Glutaraldehyde cross-linking of lectins to marker enzymes: protection of binding site by specific sugars

The role of bound specific sugars in protecting the sugar binding activity of several galactose binding proteins during their covalent conjugation to horse radish peroxidase by glutaraldehyde-mediated cross-linking was examined by: a) affinity matrix binding of the conjugate, b) enzyme linked lectin assay and c) hemagglutination assay. During conjugation using 1% glutaraldehyde, protection of jack fruit (Artocarpus integrifolia) lectin (jacalin) activity depended on concentration of specific sugar present during conjugation; optimum protection was offered by 50 mM galactose. This indicated the presence of one or more primary groups at the binding site of jacalin, which is (are) essential for sugar binding. On the other hand, such essential amino group(s) was not indicated at the sugar binding site of the peanut lectin, bovine heart galectin or of the human serum anti alpha-galactoside antibody, since exclusion of sugar during their conjugation to HRP did not diminish sugar binding activity. The differential behavior is discussed in the light of reported differences in sugar specificities. Results indicated that sugar mediated blocking of active site may be used in characterization of the latter in lectins.     

New GlcNAc/GalNAc-specific lectin from the ascidian Didemnum ternatanum

Previously we isolated GlcNAc-specific lectin (DTL) from the ascidian Didemnum ternatanum by affinity chromatography on cross-linked ovalbumin. Here we report the purification and characterization of new D-GlcNAc/D-GalNAc-specific lectin DTL-A from the same ascidian. This lectin was isolated from non-bound cross-linked ovalbumin fraction and further was purified by gel filtration on Sepharose CL-4B, affinity chromatography on GlcNAc-agarose and gel filtration on Superdex 200. SDS-polyacrylamide gel electrophoresis and gel filtration of purified lectin on Sepharose CL-4B indicates that it exists as large aggregates in the native state. Investigations of the carbohydrate specificity of DTL-A by enzyme-linked lectin assay suggest the multi-specificity of this lectin. DTL-A binds BSM, asialo-BSM as well as heparin and dextran sulfate. The binding of DTL-A to BSM was inhibited by monosaccharides D-GlcNAc and D-GalNAc, their alpha- but not beta-anomers. Among polysaccharides and glycoconjugates, DTL-A binding to BSM was effectively inhibited by BSM, asialo-BSM, pronase-treated BSM and synthetic alpha-D-GalNAc-PAA. Fetuin and asialofetuin showed a much lower inhibitory potency, heparin and dextran sulfate were noninhibitory. On the other hand, DTL-A binding to heparin was effectively inhibited by dextran sulfate, fucoidan, whereas BSM showed insignificantly inhibitory effect. DTL-A binding to heparin was not inhibited by D-GlcNAc and D-GalNAc.     

The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose

We examined the carbohydrate-binding potential of the C-type lectin-like receptor Dectin-2 (Clecf4n). The carbohydrate-recognition domain (CRD) of Dectin-2 exhibited cation-dependent mannose/fucose-like lectin activity, with an IC(50) for mannose of approximately 20 mM compared to an IC(50) of 1.5 mM for the macrophage mannose receptor when assayed by similar methodology. The extracellular domain of Dectin-2 exhibited binding to live Candida albicans and the Saccharomyces-derived particle zymosan. This binding was completely abrogated by cation chelation and was competed by yeast mannans. We compared the lectin activity of Dectin-2 with that of two other C-type lectin receptors (mannose receptor and SIGNR1) known to bind fungal mannans. Both mannose receptor and SIGNR1 were able to bind bacterial capsular polysaccharides derived from Streptococcus pneumoniae, but interestingly they exhibited distinct binding profiles. The Dectin-2 CRD exhibited only weak interactions to some of these capsular polysaccharides, indicative of different structural or affinity requirements for binding, when compared with the other two lectins. Glycan array analysis of the carbohydrate recognition by Dectin-2 indicated specific recognition of high-mannose structures (Man(9)GlcNAc(2)). The differences in the specificity of these three mannose-specific lectins indicate that mannose recognition is mediated by distinct receptors, with unique specificity, that are expressed by discrete subpopulations of cells, and this further highlights the complex nature of carbohydrate recognition by immune cells.     

Relationship of prolactin receptors to concanavalin A binding.

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar alpha-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 mu/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (Kd) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (Kd approximately equal to 6.6 . 10(-8) M. At high lectin concentrations, low affinity (Kd approximately equal to 6.7 . 10(-5) M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 microgram/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by alpha-methyl-D-mannopyranoside.     

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.     

A new lectin from the sea worm Serpula vermicularis: isolation, characterization and anti-HIV activity

A GlcNAc-specific lectin was isolated from the sea worm Serpula vermicularis (SVL) (Annelida) and purified by ion-exchange, affinity and gel permeation chromatography. SVL was a homotetrameric protein with native molecular mass of about 50 kDa, and consisted of identical subunits of 12.7 kDa. The carbohydrate content of 1.9% suggested that the lectin was a glycoprotein, and mainly composed by aspartic and glutamic acids, glycine, valine and serine; with relatively lower content of basic amino acids and cysteine. The first 15 residues of the N-terminal region were determined as ADTPCQMLGSRYGWR. It was stable at pH 6-9 and at temperatures up to 40 degrees C. SVL was Ca(2+)-independent lectin that agglutinated native and trypsinized human erythrocytes. Hapten inhibition studies indicated that SVL showed binding specificity only for N-acetyl-d-glucosamine and its derivatives among the monosaccharides tested and required the presence of hydroxyl group at the C-3 of GlcNAc. The presence of hydrophobic p-nitrophenyl aglycone improved inhibitory potency of N-acetyl-d-glucosamine. Ovomucoid and ovalbumin were found to be inhibitors among the glycoproteins used for inhibition assay. The anti-HIV-1 (human immunodeficiency virus) activity of SVL in vitro was determined: SVL inhibited the production of viral p24 antigen and cytopathic effect induced by HIV-1. The EC(50) values were 0.23 and 0.15 microg x mL(-1) respectively.     

Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves

Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.     

Time-dependent lectin binding to isolated receptors in model membranes

Binding of lectins to two integral membrane glycoproteins has been measured in lipid bilayer model membranes and in their cell of origin with an eye to clarifying the basis of time-dependence in such processes. Specific binding was monitored as a function of time using an assay that involved membrane exposure to radiolabelled wheat-germ agglutinin or concanavalin A, and subsequent differential centrifugation to remove unbound material. Qualitatively, the time dependence of lectin binding to the isolated receptors in lipid bilayers was found to be similar to that for the same receptors in the intact cell - hence the phenomenon does not depend for its existence upon receptor interaction with other specific native membrane components. Quantitatively, the time-course was sensitive to structural features of the model membrane involved. The results may be understood by viewing the glycopeptide headgroups as deformable structures which rearrange as a direct result of lectin attachment, and it would appear that rearrangement is essential for high-affinity binding. Model membrane structure was examined by light microscopy and freeze-etch electron microscopy in an attempt to assess the applicability of this type of study to a more detailed analysis of the processes involved in lectin binding. Although the freeze-etch technique is a promising one, it was concluded that heterogeneity in receptor arrangement within the lipid bilayer is the most important limitation to correlation of binding curves with membrane structure.     

Purification and molecular characterization of a sialic acid specific lectin from the phytopathogenic fungus Macrophomina phaseolina

A lectin was isolated and purified from the culture filtrate of the plant pathogenic fungus Macrophomina phaseolina by a combination of ammonium sulfate precipitation, affinity chromatography on fetuin-Sepharose 4B and ion-exchange chromatography on DEAE-A 50. The lectin designated MPL was homogeneous by PAGE and HPLC and a monomeric protein with a molecular weight of approximately 34 kDa as demonstrated by SDS-PAGE. It is a glycoprotein and agglutinated human erythrocytes regardless of the human blood type. Neuraminidase treatment of erythrocytes reduced the agglutination activity of the lectin. It is thermally stable and exhibits maximum activity between pH 6 and 7.2. Its carbohydrate binding specificity was investigated both by hapten inhibition of hemagglutination and by enzyme-conjugated lectin inhibition assay. Although, M. phaseolina lectin bound sialic acid, it exhibited binding affinity towards neuraminyl oligosaccharides of N-linked glycoproteins, alpha-Neu5Ac-(2-->3)-beta-Gal-(1-->4)-GlcNAc being maximum.     

Effect of Guanosine 5''-O-(3-Thiotriphosphate) and Calcium on γ-Aminobutyric AcidB Binding as a Function of Postnatal Development

We have examined the development of gamma-aminobutyric acidB (GABAB) receptors in rat cerebrum using a binding assay that has achieved specific binding levels of approximately 50% with the GABAB ligand (-)-[3H]baclofen. As early as postnatal day 1, GABAB receptors are present and are linked to both calcium- and guanosine triphosphate-binding protein (G protein)-regulatory sites, as indicated by the stimulation of binding by calcium and the inhibition of binding by the guanine nucleotide guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). However, whereas the EC50 for the calcium effect was at a mature value in the neonate, the IC50 for the inhibition of binding by GTP gamma S was not, and declined more than two orders of magnitude by adulthood. Moreover, while many previous studies had shown that manipulation of G proteins by guanine nucleotides affects receptors affinity rather than density, our saturation analysis of binding suggests that calcium affected GABAB receptor density rather than affinity. The results therefore suggest that calcium and the manipulation of G proteins by GTP gamma S may affect the GABAB receptor by different mechanisms.     

Partial purification of dopamine D2 receptors using lectin affinity columns

Dopamine D₂ receptors , detected by [³H]spiperone Dopamine D₂ receptors , detected by [³H]spiperone binding, were solubilized from bovine caudate nucleus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmaco-logical properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D₂ dopamine receptor is therefore a glycoprotein. binding, were solubilized from bovine caudate nucJeus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmacological properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D₂ dopamine receptor is therefore a glycoprotein.     

The distribution and localization of the fucose-binding lectin in rat tissues and the identification of a high affinity form of the mannose/N-acetylglucosamine-binding lectin in rat liver

A small-scale affinity chromatographic procedure was developed to screen for the presence of fucose and mannose/N-acetylglucosamine-binding lectins in small amounts of rat tissues. Of all tissues examined, only the liver contained the fucose-binding lectin, whereas both liver and blood serum contained the mannose/N-acetylglucosamine lectin. By means of immunocytological methods using antibodies to hepatic lectins, the fucose lectin was shown to be uniquely present in Kupffer cells and absent in all other types of rat macrophages examined. The binding and uptake of different neoglycoproteins by nonparenchymal cell fractions of liver indicated that the fucose-binding lectin was either not responsible for the uptake or that more than one lectin was acting. With the identification of another lectin (Mr = 180,000) by the above screening procedure for hepatic lectins and the results of studies in the following paper (Haltiwanger, R.S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7440-7444) two lectins appear to be involved. A small amount of the hepatic mannose/N-acetylglucosamine lectin was found by the above screening procedure to have a higher affinity for L-fucosyl-bovine serum albumin-Sepharose than the majority of the lectin in hepatocytes. This lectin, called the high affinity form, was purified and its properties examined. On a weight basis the high affinity form bound 7-12 times more ligand than the normal form. Its Ka for L-fucosyl-bovine serum albumin was 2.3 X 10(9) M-1 compared to 3.5 X 10(8) M-1 for the normal form. Moreover, the concentrations of monosaccharides required to inhibit the high affinity form were about 3 times less than those required to inhibit binding of the normal form. The two forms, however, have identical molecular weights (32,000) under reducing and nonreducing conditions, bind anti-lectin antibodies in the same way, and give identical peptide maps after V-8 protease digestion. The structural basis for the different binding affinities of the two forms remains unknown.     

Mannose-specific lectin from the mushroom Hygrophorus russula

A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate-polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5?kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528?bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Binding characteristics of N-acetylglucosamine-specific lectin of the isolated chicken hepatocytes: similarities to mammalian hepatic galactose/N-acetylgalactosamine-specific lectin

Binding characteristics of N-acetylglucosamine- (GlcNAc) specific lectin on the chicken hepatocyte surface were probed by an inhibition assay using various sugars and glycosides as inhibitors. Results indicated that the binding area of the lectin is small, interacting only with GlcNAc residues whose 3- and 4-OH's are open. The combining site is probably of trough-type, since substitution with as large a group as monosaccharide is permitted on the C-6 side of GlcNAc, and on the C-1 side, the aglycon of GlcNAc can be very large (e.g., a glycoprotein). These binding characteristics are shared with the homologous mammalian lectin specific for galactose/N-acetylgalactosamine, suggesting that tertiary structure of the combining area of these two lectins is similar. This is understandable, since there is approximately 40% amino acid sequence identity in the carbohydrate recognition domain of these two lectins [Drickamer, K., Mannon, J. F., Binns, G., & Leung, J. O. (1984) J. Biol. Chem. 259, 770-778]. A series of glycosides, each containing two GlcNAc residues separated by different distances (from 0.8 to 4.7 nm), were synthesized. Inhibition assay with these and other cluster glycosides indicated that clustering of two or more GlcNAc residues increased the affinity toward the chicken lectin tremendously. Among the ligands containing two GlcNAc residues, the structure which allows a maximal inter-GlcNAc distance of 3.3 nm had the strongest affinity, its affinity increase over GlcNAc (monosaccharide) amounting to 100-fold. Longer distances slightly diminished the affinity, while shortening the distance caused substantial decrease in the affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding characteristics of galactoside-binding lectin (galaptin) from human spleen

Binding characteristics of human spleen soluble galactoside-binding protein (galaptin) were studied using simple galactosides, galactose-terminated disaccharides, cluster glycosides containing up to 6 terminal lactosyl residues, bovine serum albumin derivatives containing 7 to 40 lactosyl residues, desialylated serum glycoproteins, and glycopeptides derived thereof as inhibitors in a newly developed binding assay. In this assay, aminohexyl lactoside was attached to divinyl sulfone-activated Sepharose, which was then used to bind 125I-galaptin. Similarly derivatized Sepharose containing mannoside served as a control. The assay is sensitive, maintains linearity in the concentration range of 125I-galaptin tested, and has very low nonspecific binding. The following new findings were made. 1) All the alpha-D-galactopyranosides with non-sugar aglycon were better inhibitors than the corresponding beta-D-galactopyranoside. 2) The S-galactosides were better inhibitors than the corresponding O-galactosides, regardless of the anomeric configuration. 3) Many Gal beta 1-4- and Gal beta 1-3-linked disaccharides were tested. Although the galaptin did not appear to recognize N-acetylglucosamine as a monosaccharide, the presence of this sugar penultimate to galactose increased the binding affinity by as much as 500-fold, as was the case for N-acetyllactosamine. Of a particular importance is the presence of an equatorial 3-OH group on this sugar. We synthesized the 3-deoxy derivative of N-acetyllactosamine and found that it had 50-fold lower binding affinity compared to N-acetyllactosamine. 4) The binding sites of this lectin do not seem to be operating in a cooperative fashion, since synthetic lactose-containing divalent ligands with various inter-galactose distances did not increase the binding affinity significantly.     

Purification and characterization of Dolichos lablab lectin

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin- Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.      

Functional incorporation and preferred orientation of phytohemagglutinin receptor glycoproteins in phospholipid vesicles

Porcine lymphocyte Phaseolus vulgaris phytohemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography have been reassembled into vesicles made of phosphatidylcholine and phosphatidylserine by detergent (dodecyltrimethylammonium bromide) dialysis. The receptor glycoproteins were incorporated into the lipid vesicles in a nonselective manner with a yield of 65-70%. Vesicles containing the glycoproteins were sealed as evidenced by their impermeability to calcium ions, using quin 2 trapped inside the vesicles. The vesicles were agglutinated by PHA, suggesting that the saccharidic moiety of the reconstituted glycoproteins was, at least in part, oriented towards the extravesicular medium. This observation was further supported by the fact that the vesicles bound 125I-labeled PHA in a specific and saturable manner. At maximum amount of lectin bound, a ratio of 1.01 +/- 0.05 microgram of PHA per microgram glycoprotein incorporated was measured. When the binding data were analyzed by Scatchard plot, a downward concave profile was observed, suggestive of a positive cooperativity at low concentrations of lectin. The orientation of the reconstituted lectin receptor glycoproteins was determined by proteolytic treatments of labeled glycoproteins. The combined action of trypsin and chymotrypsin released, in the 120,000 X g supernatant, approximately 80% of label when 125I-tagged PHA receptor glycoproteins were incorporated into the vesicles. When the oligosaccharidic moieties of the receptor glycoproteins were specifically labeled, the simultaneous action of the two enzymes released approximately 70% of tritium labeling present in the reconstituted system. Taken together, these results suggest that the reconstituted PHA receptors are preferentially oriented into the phospholipid vesicles. The reconstituted PHA receptor glycoproteins competed effectively with cellular receptors in the assay of PHA-induced porcine lymphocyte activation. A 50% inhibition of [3H]thymidine incorporation was observed when 1 microgram of glycoproteins in vesicles was added to the cultured cells, whereas vesicles alone had no effect at this (equivalent) concentration.     

Affinity modulation of human placental insulin and insulin-like growth factor receptors by lectins

The ability of plant lectins to modify the interactions of the insulin receptor (IR) and insulin-like growth factor (IGF) receptors (IGFRs) with their ligands was investigated. The lectins profoundly affected the competition-binding curves for (125)I-labelled IGF-I and insulin, causing an increase in the affinity of placental IGF1R and IR towards their ligands. This increment was of such a magnitude that it could affect the receptors' specificity towards these ligands. The lower the ligand concentration, the greater was the lectin-induced affinity shift, which suggests potential physiological significance of the effect. The affinity modulation occurred in a lectin-specific and dose-dependent manner. In contrast to IGF1R and IR, the binding of (125)I-labelled IGF-II to its receptors resisted lectin modulation. Here we provide evidence of the possibility of external modulation of the affinity of placental IGF1R and IR via interactions of the receptors' carbohydrate moieties with lectins. The existence of modulators that would selectively inhibit or enhance the binding of IGFs or insulin to their corresponding receptors may have important implications for placental cell responses to these molecules.     

Neoglycoenzymes: a versatile tool for lectin detection in solid-phase assays and glycohistochemistry

Carbodiimide-mediated coupling of p-aminophenyl glycosides to a naturally nonglycosylated enzyme yields a neoglycoenzyme. This compound combines inherent enzymatic activity with synthetically conferred ligand properties to lectins. Appropriate choice of the ligand allows custom-made synthesis to reliably detect various types of lectins. To exemplify practical applications of this class of compounds, glycosylated bacterial beta-galactosidase has been employed to quantitate plant lectins, immobilized on plastic surfaces as well as on nitrocellulose. Competitive inhibition by specific sugar ascertained the dependence of binding on protein--carbohydrate interactions. In view of lectins as tools, a sandwich lectin-binding assay for high mannose-type glycoprotein detection has been modified to principally facilitate wide application to other lectin-reactive sugar chains by introducing the neoglycoenzyme. In addition to lectin determination in solid-phase assays, neoglycoenzymes allow one to glycohistochemically localize endogenous lectins in tissue prints and tissue sections with a minimum number of steps. This nonradioactive, rapid, sensitive, and convenient assay concept, based on conjugation of a ligand to an enzyme with maintenance of its receptor-binding activity, may find extended application beyond lectinology in receptor analysis.