Glycoproteins in the whorls of membrane produced by oligodendroglia in culture

Two glycoproteins of 99 kDa and 77 kDa which exhibit intense binding to wheat germ agglutinin have been purified from the whorls of membrane produced by oligodendroglia in culture. The whorls of membrane were isolated by gradient centrifugation from purified bovine oligodendroglia maintained in culture. The two glycoproteins were solubilized from the membranes using a non-ionic detergent and purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography, and SDS-polyacrylamide pore gradient gel electrophoresis. HPLC peptide mapping of the 99-kDa and 77-kDa glycoproteins revealed structural differences between the two proteins. Peptide mapping suggested that the 99-kDa glycoprotein from the whorls of membrane may be homologous to that from the plasma membranes. The 77-kDa glycoproteins from both sets of membrane may also be structurally related. Lectin binding studies showed that both glycoproteins from the whorls of membrane bound to wheat germ agglutinin, succinylated wheat germ agglutinin, concanavalin A, and lentil lectin, indicating the presence of high mannose and hybrid type oligosaccharide side-chains.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Purification and characterization of two glycoproteins from oligodendroglial plasma membranes

Two major glycoproteins of 99 kDa and 77 kDa have been purified from oligodendroglial plasma membranes. These two glycoproteins exhibit intense binding to the lectin, wheat germ agglutinin. The 99-kDa and 77-kDa glycoproteins were purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography and SDS-polyacrylamide pore gradient gel electrophoresis. Re-electrophoresis of excised gel slices containing the two glycoproteins demonstrated their apparent homogeneity. The isoelectric points of the 99-kDa and 77-kDa glycoproteins were 6.15 and 6.00, respectively. Peptide mapping revealed structural differences between the two glycoproteins. Lectin binding studies with radiolabeled succinylated wheat germ agglutinin demonstrated that the binding of the 99-kDa and 77-kDa glycoproteins to wheat germ agglutinin was due to N-acetyl-D-glucosamine residues in the oligosaccharide side-chains.     

Purification and characterization of plasma membrane fractions from cultured pituitary cells

Highly purified plasma membrane fractions have been prepared from GH₃ pituitary cells grown in suspension cultures. These membrane fractions have been obtained by differential and sucrose gradient centrifugation and were characterized in terms of their lipid content, marker enzyme analysis and the binding of ³H-labelled thyrotropin-releasing hormone (TRH) to its receptor. Alkaline phosphatase and 5′-nucleotidase activities were enriched 12- to 15-fold in the plasma membrane fraction with somewhat greater enrichment (28-fold) of the specific binding component for [³H]TRH, with a specific activity of 2286 fmol [³H]TRH bound per mg protein. A single class of binding sites for TRH was observed with an apparent dissociation constant of 18 nM, a value similar to that observed for intact cells. No detectable TRH binding to the nuclear fraction was observed that could not be ascribed to residual plasma membrane contamination. By electron microscopy, these fragments appeared to be sealed vesicles with an average diameter of approximately 1800 Å. The binding of ¹²⁵I-labelled wheat germ agglutinin was used as a marker for plasma membrane purification. In addition to specific binding to this membrane fraction, specific binding was also observed in the nuclear fraction. Studies with fluorescein-labelled wheat germ agglutinin revealed that, in fixed cells, fluorescence was restricted to the plasma membrane. However, if the cells were treated with Triton before labelling, most of the fluorescence was then associated with the cell nucleus. Hence, the use of wheat germ agglutinin binding as a specific plasma membrane marker must be reevaluated.     

Lectin binding to the porcine and human ileal receptor of intrinsic factor-cobalamin

The purified porcine recpptor for the intrinsic factor-cobalamin complex bound to concanavalin A, lentil lectin and wheat germ lectin covalently coupled to Sepharose and was eluted with the corresponding soluble sugars. In contrast, human intrinsic factor bound efficiently to concanavalin A, to some extent to lentil lectin, but only slightly to wheat germ agglutinin. The binding of IF-Cbl to the receptor was inhibited when the receptor was pre-incubated with soluble wheat germ aglutinin, with an inhibition constant estimated to be 1.9 mol/l. After transfer of the purified receptor from SDS-PAGE to Immobilon, ligand blotting of the purified receptor with iodinated lectin showed that concanavalin A and lentil lectin bound to three (75, 56 and 43 kDa) components but that wheat germ agglutinin bound only to the 75 kDa component. These results showed that the subunit of the receptor could bind to wheat germ agglutinin, resulting in an inhibition of its binding with intrinsic factor. Both binding sites of intrinsic factor and of wheat germ agglutinin could be located near to each other.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various various lectins is Ricinuscommunis > wheat germ concanavalin A soybean >Limuluspolyphemus. No agglutination was noted for Ulex europaeus. Using ¹²⁵I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites as sites for concanavalin A and soybean lectins. Sodium deoxy-cholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity colums. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Effect of trypsinization on lectin binding to germ cells from ICR and T/t6 mice

Flow cytometry was used to quantify the binding of fluorescein isothiocyanate (FITC)-labeled lectins to testis cells from ICR and T/t6 mice before and after trypsin treatment. Soybean agglutinin, wheat germ agglutinin, and concanavalin A bound well to testis cells of both mouse strains. Limax flavus agglutinin (LFA) bound very slightly and Ulex europeas agglutinin (UEA) did not bind at all. Trypsinization increased binding of soybean agglutinin and decreased binding of wheat germ agglutinin in both mouse strains, providing evidence for masked carbohydrate-binding sites on the surface of germ cells. It did not affect binding of the other lectins. Trypsin treatment was an attempt to increase lectin binding, particularly the binding of LFA and UEA to the reported T/t-specific carbohydrates, sialic acid, and L-fucose, respectively. These studies indicate that the T/t6 locus alleles do not alter the surface carbohydrate content of testis cells sufficiently to be detected by lectin-binding differences.     

Properties of succinylated wheat-germ agglutinin.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.     

Qualitative and quantitative interactions of lectins with untreated and neuraminidase-treated normal, wild-type, and temperature-sensitive polyoma-transformed fibroblasts.

The lectin receptors of confluently grown hamster BHK, wild type polyoma virus transformed PyBHK, and temperature-sensitive polyoma transformed ts3-PyBHK fibroblasts were investigated using cell agglutination, quantitative (125I)lectin binding, and ferritin-lectin labeling. PyBHK and permissively grown ts3-PyBHK cells agglutinated more strongly with Ricinus communis I agglutinin (RCA-I)compared to BHK and nonpermissively grown ts3-PyBHK, although saturation binding of (125I)RCA-I to these cells at 4 degrees resulted in a twofold difference in lectin-binding sites on BHK and nonpermissively grown ts3-PyBHK cells (1.0-1.3 x 10 7 sites/cell) compared to PyBHK and permissively grown ts3-PyBHK (0.4-0.6 x 10 7 sites/cell). These cells bound equivalent amounts of (125I)concanavalin A (0.8-1 x 10 7 sites/cell) and (125I)wheat germ agglutinin (1-2.2 x 10 7 sites/cell). Under these binding conditions little endocytosis occurred, as judged by the subsequent release of greater than 90% cell-bound (125I)RCA-I by the RCA-I inhibitor lactose and localization of ferritin-RCA-I exclusively to the extracellular plasma membrane surface. However, if the binding is performed at 22 degrees, only 50% of the bound lectin can be removed by lactose, and ferritin-RCA-I is localized inside the cell within endocytotic vesicles. The relative mobility of RCA-I receptors was examined on ts3-PyBHK cells by the ability of ferritin-RCA-I to induce clustering of its receptors at 22 degrees. RCA-I receptors on permissively grown ts3-PyBHK cells appeared to be more mobile than on nonpermissively grown cells. BHK and PyBHK cells were treated with neuraminidase, and the resulting enzyme-treated cells were assayed for lectin agglutinability and quantitative binding of RCA-I, concanavalin A, and wheat germ agglutinin. Neuraminidase treatment resulted in decreased concanavalin A and wheat germ agglutinability and a slight increase in RCA-I agglutinability. The enzyme-treated BHK and PyBHK cells bound less (125I)wheat germ agglutinin (2.8 x 10 6 and 2.2 x 10 6 sites/cell, respectively) and 2.5 and 6.2 times more (125I)RCA-I (2.5-3 x 10 7) and 3.5-4 x 10 7 sites/per cell, respectively). There was no change in the number of concanavalin A binding sites after neuraminidase treatment. The increase in RCA-I binding sites approximated the decrease in wheat germ agglutinin binding sites indicating that the predominant penultimate oligosaccharide residue to sialic acid on these cells is D-Gal.     

Quantitation of lectin binding sites in human colon mucins by use of peanut and wheat germ agglutinins

We have developed a novel method for quantitation of lectin binding sites in mucins derived from colon tissues. Binding of peanut agglutinin and wheat germ agglutinin was measured in extracts from normal and malignant human colon epithelium. Binding of wheat germ agglutinin was used as an estimate of the total mucin present in the tissue extract. Peanut agglutinin was found to bind to mucin from normal colon, but at levels that may be difficult to appreciate by fluorescence microscopy. The yield of mucin extracted from colon cancer was more variable than that from normal colon, and the binding ratio of peanut agglutinin to wheat germ agglutinin was greater in extracts from tumors than in normal tissues. Our findings confirm the histological observation that peanut agglutinin binds more avidly to mucins from colon cancer than to those from normal colon. The finding of peanut agglutinin binding sites in mucins front normal colon was not expected. The quantitative technique may have detected small numbers of binding sites not readily appreciable by fluorescence microscopy. Alternatively, the chromatographic method for measuring lectin binding may be sufficiently sensitive to detect nonspecific binding of the lectin to terminal galactose residues other than the Thomsen-Friedenreich antigen.     

Lectin-induced inhibition of plasma membrane 5′-nucleotidase: Sensitivity of purified enzyme

To determine whether the lectin-induced inhibition of plasma membrane 5′-nucleotidase resulted from direct interaction of the lectin with the enzyme or indirectly from a membranous change due to lectin binding to other membrane glycoproteins, the enzyme was purified and its sensitivity tested in the absence of other membrane components. A 5000 fold purification was achieved by solubilization in Lubrol PX followed by gel filtration (Sephadex G-100), anion exchange (DEAE-Biogel A) and selective adsorption (hydroxylapatite) chromatography. The purified enzyme was even more sensitive to inhibition by high concentrations of concanavalin A, wheat germ agglutinin or Rincinus communis agglutinin than was the membrane-bound enzyme indicating that inhibition is due to direct binding of the lectins to the glycoprotein enzyme itself. Divalent succinyl Con A inhibited neither form of the enzyme suggesting the need for crosslinking for inhibition by the native lectin. The purified enzyme could not be activated by low concentrations of lectins which stimulated the membrane bound enzyme.     

Lectin receptors in the salivary glands of the rat during postnatal ontogenesis

Distribution of lectin-binding sites in rat submandibular and sublingual salivary glands during postnatal development has been investigated. Lectin preparations include con A, lentin lectin, castor beans agglutinin, peanut, soybean and Sophora japonica agglutinins, wheat germ agglutinin and lectin from the bark of Laburnum anagyroides. The direct and indirect peroxidase techniques are used. According to the similarities of histochemical patterns, all lectins are divided into four groups. Besides the general patterns of lectin binding sites, some details are noted. Lectins of peanut and Sophora japonica possess an extremely high affinity to mast cells, con A, lens lectin, castor beans and wheat germ agglutinins--to serous demilunes cells. Laburnum lectin--to salivary ducts epithelia in adult rat salivary glands. Lentin lectin, con A and Laburnum lectin preferentially stain cells with specific granularity in granular ducts at early stages of postnatal development. Considering the character of staining, we propose for further histochemical investigations of the salivary glands lentin lectin, peanut agglutinin, wheat germ agglutinin and Laburum anagyroides lectin.     

Characterization of cell membrane and sarcoplasmic reticulum from bovine uterine smooth muscle

Subcellular fractions, enriched in sarcoplasmic reticulum or in cell membrane, were separated from one another. Starting material was a microsomal pellet (15–40 × 1000g) obtained by differential centrifugation from the uteri of close-to-term pregnant cows. A microsomal fraction enriched in ATP-dependent calcium accumulation was shown to contain sarcoplasmic reticulum and cell membrane. Only 8% or less of the protein in this fraction could be recovered, using affinity chromatography on Sepharose 6MB wheat germ agglutinin. The small yields did not allow extensive characterization. A method was developed to separate sarcoplasmic reticulum from cell membrane using discontinuous sucrose density gradient centrifugation. Protein was collected at the 24–28, the 28–33, and the 33–45% sucrose interfaces. Characterization was by enzyme assays and by specific receptor assay. ATP-dependent calcium accumulation was fourfold greater in the 24–28% sucrose layer than in the 33–45% layer. In contrast, 5′-nucleotidase was more than threefold as high in the 33–45% sucrose layer as in the 24–28% layer. Ouabain-inhibited p-nitrophenylphosphatase doubled and ouabain-inhibited Na,K-ATPase tripled in the 28–33% layer, compared with the 24–28% layer, specific ouabain binding was also doubled in the 28–33% sucrose layer. ¹²⁵I-Labeled wheat germ agglutinin binding was greatest in the 33–45% sucrose layer. It is concluded that the 24–28% layer consists primarily of sarcoplasmic reticulum, whereas the 28–33 and the 33–45% layers are concentrated in the cell membrane. Specific prostaglandin (PGE₂) binding was found to be a property of the cell membrane.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

The Involvement of a Class of Cell Surface Glycoconjugates in Pseudoplasmodial Morphogenesis in Dictyostelium discoideum

Pseudoplasmodia of the cellular slime mold Dictyostelium discoideum were mechanically dissociated into cells and treated with the lectin, wheat germ agglutinin, or an antipseudoplasmodial cell antibody. Morphogenesis of the agglutinates and cell differentiation were reversibly interrupted by binding of these proteins to the cell surface. This demonstrates that one or more wheat germ agglutinin receptors and the pseudoplasmodial antigen on the cell surface of these cells play a critical role in development after aggregation.     

Biochemical fractionation and characterization of proteins from Golgi-enriched membranes.

Fractions enriched in Golgi membranes were prepared from rat liver by sucrose gradient ultracentrifugation. These enriched membranes were further subfractionated on the basis of their solubilities in EGTA, 150 mM sodium carbonate, pH 11.5, sodium deoxycholate, Triton X-100, or sodium dodecyl sulfate. This led to isolation of peripheral, luminal, and integral membrane proteins of the Golgi-enriched membranes. Luminal and membrane proteins were further purified by wheat germ agglutinin and concanavalin A lectin affinity chromatographies. Some proteins from these lectin columns were resolved by preparative gel electrophoresis and microsequenced. Subsequently, antibodies were produced for two proteins by immunization of either mice or rabbits. Immunofluorescence microscopy suggests that these proteins are confined to Golgi apparatus-like structures. The protocol described is well suited for the study of organelle structure and function.     

Characterization of surface glycoproteins on Schistosoma mansoni adult worms by lectin affinity chromatography

Adult Schistosoma mansoni were radiolabeled by direct radioiodination using the Bolton-Hunter reagent or by metabolic labeling using radioactive hexose precursors. Tegumental material was extracted by freeze-thaw or by incubation in the non-ionic detergent Nonidet P-40, then applied to chromatography columns containing the following immobilized lectins: Con A, lentil lectin, wheat germ agglutinin, soybean agglutinin and the agglutinins from Ricinus communis and Helix pomatia. SDS-PAGE analysis of the sugar eluates from these columns revealed the presence of 15 glycoproteins with apparent molecular weights greater than or equal to 300,000, 215,000, 168,000, 152,000, 134,000, 122,000, 108,000, 83,000, 58,000, 53,000, 46,000, 41,000, 34,000, 30,000 and 23,500. Many of the glycoproteins reacted with more than one lectin. Information about carbohydrate content and lectin binding provides a preliminary characterization of the tegumental glycoprotein antigens of adult worms.     

Incorporation of acylated wheat germ agglutinin into liposomes

Purified wheat germ agglutinin (WGA) was derivatized with palmitic acid at an average stoichiometry of one fatty acid per dinner. Palmitoyl WGA was readily incorporated into liposomes with a cholate-dialysis method. Liposome-bound WGA caused agglutination of red blood cells at a concentration eight-fold lower than that of the native lectin. Furthermore, enhanced binding of liposome-bound WGA to mouse spleen cells was also observed. Potential applications of the liposome-bound lectin are discussed.     

Lectin receptors in Trypanosoma cruzi. An N-acetyl-D-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 131I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent Mr 85000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unaffected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since N-acetyl-D-glucosamine inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface N-acetyl-D-glucosamine-specific receptor activity.