Studies on phytohemagglutinins. XXV. Isolation and characterization of hemagglutinins of the spindle tree seeds (Evonymus europaea L.).

A mixture of isophytohemagglutinins has been isolated from the fleshy arils of the spindle tree seeds (Evonymus europaea L.) by fractional precipitation of the saline extract of the arils by (NH4)2SO4 at a 0.40% saturation. Successive preparative disc electrophoresis on polyacrylamide gel affords separation of one slower moving component, phytohemagglutinin I, from the mixture of other isophytohemagglutinins that have a very similar electrophoretic mobility. Phytohemagglutinin I has a sedimentation coefficient Sw,20 of 7.1 S and an approximate mol. wt of 127 000. Amino acid analysis shows a high amount of aspartic acid, alanine and glycine but also significant amounts of serine, threonine, cysteine and methionine. Aspartic acid is the only N-terminal amino acid found by the dansylation technique. Phytohemagglutinin I contains glucosamine and 4.7% neutral sugar. Its approximate pI in citrate/phosphate buffer is 4.4-4.5. The metal content amounts to 0.250% Ca, 0.019% Mg, 0.034% Zn and 0.026% Cu. Mn is not present. Ultracentrifugation analysis reveals homogeneity in the sedimentation behavior of the mixture of isophytohemagglutinin, an Sw,20 of 7.1 S and an approximate mol. wt of 119 000. The mixture has an amino acid composition closely resembling that of phytohemagglutinin I and an identical pI but contains only 1.9% neutral sugar. Two N-terminal amino acids were shown to be present, aspartic acid and tyrosine. With the exception of Cu which is absent, the metal content is almost the same as that of phytohemagglutinin I. Both phytohemagglutinin I and the mixture are devoid of anti-A1 activity and show detectable anti-H, anti-B and anti-A2 erythroagglutinating activity in approximate limit concentrations of 2.5, 5 and 10 mug/ml, respectively. This activity is not influenced by the presence of EDTA, Ca2+ or Mg2+, but is stimulated by Zn2+. Mn2+ and Co2+ have an inhibitory effect. None of the simple sugars tested inhibited the hemagglutination reactions.

Studies on phytohemagglutinins. XXIV. Isoelectric point and hybridization of the pea (Pisum sativum L.) isophytohemagglutinins.

The isoelectric point of the two pea isophytohemagglutinins varies from pH 5.7 to pH 8.4 depending on the composition of the buffer used. Isoelectric focusing reveals three main molecular species with pI at pH 5.90, 6.35 and 7.00. Molecular species with pI at pH 5.9 and 7.0 correspond to the two pea isophytohemagglutinins which can be obtained by DEAE-cellulose chromatography (Entlicher, G., Kostír, J.V. and Kocurek, J. (1970) Biochim. Biophys. Acta 221, 272-281). A molecular species with pI at pH 6.35 is formed from the two pea isophytohemagglutinins by hybridization. According to the hybridization pattern and subunit composition of the pea isophytohemagglutinins the subunit composition AABB, AACC and AABC can be proposed for the three molecular species with respect to ionic properties of the subunits.

Studies on lectins. XXXVII. Isolation and characterization of the lectin from Jimson-weed seeds (Datura stramonium L.).

The lectin of Jimson-weed seeds (Datura stramonium L.) was isolated by affinity chromatography on a polysaccharide mixture from mycelium of Aspergillus niger. The lectin yields two bands on disc electrophoresis, it has sedimentation coefficient s20,w = 3.8 S and its apparent molecular weight estimated by thin layer gel chromatography is 120,000. The lectin reduced with mercaptoethanol yields on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate three zones corresponding to subunits of molecular weight 72,000, 45,000 and 25,000. The lectin contains large amounts of cystine, glycine, 6.3% of hydroxyproline residues, 4.5% glucosamine and 28% of neutral sugar, predominantly arabinose. The lectin is nonspecific in human erythrocyte ABO system, it is not inhibited by simple sugars but is inhibited by a partial hydrolysate of chitin-containing mixture of polysaccharides from Aspergillus niger.

Studies on lectins. XXXVI. Properties of some lectins prepared by affinity chromatography on O-glycosyl polyacrylamide gels.

A number of lectins has been purified by affinity chromatography on O-glycosyl polyacrylamide gels. The lectins isolated (and the particular sugar ligands used in the affinity carriers) are as follows: Anguilla anguilla, serum (alpha-L-fucosyl-), Vicia cracca, seeds; Phaseolus lunatus, seeds; Glycine soja, seeds; Dolichos biflorus, seeds; Maclura pomifera, seeds; Sarothamnus scoparius, seeds; Helix pomatia, ablumin glands; Clitocybe nebularis, fruiting bodies (all N-acetyl-alpha-D-galactosaminyl-); Ricinus communis, seeds (beta-lactosyl-); Ononis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all alpha-D-galactosyl-), Canavalia ensiformis, seeds, (i.e., concanavalin A) (alpha-D-glucosyl-). Physicochemical properties of Glycine soja, Dolichos biflorus, Phaseolus lunatus, Helix Pomatia and Ricinus communis lectins corresponded well to properties of the preparations studied earlier by other workers. For the other purified lectins the essential physiochemical data (sedimentation coefficient, molecular weight, subunit composition, electrophoretic patterns, amino acid composition, carbohydrate content, isoelectric point) were established and their precipitating, hemagglutinating and mitogenic activities determined.

Studies on phytohemagglutinins. XXVI. A new type of a lentil hemagglutinin isolated from Lens esculenta Moench., subsp. microsperma (Baumg.) Barulina.

In seeds of the lentil Lens esculenta Moench., subsp. microsperma (Baumg.) Barulina, a nonspecific phytohemagglutinin was found, the properties of which differ from those of the previously described lentil hemagglutinins. The phytohemagglutinin is not adsorbed to the Sephadex matrix; its hemagglutinating activity is different towards erythrocytes of different human blood groups of the ABO system. The isolated phytohemagglutinin preparation is homogeneous by disc polyacrylamide and starch gel electrophoreses and by gel chromatography on Bio-Gel P-100. Ultracentrifugation of the phytohemagglutinin yields a single symmetrical peak with S20,W of 3.9 S. From the sedimentation data, a molecular weight of 53 300 was calculated. Phytohemagglutinins of similar properties were shown to be present also in other cultivars of Lens esculenta, subsp. microsperma.

Studies on lectins. XLI. Isolation and characterization of a blood group B specific lectin from the role of the powan (Coregonus lavaretus maraena).

A B-specific lectin from the roe of the powan (Coregonus lavaretus maraena), a fish of the Salmonidae family, was isolated by affinity chromatography on O-alpha-D-galactosyl polyacrylamide gel. From 630 g of the lyophilized roe, 346 mg of pure lectin was obtained in a single isolation step. The lectin is electrophoretically homogeneous, its sedimentation coefficient s20,w is 2.9S and molecular weight 25 000. The molecular weight of the subunits estimated by electrophoresis in the presence of dodecyl sulfate is 27 000 for both reduced and nonreduced substance. The lectin contains a large amount of cysteine, has a small content of aromatic amino acids, 10.4% of neutral sugar and 0.145% of Zn. It agglutinates specifically human B-group erythrocytes; the agglutination is stimulated by Zn2+ and Mg2+ ions and partially inhibited by EDTA. The most efficient carbohydrate inhibitors are methyl alpha-L-rhamnoside (6 micrometer), L-rhamnose (12 micrometer) and raffinose (0.1 mM). The association constant of the complex lectin . L-rhamnose is KA = 9.5 . 10(2) M-1, as determined by fluorimetric titration.

Studies on lectins. XXXVIII. Isolation and characterization of the lectin from black locust bark (Robinia pseudacacia L.).

The lectin of black locust (Robinia pseudacacia) bark was isolated by specific adsorption on formaldehyde-fixed human erythrocytes and elution with a borate solution. The lectin is homogeneous on disc electrophoresis and ultracentrifugation (s20,w = 5.8 S) but yields three bands on isoelectric focusing. It has a molecular weight of approximately 110,000 and consists of two types of subunit (mol. wt 29,000 and 31,500). Its pI is approximately 5.9; it contains high amounts of aspartic acid, threonine and serine, no cysteine and very little methionine. Also 7.2% of covalently bound neutral sugar and 0.47% of glucosamine are present. The lectin is nonspecific in agglutination of human erythrocytes, it is inhibited by high concentrations of N-acetyl-D-galactosamine and is mitogenic in rabbit lymph node lymphocytes.

Studies on lectins. XLIII. Isolation and characterization of the lectin from restharrow roots (Ononis hircina Jacq.).

From 1 kg of dried Ononis hircina Jacq. roots 36 mg of a lectin were isolated by affinity chromatography on O-beta-lactosyl polyacrylamide gel. The lectin is homogeneous as judged by ultracentrifugal analysis (S20,W=6.2 S), polyacrylamide disc electrophoresis at pH 8.6 or 4.5, gel filtration on thin layers of Sephadex G-200 (Mr = 110 000) and dodecyl sulfate electrophoresis (Mr of subunits 31 000, both in presence and absence of mercaptoethanol) and disc dodecyl sulfate electrophoresis (pH 9.5). The lectin contains much aspartic and glutamic acids, serine and threonine and also 7.2% of neutral sugar. It is relatively specific for human type O erythrocytes that are agglutinated at a minimal lectin concentration 0.3 microgram/ml. The erythroagglutinating activity is not stimulated by Ca2+, Zn2+, Mg2+, Mn2+, Co2+ or Ni2+ salts; it is inhibited most effectively by N-acetyl-D-galactosamine and a number of D-galactose derivatives. Dissociation constants of several lectin . sugar complexes were estimated by affinity electrophoresis. The lectin is not mitogenic in rabbit lymph node lymphocytes.

Studies on lectins. XXXV. Water-soluble O-glycosyl polyacrylamide derivatives for specific precipitation of lectins.

By copolymerization of acrylamide and allyl glycosides of various sugars, O-glycosyl derivatives of polyacrylamide copolymers were prepared. The sugar content of the copolymers can be varied in the range 0--40%, their sedimentation coefficient shows the vales of 2.5-5.7 S; the molecular weight of an O-alpha-D-mannopyranosyl polyacrylamide copolymer (29% mannose, so20,w = 2.9 S) was estimated as 44 500. Copolymers with incorporated glycosyl residues interacting specifically with lectins yield precipitates with them upon immunodiffusion in cellulose acetate. The quantitative precipitin curves obtained with these copolymers are similar to those produced by quantitative precipitation of lectins with natural polysaccharides. The copolymers may serve as model substances of natural polysaccharides.

Studies on lectins. XL. O-glycosyl derivatives of Spheron in affinity chromatography of lectins.

Free monosaccharides can be used for direct glycosylation of Spheron, a spherical macroporous hydroxyalkyl methacrylate-ethylene dimethacrylate copolymer, in a reaction that proceeds at room temperature in dioxane medium under catalysis of dry HCl or BF3. Derivatives of L-fucose, D-galactose, D-glucose, D-mannose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine thus prepared from Spheron beads have been shown to be efficient affinity carriers in isolation of lectins from seeds of Canavalia ensiformis D.C. (concanavalin A), Dolichos biflorus L., Glycine soja (L.) Sieb. et Zucc., Lens esculenta Moench, Ricinus communis L., Ulex europaeus L. and from albumin glands of the garden snail Helix pomatia L.

Studies on phytohemagglutinins. XXVII. A study of the pea lectin binding site.

Under defined mild conditions the reaction of the pea lectin with 2-nitrophenylsulfenyl chloride results in sulfenylation of only 2 of the 10 tryptophan residues of the lectin molecule with simultaneous loss of biological activity. Both sulfenylated tryptophan residues belong to the two heavy subunits of the lectin. Enzymic hydrolysis and separation of the tryptic peptides yields only one homogeneous yellow peptide containing the modified tryptophan residue. The isolated peptide has the following sequence (NPS, nitrophenylsulfenyl): HAsp-Val-Val-Pro-Glu-(2-NPS-Trp)-Val-ArgOH. The octapeptide is either directly a part of the pea lectin binding site or it plays an important role in maintaining the tertiary structure of the binding site. According to the amino acid composition and amino acid sequence, the octapeptide isolated from the pea lectin is almost identical with that part of the peptide chain of concanavalin A near to which the location of the sugar binding site is supposed to be.

Studies on lectins. XXXI. Determination of dissociation constants of lectin. Sugar complexes by means of affinity electrophoresis.

A modification of affinity electrophoresis in polyacrylamide gels containing immobilized sugar residues is described. The immobilization of sugar residues is achieved by addition of a water soluble O-glycosyl polyacrylamide copolymer to the polymerization mixture, which serves for the preparation of gels for commonly used discontinuous polyacrylamide gel electrophoresis. The electrophoretic mobility of lectins in affinity gels containing specific immobilized sugar decreases with increasing sugar concentration. The addition of free sugar into the affinity gel abolishes retardation of the electrophoretic mobility caused by immobilized sugar. The relationship between electrophoretic mobility of lectins and concentration of immobilized and free sugar was used for the calculation of dissociation constants of the various complexes lectin-immobilized sugar and lectin-free sugar.

Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

Studies on phytohemagglutinins. XXVIII. Chemical modifications of the lectin from seeds of the lentil (Lens esculenta Moench.)

The reaction of lentil lectin with maleic anhydride yields maleyl derivatives with a degree of modification depending on the amount of used maleic anhydride. Hemagglutinating activity of maleyl derivatives decreases with the increasing number of modified lysine residues, but even at a maleylation of 90% of free -NH2 groups, the maleyl derivative of the lectin still retains a hemagglutinating activity. Maleylation of the lentil lectin is not accompanied by dissociation; the chemically modified lectin also retains its bound Mn. Maleyl derivatives of the lectin retain the ability to interact specifically with polysaccharides (Sephadex, mannan). After removal of maleyl groups in acidic medium, the hemagglutinating activity does not differ from that of the native lectin. Acetylation of the lentil lectin with N-acetylimidazole results in formation of a modified protein containing 3-5 acetylated -OH groups of the tyrosyl residues out of the total of 15 groups and 7-17 acetylated -NH2 groups out of the total of 26 free -NH2 groups, depending upon the reaction conditions. The presence of D-mannose in the reaction mixture does not affect the extent of modification of tyrosyl groups, but it decreases the modification of -NH2 groups. Acetyl derivatives of the lentil lectin do not possess hemagglutinating activity, but they retain the ability to interact with polysaccharides and to bind to erythrocytes. Acetylation does not affect the content of protein bound Mn. After de-O-acetylation, the hemagglutinating activity of the modified protein increases to the value of the native protein.

Studies on lectins. XXXIX. S-Glycosyl polyacrylamide gels for affinity chromatography of lectins.

Hydrophilic water-insoluble gels suitable for affinity chromatography of lectins have been prepared by copolymerization of acrylamide, N,N'-methylene bisacrylamide and alkenyl 1-thioglycosides. Water-soluble copolymers of analogous type have been obtained by omitting the cross-linking agent, N,N'-methylene bisacrylamide. In affinity chromatography of the Ricinus communis lectin it could be shown that the capacity for the lectin of the water insoluble copolymers was more than four times higher in copolymers having the S-beta-D-galactosyl ligand attached through a methylene bridge than in derivatives with a nonamethylene spacer. None of the insoluble S-beta-D-glycosyl copolymers prepared could be shown usable as affinity adsorbent for glycosidases though the corresponding soluble copolymers inhibited the activity of the enzymes.

Studies on lectins. LI. The role of Mn2+ in the activity of the soybean lectin.

In contradistinction to previous reports, the lectin of soybeans (Glycine soja) has been shown to retain its erythroagglutinating activity after complete removal of manganese from its molecule. The applied demetallization procedures (dialysis against 0.1 M HCl or 1M acetic acid and against 0.1 M EDTA following by dialysis against 1 M acetic acid) had no effect on the stability of the lectin subunit structure, as proved by ultracentrifugation analysis or thin-layer chromatography on Sephadex G-200 Superfine. Affinity electrophoresis on polyacrylamide gel containing immobilized alpha-D-galactosyl residues and affinity chromatography on alpha-D-galactosyl derivative of Separon have shown that the interaction of the demetallized lectin preparations does not differ from that of the native lectin in alkaline media but is decreased in acidic media. Demetallized preparations of soybean lectin can be fully reactivated by dialysis against solutions containing Mn2+, Zn2+ or Cd2+.

Studies on lectins. XLIV. The pH dependence of lectin interactions with sugars as determined by affinity electrophoresis.

The pH dependence of association constants of the lectin-sugar complexes was determined by means of affinity electrophoresis. All the lectins studied (from the seeds of Dolichos biflorus, Glycine soja, Lens esculenta and Vicia cracca and of the fruiting body of Marasmius oreades) were characterized by a similar course of pH dependence of the association constants, with the maximum values at pH 7--9. For concanavalin A and the L-fucose binding Ulex europaeus lectin only the association constants at three selected pH values were determined. Concanavalin A does not interact with immobilized alpha-D-mannosyl residues at pH 2.3. The association constants vs. pH curves measured for lectins isolated from two different lentil varieties slightly differ in accordance with the differences observed in the interaction of these lectins with the Sephadex gel.

Studies on lectins. XLVII. Some properties of D-galactose binding lectins isolated from the seeds of Butea frondosa, Erythrina indica and Momordica charantia.

The lectins from the seeds of Butea frondosa, Erythrina indica and Momordica charantia were isolated by affinity chromatography on O-alpha-D-galactosyl polyacrylamide gels. The Butea frondosa lectin has sedimentation coefficient S20,W = 6.7 S, its molecular weight is 141 000 and it consists of noncovalently associated subunits of molecular weights 36 000 and 65 000. The lectin contains two 1/2-cystine and 3--4 methionine residues per molecule, 4.3% neutral sugar and 0.5% glucosamine. Threonine is the single N-terminal amino acid residue. The lectin from Erythrina indica seeds has a sedimentation coefficient S20,W = 4.0 S, its molecular weight is 66 200 and it consists of two kinds of subunit (molecular weights 30 000 and 34 000, respectively), which are noncovalently associated. The lectin is devoid of cystein, has a neutral sugar content 5.6% and glucosamine content 0.46%. It contains probably one Mn atom per subunit. The N-terminal amino acid sequence is Val-Glu-Val-Leu-(Phe)-Phe-(Ala)-Phe-. The lectin from Momordica charantia seeds has a sedimentation coefficient S20,W = 6.5 S, its molecular weight is 129 000. It interacts weakly with Sephadex G-200. The lectin consists of subunits of molecular weights 29 000, 32 000 and 36 000, respectively, held together by cystine bridges. Its content of cystine and methionine residues is relatively high, 4.1% neutral sugar and 0.5% glucosamine form the carbohydrate moiety. Two N-terminal amino acids (asparagine and valine) are present.

Localization and characterization of the carbohydrate-binding site of the porcine lymphocyte mannan-binding protein.

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.

Lactosamine type asialooligosaccharide recognition in NK cytotoxicity.

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.