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Water-soluble poly(acrylamide-allylamine) copolymers containing covalently bound amino groups, prepared by copolymerization of acrylamide and allylamine, can be used as general carriers for coupling of different types of saccharides or saccharide derivatives. The water-soluble macromolecular carbohydrate derivatives can be easily labelled and used in various solid-phase techniques to study protein-saccharide interaction. Two types of coupling reaction were used to prepare polyacrylamide derivatives of saccharides: reductive amination was applied to couple the reducing disaccharides and a carbodiimide reaction was used to couple heparin via its carboxyl groups to the amino groups of the poly(acrylamide-allylamine) derivative. Peroxidase labelled or biotinylated derivatives were shown to be useful in studies on the binding properties of lectins and proteins from boar seminal plasma.

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Membrane lectins of mammalian large granular lymphocytes are thought to be important receptors in their non-major-histocompatibility complex-restricted activation. A triantennary desialylated oligosaccharide has been reported as the most effective triggering structure [Pospísil M., Kubrycht J., Bezouska K., Táborský O., Novák M. & Kocourek J. (1986) Immunol. Lett. 12, 83-90] while its cell surface receptor has recently been identified in pig natural killer cells as a 205-kDa membrane lectin resembling the proteins of the leukocyte common antigen family (LCA). In this study we have prepared 4-azidophenyl (photoactivatable) and 4-hydroxyphenyl (radio-iodinatable) derivatives of triantennary oligosaccharides by a new procedure which allows the natural conformation of the N-glycosidic linkage between the oligosaccharide and the respective labeling group to be retained. We used these high-affinity ligands to investigate the oligosaccharide-combining site of the 205-kDa lectin. Photoaffinity labeling of the whole cells and solubilized proteins confirmed that a 205-kDa polypeptide constitutes the major cell-surface calcium-independent receptor for triantennary oligosaccharides in pig lymphocytes. Isolation and manual sequencing of two ligand-labeled and eleven other peptides proved that the 205-kDa lectin represents a member of the LCA family expressing exons 4 and 6 during alternative splicing and that the high-affinity binding site is localized in the N-terminal 70-kDa extracellular domain. Binding studies with radiolabeled oligosaccharides and the above carbohydrate-recognition domain subjected to various chemical and enzymatic treatments indicated that the binding of oligosaccharides might be significantly modulated by sialylated O-glycosidically linked lineage-specific carbohydrate epitopes localized within this domain. Affinity chromatography of LCA isolated by conventional methods on immobilized oligosaccharides revealed that only a fraction of these cell-surface glycoproteins expressed high-affinity binding sites for the oligosaccharide ligands. Thus, N-linked oligosaccharide moieties of cell-surface glycoproteins seem to represent possible ligands of LCA that may be important in intercellular adhesion and oligosaccharide-mediated activation of lymphocytes.

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Mannan-binding proteins found in the liver and serum of several vertebrate species are supposed to play an important role in the intracellular transport of glycoproteins, as well as in several protective reactions including complement activation and elimination of various pathogens. To study these protective functions at molecular level it is necessary to understand the fine oligosaccharide specificity and mutual relation among various forms of these soluble lectins. We have isolated mannan-binding protein as peripheral membrane proteins of porcine lymphocytes. This lectin was purified to homogeneity and shown to possess many properties in common with the well studied rat liver proteins (mol. mass, subunit composition and general organization of the molecule). Binding studies performed with three series of defined oligosaccharides (high mannose, hybrid type, and complex) on native lectin molecules as well as isolated carbohydrate-binding domains revealed distinctive features of this mannan-binding protein, including its impaired ability to bind the oligosaccharide ligand after reduction and decyclization at core N-acetyl-D-glucosamine 1.

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Mammalian endogenous carbohydrate-binding proteins (lectins) play fundamental roles in a variety of mechanisms of interactions both at the molecular and cellular levels. We have investigated the binding of one of them (human brain lectin) to soluble acrylamide copolymerized with derivatives of either lactose (O-beta-lactosyloxyallylallylaminoacrylamide copolymer) or D-mannose (D-alpha-mannosyloxyallylallylaminoacrylamide copolymer) in direct enzyme affinoassays, in an attempt to develop simple procedures for detection and estimation of its carbohydrate-binding activity. Biotinylated plant lectins were utilized as reference standards. Affinoassays employed the polymer dotted on nitrocellulose and the polymer coated on microtiter plates as well as detection of bound biotinylated lectin by streptavidin/horseradish peroxidase reagent. Both assays provided reproducible binding, inhibitable by specific sugars. The microtiter plate assay is well suited to sensitive detection of the negative endogenous lectin by competition with biotinylated brain lectin. We conclude that the use of derivatized acrylamide in dotting and microtiter plate assays may prove practical for detection of endogenous lectins and that such polymers may serve as model substances in the study of biological partners of these carbohydrate-binding proteins.

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1. A cortical granule lectin was isolated from vitellogenic oocytes of a bony fish, the roach Rutilus rutilus. 2. The lectin agglutinates human erythrocytes, is specific for L-rhamnose and is composed of different polypeptide subunits. 3. The lectin is the first lectin of animal origin that inhibits protein-synthesis by a rabbit reticulocyte lysate, and is also mitogenic for human lymphocytes.

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Binding of egg-white glycoproteins and their oligosaccharides to hexameric solubilized form of the chicken hepatic lectin and the monomeric soluble fragment containing the carbohydrate-recognition domain has been investigated by several techniques. Ligand blotting revealed significant differences in binding to two forms of the lectin only for glycoproteins bearing multiple N-linked oligosaccharide moieties in their molecule (riboflavin-binding glycoprotein, avidin or ovomucoid). Inhibition studies indicated that inhibitory potency in a series of linear and branched N-acetyl-D-glucosamine-terminated oligosaccharides is critically dependent on the number and spatial arrangement of the terminal monosaccharide residues for both forms of the lectin. Direct binding of 4-hydroxyphenyl-derivatized radioiodinated oligosaccharides measured by equilibrium dialysis and frontal affinity chromatography points to the existence of two N-acetyl-D-glucosamine-combining sites per one subunit of the lectin, as has been recently reported for the rabbit and rat liver lectin [Lee & Lee (1988) Biochem. Biophys. Res. Commun. 155, 1444-1452]. Highly branch (penta-antennary) oligosaccharides interact with more than one subunit of the hexameric form of the lectin and thus resemble the more complex interaction of the whole glycoprotein.

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Polyacrylamide gel electrophoresis in an acidic buffer system was used to study the electrophoretic behaviour of two forms of alpha-D-galactosidase from seeds of soy bean (Glycine soja) and mung bean (Vigna radiata). The interaction of the enzymes with saccharides was monitored by affinity electrophoresis; for the preparation of affinity gels, water-soluble O-glycosyl polyacrylamide copolymers and polysaccharides were used. alpha-D-Galactosidases from both sources interact with immobilized alpha-D-galactosyl residues. On the basis of the results of affinity electrophoresis performed in the presence of various free sugars, dissociation constants for the complexes between alpha-D-galactosidase and free sugars were calculated.

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Inhibition of pig NK cell activity by asialooligosaccharides (aOS) isolated from human serum glycoproteins was investigated. Train-tennary aOS (aOSIII) of ceruloplasmin was found to be the most potent inhibitor up to the concentration 0.1 micrograms/ml, which is in agreement with its highly specific binding to NK-activity-enriched pig lymphocytes (with a morphology similar to human large granular lymphocytes (LGL]. Only lectins with the specificity to Gal(beta 1----4)GlcNAc or Gal(beta 1----3)GalNAc structures exhibited inhibition of NK cytotoxicity. F(ab)2 fragments of rabbit antibodies against pig spleen membrane lectin cross-reacting with the pig liver membrane lectin completely inhibited NK activity when preincubated with the effectors or present in the incubation mixture during the assay. These data suggest that lectin receptors on cells of pig NK-activity-enriched fraction specific for aOSIII and antigenically related to membrane lectins isolated from pig spleen and liver, are involved in the NK recognition of several xenogeneic targets.

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The interaction of alpha-D-galactosidases from Vicia faba seeds with saccharides was studied by means of affinity electrophoresis on polyacrylamide gel in an acidic buffer system. For the preparation of affinity gels, water-soluble O-glycosyl polyacrylamide copolymers and polysaccharides were used. alpha-D-Galactosidases interact with immobilized O-alpha-D-galactosyl residues and glycogen, but no interaction was observed with immobilized O-alpha-D-mannosyl residues. On the basis of the results of affinity electrophoresis performed in the presence of various free sugars, dissociation constants of the various alpha-D-galactosidase-free sugar complexes were calculated.

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The lectins of fruiting bodies of Xerocomus chrysenteron and Lactarius lignyotus were purified on Sepharose 4B containing immobilized fetuin. Both lectins agglutinate human erythrocytes nonspecifically at limit concentrations of 15 micrograms/mL. Their erythroagglutinating activities are not inhibited by simple sugars; desialyzed fetuin, desialyzed glycoprotein from edible bird's nest, and desialyzed mucin from porcine submaxillary glands are the most effective inhibitors.

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Oligosaccharides with four different types of branching were prepared from purified human transferrin, alpha 2-macroglobulin, caeruloplasmin and alpha 1-acid glycoprotein and labelled with NaBH3 3H. Binding of these oligosaccharides to rat liver plasma membrane, rat leucocytes, pig liver plasma membranes and pig leucocyte plasma membranes was investigated. A striking dependence of binding on oligosaccharide branching was observed. The values of apparent association constants Ka at 4 degrees C vary from 10(6) M-1 (biantennary structure) to 10(9) M-1 (tetra-antennary structure) in the liver, whereas in the leucocytes the Ka values were found to be of reversed order, from 1.8 X 10(9) M-1 for biantennary to 2.2 X 10(6) M-1 for tetra-antennary structures. The binding is completely inhibited by 150 mM-D-galactose, but 150 mM-D-mannose has almost no effect on binding. Leucocyte plasma membranes bind preferentially 125I-asialoglycoproteins with biantennary oligosaccharides, thus completing the specificity pattern of the hepatic recognition system for desialylated glycoproteins. Possible physiological roles of these two complementary recognition systems under normal and pathological conditions are discussed.

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A selective affinity-adsorbent for the extracellular endo-D-galacturonanase (E.C. 3.2.1.15) of Aspergillus niger was prepared by covalent coupling of tri(D-galactosiduronic acid) to Separon, a poly(hydroxyalkyl methacrylate) gel. Complexing of the enzyme with the adsorbent is pH dependent; maximal interaction occurs at the optimum pH for enzyme activity. The enzyme was quantitatively displaced from the adsorbent either by changing the pH or by bioelution with soluble tri(D-galactosiduronic acid) or other substrate. Within the range of substitution of Separon examined [content of tri(D-galactosiduronic acid) 1.7-6.7%] the amount of endo-D-galacturonanase retained was proportional to the content of affinity ligand. Under the same conditions, unsubstituted carrier did not complex with endo-D-galacturonanase. The dissociation constant of the affinity complex, as determined by zonal analysis, kinetic measurements, and by means of the adsorption isotherm KL (0.54 mmol.L-1), is close to the value (KI 0.44 mmol.L-1) obtained by the two first methods with soluble tri(D-galactosiduronic acid). The results show that adsorption of endo-D-galacturonanase on tri(D-galactosiduronic acid)-Separon is due exclusively to active-site-directed interaction with bound affinity-ligand.

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The lectin of the Indian bean or lablab (Dolichos lablab L.) was purified by affinity chromatography on two types of affinity carriers: O-alpha-D-mannopyranosyl-Separon and Separon-bound ovomucoid. The lectin is homogeneous in the ultracentrifuge: S20, w = 6.14 S, Mr = 110 000; the molecule appears to comprise two pairs of two types of subunits (Mr 16 000 and 40 000), and contains 2% neutral sugar and 0.2 Mn and 0.5 Zn atom respectively. The lectin agglutinates human erythrocytes non-specifically with regard to ABO grouping at a limit concentration of 8 micrograms/ml, and this activity is inhibited most effectively by N-acetyl-D-glucosamine, methyl alpha-D-mannopyranoside and ovomucoid, but not by free D-mannose.

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The occurrence of endogeneous lectins in the ovaries of four fish species has been studied by indirect immunofluorescence staining with antibodies against individual lectins. Paraffin sections of the ovary of perch (Perca fluviatilis L.) were treated with an antibody against perch lectin. In cryostat sections of the tench (Tinca tinca L.) ovary, the L-rhamnose-specific lectin "I" was detected with a specific antibody. In cryostat sections of both roach (Rutilus rutilus L.) and rudd (Scardinius erythrophthalmus L.) ovaries, lectins were localized using a single antibody against roach lectin. The isolation of tench lectins is briefly described. In the fish species employed for this study, lectins are associated exclusively with the content and surrounding membrane of cortical vesicles situated within the cytoplasm of maturing oocytes. The positive reaction with lectin antibody was observed almost immediately after the formation of the first cortical vesicles in the peripheral cytoplasm of early previtellogenic oocytes. Their lectin content increases during the later stages when cortical granules fill the whole cytoplasm before moving towards the cell periphery, as the oocyte starts to accumulate yolk. The presence of lectins within cortical vesicles is significant also in view of the polysaccharide content of these structures. In the vitellogenic oocytes lectins seem to move towards the cell periphery and accumulate beneath the plasma membrane. Our observations are discussed in view of the present ideas on the intracellular function of lectins, and with respect to the role of cortical vesicles in fertilization and in post-fertilization modifications of the egg envelopes.

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Rye germ lectin was isolated by extraction of defatted rye germ, fractionation of the extract by ammonium sulfate precipitation, affinity chromatography of the active substances on chitin-beta-glucan and gel filtration on Sephadex G-50. The lectin shows erythroagglutinating activity at a minimum concentration of 2.5 micrograms/ml. The erythroagglutinating activity is the same against human red blood cells of all types of the ABO system and is inhibited by N-acetyl-D-glucosamine. The lectin has no mitogenic activity against mouse splenic lymphocytes. According to the results of polyacrylamide gel electrophoresis the lectin obtained is a mixture of three very similar isolectins of equal erythroagglutinating activity. Sedimentation analysis indicates homogeneity of the lectin preparation; the molecular weight was 56000 as estimated by sedimentation equilibrium. In the presence of urea and sodium dodecyl sulfate the lectin dissociates into 2 types of subunits with molecular weights of 35000 and 19000. The rye germ lectin contains about 2% of neutral sugar and 1% of D-glucosamine. The amino acid composition of the lectin is characterized by a very high content of glycine and half cystine and a low content of apolar amino acids. N-terminal amino acids of the lectin are apparently blocked.

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