Developmental regulation of neuraminidase-sensitive lectin-binding glycoproteins during myogenesis of rat L6 myoblasts.

Intact monolayers of L6 myoblasts were treated with neuraminidase, with the aim of selectively removing sialic acid residues of cell-surface glycoproteins. Neuraminidase treatment unmasked binding sites for Ricinus communis agglutinin I and peanut agglutinin, thus allowing the identification of the major binding proteins for these lectins. For Ricinus communis agglutinin I these neuraminidase-sensitive glycoproteins had apparent Mr values of 136000, 115000, 87000, 83000 and 49000. For peanut agglutinin the major neuraminidase-sensitive glycoproteins had apparent Mr values of 200000, 136000, 87000 and 83000. We found highly reproducible, developmentally regulated, changes in the lectin-binding capacity of certain of these glycoproteins as L6 myoblasts differentiated into myotubes. Coincident with myoblast fusion there was a co-ordinate decrease in Ricinus communis agglutinin I binding by glycoproteins of apparent Mr of 136000 and 49000. There was also a co-ordinate shift in mobility of the broad band of glycoprotein, centred at an apparent Mr of 115000 in myoblasts, to a new average apparent Mr of 107000 in mid-fusion cultures and myotube cultures. Peanut agglutinin binding by the major protein of apparent Mr 136000 also decreased at the mid-fusion stage of myogenesis, and was barely detectable in 7-day-old fused cultures. These developmentally regulated changes in neuraminidase-sensitive glycoproteins were all inhibited by growth of myoblasts in 6.4 microM-5-bromo-2'-deoxyuridine, indicating that they are associated with myoblast differentiation. In contrast, an increase in fibronectin was seen in mid-fusion cultures, which was not inhibited by growth of myoblasts in 5-bromo-2'-deoxyuridine. This initial increase in fibronectin is, therefore, unlikely to be directly related to myoblast fusion or differentiation.     

The preparation of rat liver lysosome membranes. Several membrane proteins contain complex oligosaccharides

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze-thaw shock and membranes were isolated. The release of beta-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.     

Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents

A rapid and convenient method was established for analysis of the N-linked carbohydrate chains of glycoproteins on nitrocellulose sheets. Proteins were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, reacted with peroxidase-coupled lectins, and detected by color development of the enzyme reaction. Four glycoproteins having N-linked oligosaccharide chains were used as test materials: Taka-amylase A (which has a high-mannose-type chain), ovalbumin (high-mannose-type chains and hybrid-type chains), transferrin (biantennary chains of complex type), and fetuin (triantennary chains of complex type and O-linked-type chains). Concanavalin A interacted with Taka-amylase A, transferrin, and ovalbumin but barely interacted with fetuin. After treatment of the glycoproteins on a nitrocellulose sheet with endo-beta-N-acetylglucosaminidase H, transferrin reacted with concanavalin A but Taka-amylase A and ovalbumin did not. Wheat germ agglutinin interacted with Taka-amylase A but not ovalbumin; therefore, they were distinguishable from each other. Fetuin and transferrin were detected by Ricinus communis agglutinin or peanut agglutinin after removal of sialic acid by treatment with neuraminidase or by weak-acid hydrolysis. Erythroagglutinating Phaseolus vulgaris agglutinin detected fetuin and transferrin. Thus, the combined use of these procedures distinguished the four different types of N-linked glycoproteins. This method was also applied to the analysis of membrane glycoproteins from sheep red blood cells. The terminally positioned sugars of sialic acid, alpha-fucose, alpha-galactose, and alpha-N-acetylgalactosamine were also detected with lectins from Limulus polyphemus, Lotus tetragonolobus, Maclura pomifera, and Dolichos biflorus, respectively.     

Glycoproteins histochemistry of the gills of Odontesthes bonariensis (Teleostei, Atherinopsidae)

The histochemistry of glycoproteins (GP) in the mucous cells of the gills of the silverside Odontesthes bonariensis was identified with: (1) oxidizable vicinal diols; (2) sialic acid and some of their chain variants, carbon 7 ((7) C), carbon 8 ((8) C) or carbon 9 ((9) C); (3) sialic acid residues without O-acyl substitution and with O-acyl substitution at (7) C, (8) C or (9) C; (4) carboxyl groups and (5) sulphate groups. A battery of seven biotinylated lectins allowed GPs sugar residues to be distinguished. Mucous cells showed the presence of neutral, sulphated and sialylated GPs. Dolichos biflorus agglutinin (DBA) and Glycine max agglutinin (SBA) showed strong positive staining; Arachis hypogaea agglutinin (PNA), Ricinus communis agglutinin-I (RCA-I) and Triticum vulgaris agglutinin (WGA) showed moderate staining, while Ulex europaeus agglutinin-I (UEA-I) was completely negative.     

Localization of prostatic glycoconjugates by the lectin-gold method.

The glycoconjugates of the lateral prostate were examined ultrastructurally by lectin-gold histochemistry in combination with a low-temperature embedding technique using Lowicryl K4M. The binding patterns of concanavalin A, wheat germ agglutinin, Griffonia simplicifolia, soybean agglutinin, peanut agglutinin, Ricinus communis agglutinin isolectin I, Griffonia simplicifolia isolectin B4, Ulex europaeus isolectin I and Phaseolus vulgaris agglutinin P have been documented in the subcellular compartments of the lateral prostate. The results show that the granular endoplasmic reticulum (GER) is rich in glycoproteins with mannosyl residues while the Golgi cisternae, secretory granules and microvilli are less so. The mannose (Man) and N-acetylglucosamine (GlcNAc) residues present in the GER of the epithelial cells may be associated with the initial assembly of the N-linked oligosaccharides of glycoproteins. The secretory granules exhibited different reactivities to lectins. Most of the lectin-binding sites confined to the limiting membranes may play a role in the transport of plasmalemma glycoconjugates to the apical plasma membrane. The epithelial Golgi stack is rich in GlcNAc, galactose (Gal), N-acetylgalactosamine (GalNAc) and sialic acid residues, and a compartmental organization of the Golgi stack is apparent which might be associated with the sequential addition of sugar residues to the oligosaccharides. The plasma membrane contains abundant Man, GlcNAc, Gal, GalNAc and complex carbohydrates, especially in the microvilli, and a differential lectin labelling was noted between the apical and basolateral plasma membrane. The present study showed that fucose-containing glycoconjugates were detected in the apical plasma membrane of the lateral prostate. The stromal extracellular matrices as well as the epithelial basement membranes demonstrated weak lectin reaction. Man, GlcNAc, Gal residues and complex sugars were also noted in the stromal tissues of the lateral prostate including the extracellular matrix, capillaries and smooth muscle.     

Chemical modification studies on Ricinus communis (Castor Bean) agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.     

Binding site of the rat liver specific monoclonal antibody

A rat liver-specific antigen (RLSA) lost its binding ability to the corresponding monoclonal antibody after treatment with N-glycanase or sialidase, which suggested that the specific binding site might be in a portion of the sugar chain containing sialic acid. The specific antigen reacted with wheat germ agglutinin, lentil lectin, erythroagglutinating phytohemagglutinin and Ricinus communis agglutinin, but not with concanavalin A or peanut agglutinin. These results suggest that the specific antigen has asparagine-linked complex-type sugar chains which might be the binding sites of the monoclonal antibody.     

Lectin affinity high-performance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II

The structural determinants required for interaction of oligosaccharides with Ricinus communis agglutinin I (RCAI) and Ricinus communis agglutinin II (RCAII) have been studied by lectin affinity high-performance liquid chromatography (HPLC). Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of silica-bound RCAI and RCAII. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. RCAI binds oligosaccharides bearing terminal beta 1,4-linked Gal but not those containing terminal beta 1,4-linked GalNAc. In contrast, RCAII binds structures with either terminal beta 1,4-linked Gal or beta 1,4-linked GalNAc. Both lectins display a greater affinity for structures with terminal beta 1,4-rather than beta 1,3-linked Gal, although RCAII interacts more strongly than RCAI with oligosaccharides containing terminal beta 1,3-linked Gal. Whereas terminal alpha 2,6-linked sialic acid partially inhibits oligosaccharide-RCAI interaction, terminal alpha 2,3-linked sialic acid abolishes interaction with the lectin. In contrast, alpha 2,3- and alpha 2,6-linked sialic acid equally inhibit but do not abolish oligosaccharide interaction with RCAII. RCAI and RCAII discriminate between N-acetyllactosamine-type branches arising from different core Man residues of dibranched complex-type oligosaccharides; RCAI has a preference for the branch attached to the alpha 1,3-linked core Man and RCAII has a preference for the branch attached to the alpha 1,6-linked core Man. RCAII but not RCAI interacts with certain di- and tribranched oligosaccharides devoid of either Gal or GalNAc but bearing terminal GlcNAc, indicating an important role for GlcNAc in RCAII interaction. These findings suggest that N-acetyllactosamine is the primary feature required for oligosaccharide recognition by both RCAI and RCAII but that lectin interaction is strongly modulated by other structural features. Thus, the oligosaccharide specificities of RCAI and RCAII are distinct, depending on many different structural features including terminal sugar moieties, peripheral branching pattern, and sugar linkages.     

Secretory glycoproteins of the rat subcommissural organ are N-linked complex-type glycoproteins. Demonstration by combined use of lectins and specific glycosidases, and by the administration of Tunicamycin

Two experimental protocols were used to investigate the secretory glycoproteins of the subcommissural organ (SCO). Protocol I: Lectins, specific exoglycosidases and immunocytochemistry were sequentially applied to the same section or to adjacent semithin sections of the rat SCO fixed in Bouin's fluid and embedded in methacrylate. Lectins used: concanavalin A (con A), wheat germ agglutinin, Limulus polyphemus agglutinin, Ricinus communis agglutinin and Arachis hypogeae agglutinin. Glycosidases used: neuroaminidase, beta-galactosidase, alpha-mannosidase, alpha-glucosidase and beta-N-acetyl-glucosaminidase. For immunocytochemistry an antiserum against bovine Reissner's fiber (AFRU) was used. Lectins and glycosidases were used in sequences that allowed the cleaved sugar residue to be identified as well as that appearing exposed as a terminal residue. This approach led to the following conclusions: (1) the terminal sugar chain of the secreted glycoproteins has the sequence sialic acid-galactose-glucosamine-; (2) the con A-binding material present in the rough endoplasmic reticulum corresponds to mannose; (3) the apical secretory granules and Reissner's fibers displayed a strong con A affinity after removing sialic acid, thus indicating the presence of internal mannosyl residues in the secreted material; (4) after removing most of the sugar moieties the secretory material continued to be strongly immunoreactive with AFRU. Protocol II: Rats were injected into the lateral ventricle with Tunica-mycin and killed 12, 24, 50 and 60 h after the injection. The SCO of rats from the last two groups showed a complete absence of con A binding sites. The results from the two experiments confirm that the secretory glycoproteins of the rat SCO are N-linked complex-type glycoproteins with the conformation previously suggested (Rodríguez et al. 1986).     

The identification of membrane glycoconjugates in Leishmania species

The membrane glycoconjugates of 8 different species of Leishmania were compared by lectin blotting. Five different lectins with various sugar specificities were examined: concanavalin A, Lens culinaris, Ricinus communis, soybean agglutinin, and peanut agglutinin. Concanavalin A and Lens culinaris reacted with every Leishmania tested. The patterns observed for these 2 lectins, as well as the various species of parasites, were different. However, a common 41,000-52,000 and a 160,000-185,000 Mr component was present in almost all the parasite isolates examined. Ricinus communis only recognized a nondiscrete galactose-containing glycoconjugate similar to Leishmania-excreted factor. Soybean and peanut agglutinins reacted with a few low molecular weight parasite components. Soybean agglutinin reacted with all the Leishmania species tested, whereas peanut lectin only recognized 3 isolates. The latter lectin bound to discrete components migrating with the dye front and with Mr's of 35,000 and 52,000. Increased glycosylation was noted on avirulent L. major promastigotes and was associated with the appearance of several new peanut agglutinin-binding glycoproteins.     

Membrane glycoproteins of the nerve growth cone: diversity and growth regulation of oligosaccharides

A subcellular fraction prepared from fetal rat brain and enriched in growth cone membranes is analyzed for its lectin-binding proteins. Growth-associated glycoproteins are identified by comparing the growth cone glycoproteins with those of synaptosomes. Protein was resolved in one- or two-dimensional gels, electroblotted, and blots probed with radioiodinated concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinins I and II. In one-dimensional gels, each lectin recognizes approximately 20 polypeptides (with substantial overlap) most of which migrate diffusely and have relatively high molecular masses (range 30-200 kD). The seven major Coomassie-staining proteins of the membrane fraction (34-52 kD) are not the major lectin-binding proteins. In two-dimensional gels, the lectin-binding proteins are either streaked across the pH gradient or exist as multiple spots, indicating broad charge heterogeneity. Seven wheat germ agglutinin- and Ricinus communis agglutinin II-binding glycoproteins are present in greater abundance in growth cone fractions compared with synaptosomes. Most notably, an acidic, sialic acid-rich protein (27-30 kD, pI 4.0; termed gp27-30) is most abundant at postnatal day 4, but absent from adult brain. The protein's very acidic isoelectric point is due, at least in part, to its high sialic acid content. Growth regulation of specific protein-linked oligosaccharides suggests that they play a special role in growth cone function. In addition, the great diversity of growth cone glycoproteins from whole brain suggests glycoprotein heterogeneity among growth cones from different neuron types.     

The cell surface of a restrictive fenestrated endothelium

The choriocapillaris is one example of a capillary bed lined by a fenestrated endothelium that is restrictive to exogenous tracers and endogenous plasma proteins. In this study we have examined the distribution of cell-surface monosaccharides utilizing biotinylated lectin-avidin ferritin cytochemistry. Receptors for wheat germ agglutinin were localized to the plasmalemma and diaphragms of some fenestrae, vesicles, and channels at the luminal endothelial front in amounts greater than seen for the other lectins employed. The absence of labeling following inhibition with N-acetylglucosamine and after tissue digestion with N-acetylhexosaminidase, but not after neuraminidase indicated that this lectin marked N-acetylglucosamine residues and not sialic acid. Wheat germ agglutinin receptors were not affected by pronase E or trypsin digestion, but were partially removed by proteinase K. The latter also removed many fenestral diaphragms. Wheat germ agglutinin receptors were cleaved with endoglycosidase D. The combined results indicate that the wheat germ agglutinin receptor is of the low-mannose type and part of a protein with hydrophobic properties. Receptors for concanavalin A (mannose) and Ricinus communis agglutinin (galactose) were also localized to the plasmalemma and endothelial diaphragms. The examination of sections at different tilt angles revealed that these lectins bound to the endothelium in a non-random distribution, encircling diaphragms of fenestrae and channels. Soybean agglutinin (N-acetylgalactosamine) marked endothelial structures sparsely. Following digestion with pronase E or trypsin, receptor sugars for the latter three lectins were completely removed, indicating their presence on protease susceptible glycoproteins. These findings demonstrate that the endothelium of the choriocapillaris bears carbohydrate moieties that are different than those described for permeable fenestrated endothelia.     

DEVELOPMENTAL STUDIES IN PORPHYRA (BANGIOPHYCEAE). II. CHARACTERIZATIO OF THE MAJOR LECTIN BINDING GLYCOPROTEINS IN DIFFERENTIATED REGIONS OF THE PORPHYRA PERFORATA THALLUS1

Detergent soluble extracts of differentiated regions of the Porphyra perforata J. Ag. thallus (holdfast, rhizoidal, vegetative and reproductive cells) were fractionated on sodium dodecyl sulfate polyacrylamide gels. Glycoproteins were identified by their lectin affinity. Extracts from all areas of the thallus contained glycoproteins, but the staining patterns were different for each region with each of the lectins tested: concanavalin A, Ulex europeaus agglutinin, Ricinus communis agglutinin, soybean agglutinin and peanut agglutinin. These data indicate that the morphologically distinct regions of the thallus also differ biochemically. Analysis of the lectin blots revealed the presence of tissue-specific glycoproteins in the five thallus areas. Such unique glycoproteins could be used as markers of differentiation in this species.     

Sialoglycoproteins of murine RAW117 large cell lymphoma/lymphosarcoma sublines of various metastatic colonization properties

A metastatic model for large-cell lymphoma/lymphosarcoma has been developed by sequential selection in vivo of the murine RAW117 cell line for enhanced liver metastasis or in vitro for loss of lectin-binding properties. The metastatic variants obtained from such selections show alterations in cell surface lectin-binding components, such as the wheat germ agglutinin (WGA)-reactive sialoglycoproteins. Detergent lysates from RAW117 cells were analyzed by polyacrylamide gel electrophoresis (PAGE) followed by reaction with 125I-labeled WGA. The [125I]WGA became bound to a diffuse band of Mr 120 000-200 000 in the gels that overlapped with the major sialoglycoprotein band revealed by the periodate-sodium borotritide labeling. However, the [125I]WGA reactivity diminished when gels were pretreated with mild acid to remove sialic acid in situ. The binding of [125I]WGA to the glycoprotein(s) was greater in the high liver-colonizing RAW117-H10 subline than in the parental RAW117-P line. Another lectin with different saccharide specificity, Ricinus communis agglutinin I (RCAI), became bound to a similar class of sialoglycoproteins, as well as to glycoproteins of lower Mr, but only when the gels were pretreated with mild acid to remove sialic acid. These differences in the relative RCAI-binding intensities after chemical removal of sialic acid were similar to those seen with WGA and indicate that differences in WGA reactivity of this class of sialoglycoproteins were not due to increased sialylation of the carbohydrate chains. Sialic acid was removed from RAW117 cells by neuraminidase treatment, and lysates were analysed for [125I]RCAI reactivity after electrophoresis. The migration of the glycoproteins was not affected by neuraminidase, indicating that the diffuseness of the major sialoglycoprotein band was not due to differences in sialylation. [125I]WGA reactivity to the sialoglycoprotein components, before and after Smith degradation in situ, strongly suggests that the oligosaccharide back-bones are highly branched and asparagine-linked. Only the high Mr portion of the diffuse sialoglycoprotein band was stained with peanut agglutinin (PNA) after in situ removal of sialic acid. To determine whether the expression of the sialoglycoprotein was causally related to liver metastasis, the amounts of sialoglycoproteins in RAW117 cells obtained by in vitro selection for increased or decreased metastasis were examined. Binding of [125I]WGA to intact cells and affinity chromatography of vectorially radiolabeled cell surface proteins on WGA-agarose were performed, and the results indicated that the in vitro selected high liver-colonizing RAW117 variants possesses high WGA r     

Detection of sialic acid residues in the axonal reticulum of rat superior cervical ganglion cells by lectin-gold cytochemistry

Highly glycosylated compounds have been demonstrated in the axonal reticulum elements of the superior cervical ganglion cells of the rat, and this is considered to suggest a connection of the reticulum with the trans Golgi side. In the present study, the axonal reticulum and the Golgi elements were further characterized by post-embedding methods of lectin-gold cytochemistry to determine their carbohydrate residues and to see, more specifically, if sialic acid residues could be detected in the axonal reticulum elements. Therefore, the affinity of neuronal cell structures for Limax flavus agglutinin (LFA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin I (RCA-I) was tested in ultra-thin sections of glycolmethacrylate-embedded material, counterstained with phosphotungstic acid (PTA) at low pH. The trans Golgi network, the Golgi-associated axonal reticulum, the reticulum within axons, the large dense-cored vesicles, and the plasma membranes were reactive for all three lectins used. We conclude that the axonal reticulum elements carry sialic acid residues, relating them to the trans Golgi network. The present results support the concept that the axonal reticulum is an extension of the trans network of the Golgi apparatus specialized for neurosecretion.     

Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine

Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains.     

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.     

Effect of sulfhydryl reagents and protease inhibitors on sodium dodecyl sulfate-heat induced dissociation of Ricinus communis agglutinin

Ricinus communis agglutinin dissociated to lower molecular weight forms when heated in sodium dodecyl sulfate in the absence of reducing agents, while ricin was little affected by such treatment. The data suggest that strong noncovalent bonds hold together two A-B heterodimers in the Ricinus communis agglutinin tetramer. Protease inhibitors such as diisopropylfluorophosphate, phenylmethansefulonyl fluoride, and EDTA, did not prevent the sodium dodecyl sulfate-heat induced dissociation; however, sulfhydryl specific reagents (N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoic acid) and p-chloromercuribenzoate) were effective. Titration of the lectins in sodium dodecyl sulfate indicated that ricin contains one sulfhydryl and Ricinus communis agglutinin four sulfhydryl groups, none of which react in the presence of 8 M urea. The sulfhydryl groups that could be titrated in the intact proteins in sodium dodecyl sulfate were on the A chains.     

Wheat germ agglutinin, concanavalin A, and lens culinalis agglutinin block the inhibitory effect of nerve growth factor on cell-free phosphorylation of Nsp100 in PC12h cells

It has been shown that in PC12 and its subclone PC12h treatment of the cells with nerve growth factor (NGF) induces a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton protein, designated in an earlier study as Nsp100, in the subsequent phosphorylation of soluble extracts from cells with (gamma-32P)ATP. In the present study, we show that plant lectins, wheat germ agglutinin (WGA), concanavalin A (Con A), and lens culinaris agglutinin (LCA), inhibit the action of NGF on Nsp100 phosphorylation in PC12h cells. Treatment of the cells with WGA, which binds to N-acetylglucosamine and sialic acid residues on glycoproteins, strongly blocked the inhibitory action of NGF on the protein phosphorylation. Con A and LCA, both of which recognize the same specific sugars (mannose, glucose), displayed only a moderate blocking effect. Unlike the native lectin, succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, and other lectins examined in this study did not inhibit the action of NGF on Nsp100. WGA-mediated inhibition of NGF action was reversed by the addition of N-acetylglucosamine and by the addition of a much lower concentration of a sialoglycoprotein, mucin, into the culture. Since the binding of succinylated WGA to N-acetylglucosamine residues of cell-surface glycoconjugates is not sufficient to prevent the action of NGF, WGA might act on sialic acid residues of the NGF receptor molecule to effect the inhibition of biological actions of NGF.     

Wheat germ agglutinin and other selected lectins increase synthesis of decay-accelerating factor in human endothelial cells.

Decay accelerating factor (DAF) is a cell-surface phosphatidylinositol-anchored protein that protects the cell from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have measured DAF on human umbilical vein endothelial cells (HUVEC) by immunoradiometric assay after its removal by phosphatidylinositol-specific phospholipase C or Nonidet P-40 detergent extraction and have previously demonstrated that DAF synthesis can be stimulated by phorbol ester activation of protein kinase C. We now report that although stimulation (4-48 h) of HUVEC with various cytokines, including TNF, IL-1, and IFN-gamma, did not alter DAF levels, wheat germ agglutinin (WGA) (5-50 micrograms/ml), a lectin specific for binding N-acetyl neuraminic acid and N-acetyl glucosamine residues, increased DAF levels fivefold when incubated with HUVEC for 12 to 24 h. The lectins Con A and PHA also stimulated DAF expression twofold, whereas a number of others including Ulex europaeus, Bandeiraea simplicifolia lectin I, and Ricinus communis agglutinin I, which bind to endothelial cells, were inactive. The increase in DAF by WGA was inhibited by N-acetyl glucosamine (10-50 mM) but by neither N-acetyl neuraminic acid nor removal of surface N-acetyl neuraminic acid with neuraminidase. However, succinylated WGA, which has unaltered affinity for N-acetyl glucosamine but not longer binds N-acetyl neuraminic acid, was inactive. These data suggest that the binding of WGA to sugar residues alone is not sufficient to trigger DAF expression and that occupation of additional, specific sites are required. The increase in DAF levels on HUVEC was blocked by inhibitors of RNA and protein synthesis. We conclude that continuous occupation by WGA of specific binding sites on HUVEC triggers events leading to DAF synthesis. This unique, long term stimulation of endothelial cells by lectins may be relevant to cell:cell interactions at the endothelium.