monitor the prevalence and kinetics of intracellular ice formation after ice nucleation in the extracellular solution. The temperature for intracellular freezing in 50% of the cells was significantly affected by both cell-cell and cell-surface interactions. There was also evidence of intercellular nucleation through cell-cell interactions. The results indicate that cell-cell and cell-surface interactions play a significant role in the low-temperature response of tissue systems.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

Galactosyl receptor in human testis and sperm is antigenically related to the minor C-type (Ca2+-dependent) lectin variant of human and rat liver

Galactosyl receptor, a cell surface Ca²⁺-dependent lectin with binding affinity for galactose, was evaluated by immunoblotting, immunoprecipitation, Northern blotting, and immunocytochemistry in human liver, testis, and sperm. Polyclonal antisera raised against the minor asialoglycoprotein receptor variant of rat hepatocytes (designated rat hepatic lectin-2/3, RHL-2/3), and its human liver-equivalent (designated H2), recognize native galactosyl receptor in the testis and sperm in immunoblotting, immunoprecipitation, and immunocytochemical experiments. An equivalent to the major hepatocyte asialoglycoprotein receptor variant (rat RHL-1 and human H1) was not detected. Human testis and sperm galactosyl receptor was resolved, after immunoprecipitation and immunoblotting, as a single protein component of molecular mass 50 kD. The single protein component in human testis and sperm contrasted with the doublet nature of rat testis and sperm galactosyl receptor, consisting of two components of molecular masses of 54 and 49 kD. Northern blotting experiments using radiolabeled H1 and H2 cDNA probes confirmed the presence of H2 mRNA and the lack of H1 mRNA in the human testis. Immunocytochemical studies detected specific antigenic sites on the entire surfaces of spermatogenic cells. However, immunoreactivity in epididymal and ejaculated sperm was confined to head surfaces overlying the acrosome. Results from these studies, and from previous studies in the rat, suggest that the testis/sperm galactosyl receptor is a C-type Ca²⁺-dependent lectin with possible roles in cell-cell interaction during spermatogenesis and sperm-zona pellucida binding at fertilization. © 1995 Wiley-Liss, Inc.     

Identification and purification of an endogenous receptor for the lectin pallidin from Polysphondylium pallidum

We report the identification and purification of an endogenous carbohydrate-containing receptor of pallidin, the cell surface lectin implicated in mediating cell-cell adhesion in the cellular slime mold Polysphondylium pallidum. The receptor is identified in an aqueous extract of crude P. pallidum membranes as a potent inhibitor of the hemagglutination activity of pallidin. The inhibitor is purified to apparent homogeneity by affinity precipitation with pallidin followed by fractionation of the solubilized precipitate on Sepharose 4B. The hemagglutination inhibitor (HAI) is metabolically radiolabeled, indicating that it is a biosynthetic product of the amoebae and not an ingested food substance. The HAI is released into the extracellular medium by living, differentiated amoebae. This release is markedly facilitated by the addition of D-galactose, a specific saccharide that binds to pallidin. Hence, the HAI appears to have an in situ association with pallidin at the cell surface. Exogenously added HAI promotes the agglutination of differentiated amoebae in a gyrated suspension at very low concentrations. The results are consistent with a model of cell-cell adhesion in which the HAI is a multivalent, extracellular aggregation factor that is recognized by pallidin molecules on adjacent cells. The HAI would then be analogues to the aggregation factors identified in marine sponges.     

A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates

We have isolated two Ca(2+)-dependent, galactose-binding polypeptides from the budding tunicate, Polyandrocarpa misakiensis. Based on their partial amino acid sequences, full-length cDNAs were cloned. One of them was identical with a tunicate C-type lectin (TC14-2) reported previously. The other was a novel C-type lectin, referred to as TC14-3. In living animals, they appeared to be coupled. This complex of lectins, when applied in vitro to tunicate multipotent cells of epithelial origin, blocked cell proliferation and induced cell aggregation. The aggregates expressed a homolog of the integrin alpha-chain and other differentiation markers specific for epithelial cells. Recombinant TC14-3 could reproduce all the activities of native lectins by itself, which was accelerated by recombinant TC14-2. The inhibitory activity of TC14-3 on cell growth was completely abolished by the addition of 50 microM D-galactose. Anti-TC14-3 monoclonal antibody showed that the antigen was expressed constitutively by the multipotent epithelial and mesenchymal cells. These results provide evidence that in P. misakiensis a C-type lectin plays a novel, cytostatic role in regulating cell growth, cell adhesion and cell differentiation during asexual reproduction.     

Plant lectins specific for N-acetyl-β-D-galactosamine

N-Acetyl-D-galactosamine in β-linkage being ubiquitous in cell surface glycoproteins, their interaction with lectins specific for this sugar moiety may be a significant event in cell adhesion phenomena. This article discusses the common β-N-acetyl galactosamine-specific lectins, with particular stress on the lectin from winged beans (Psophocarpus tetragonolobus).     

Erythro- and lymphoagglutinins of Phaseolus acutifolius

A potent lymphoagglutinin which had low affinity for red cells or fetuin and another lectin which reacted strongly with red cells and fetuin but was a poor agglutinin for lymphocytes were isolated from seeds of Phaseolus acutifolius. A number of other lectin components with intermediate activity towards these cells was also isolated. All the lectins had very similar amino acid and carbohydrate composition, sedimentation patterns, partial specific volume and molecular weight values of about 116 600 and were thus smaller than the related Phaseolus vulgaris lectins (M_r = 119 000). The lectins contained four subunits with only minor size and charge differences between the lympho- and erythroagglutinating subunits and their electrophoretic mobility in SDS gel electrophoresis was anomalously high. The existence of lympho- and erythroagglutinating subunits in two members of the genus Phaseolus supports their close morphological similarity.