Defining the carbohydrate specificities of aplysia gonad lectin exhibiting a peculiar D-galacturonic acid affinity

Aplysia gonad lectin (AGL), which has been shown to stimulate mitogenesis in human peripheral lymphocytes, to suppress tumor cells, and to induce neurite outgrowth and improve cell viability in cultured Aplysia neurons, exhibits a peculiar galacturonic acid/galactose specificity. The carbohydrate binding site of this lectin was characterized by enzyme-linked lectino-sorbent assay and by inhibition of AGL-glycan interactions. Examination of the lectin binding with 34 glycans revealed that it reacted strongly with the following glycoforms: most human blood group precursor (equivalent) glycoproteins (gps), two Galalpha1-->4Gal-containing gps, and two d-galacturonic acid (GalUA)-containing polysaccharides (pectins from apple and citrus fruits), but poorly with most human blood group A and H active and sialylated gps. Among the GalUA and mammalian saccharides tested for inhibition of AGL-glycan binding, GalUA mono- to trisaccharides were the most potent ones. They were 8.5 x 10(4) times more active than Gal and about 1.5 x 10(3) more active than the human blood group P(k) active disaccharide (E, Galalpha1-->4Gal). This disaccharide was 6, 28, and 120 times more efficient than Galbeta1-->3GlcNAc(I), Galbeta1-->3GalNAc(T), and Galbeta1--> 4GlcNAc (II), respectively, and 35 and 80 times more active than melibiose (Galalpha1-->6Glc) and human blood group B active disaccharide (Galalpha1-->3Gal), respectively, showing that the decreasing order of the lectin affinity toward alpha-anomers of Gal is alpha1-->4 > alpha1-->6 > alpha1-->3. From the data provided, the carbohydrate specificity of AGL can be defined as GalUAalpha1-->4 trisaccharides to mono GalUA > branched or cluster forms of E, I, and II monomeric E, I, and II, whereas GalNAc is inactive.     

Carbohydrate specificity of a toxic lectin, abrin A, from the seeds of Abrus precatorius (jequirity bean)

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Gal beta1-->4GlcNAc (II), Gal alpha1-->4Gal (E), and Gal beta1-->3GalNAc (T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the (D)Fuc pyranose ring to penta-saccharides and probably internal Gal alpha,beta-->; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Gal alpha1-->3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I > Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.     

Binding profile of Artocarpus integrifolia agglutinin (Jacalin)

Artocarpus integrifolia agglutinin (Jacalin) from the seeds of jack fruits has attracted considerable attention for its diverse biological activities and has been recognized as a Galbeta1-->3GalNAc (T) specific lectin. In previous studies, the information of its binding was limited to the inhibition results of monosaccharides and several T related disaccharides, but its interaction with other carbohydrate structural units occurring in natural glycans has not been characterized. For this reason, the binding profile of this lectin was studied by enzyme linked lectinosorbent assay (ELLSA) with our glycan/ligand collection. Among glycoproteins (gps) tested for binding, high density of multi-Galbeta1-->3GalNAcalpha1--> (mT(alpha)) and GalNAcalpha1-->Ser/Thr (mTn) containing gps reacted most avidly with Jacalin. As inhibitors expressed as nanograms yielding 50% inhibition, these mT(alpha) and mTn containing glycans were about 7.1 x 10(3), 4.0 x 10(5), and 7.8 x 10(5) times more potent than monomeric T(alpha), GalNAc, and Gal. Of the sugars tested and expressed as nanomoles for 50% inhibition, Tn containing peptides, T(alpha), and the human P blood group active disaccharide (P(alpha), GalNAcbeta1-->3Galalpha1-->) were the best and about 283 times more active than Gal. We conclude that the most potent ligands for this lectin are mTn, mT, and possibly P(alpha) glycotopes, while GalNAcbeta1-->4Galbeta1-->, GalNAcalpha1-->3Gal, GalNAcalpha1-->3GalNAc, and Galalpha1-->3Gal determinants were poor inhibitors. Thus, the overall binding profile of Jacalin can be defined in decreasing order as high density of mTn, and mT(alpha) > simple Tn cluster > monomeric T(alpha) > monomeric P(alpha) > monomeric Tn > monomeric T > GalNAc > Gal > Methylalpha1-->Man z.Gt; Man and Glc (inactive). Our finding should aid in the selection of this lectin for biological applications.     

Carbohydrate binding specificity of immobilized Allomyrina dichotoma lectin II

The carbohydrate binding specificity of Allomyrina dichotoma lectin II was investigated by analyzing the behavior of various complex type oligosaccharides and human milk oligosaccharides on an A. dichotoma lectin II-agarose column. Basically, the lectin interacts with the Gal beta 1----4GlcNAc group. Substitution of their terminal galactose residues by Neu5Ac alpha 2----6 will enhance their affinity to the lectin. By contraries, substitution at the C-2 or C-3 position of their terminal galactose with other sugars including sialic acid deprives their affinity to the lectin. With this characteristic, the immobilized lectin column can be used to separate complex type oligosaccharides with the Neu5Ac alpha 2----6Gal beta 1----4GlcNAc group from their isomeric oligosaccharides with the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc group, where Neu5Ac is N-acetylneuraminic acid.     

Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica.

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.     

The B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residues alpha-linked to serine or threonine residues in cell surface glycoproteins

We have examined the carbohydrate binding specificity of the B4 lectin from Vicia villosa seeds. The B4 lectin agglutinates Tn-exposed erythrocytes specifically and binds to these erythrocytes (1.4 X 10(6) sites/cell) with an association constant of 4.2 X 10(7) M-1. The concentrations of saccharides and glycopeptides of defined structure which cause 50% inhibition of B4 lectin binding to Tn-exposed erythrocytes were determined. N-Acetylgalactosamine is the best monosaccharide inhibitor, causing 50% inhibition of binding at a concentration of 0.04 mM. Other monosaccharides inhibit lectin binding in the following order of decreasing potency: N-acetylgalactosamine greater than methyl-alpha-galactopyranoside greater than p-nitrophenyl-alpha- or beta-galactopyranoside greater than methyl-beta-galactopyranoside, galactose greater than galactosamine greater than mannose, N-acetylglucosamine. The disaccharide Gal beta 1,3GalNAc causes 50% inhibition of binding at a concentration of 2.8 mM, a concentration similar to that of the p-nitrophenyl-alpha- or beta-galactopyranosides. Glycopeptides containing O-glycosidically linked oligosaccharide units are significantly more potent inhibitors of lectin binding than the oligosaccharide units alone. The most potent glycopeptide inhibitor is a fetuin glycopeptide containing two alpha-linked N-acetylgalactosamine units. This glycopeptide causes 50% inhibition of lectin binding at a concentration of 0.00034 mM and probably closely resembles the B4 lectin binding site on Tn-exposed erythrocytes.     

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.     

Recognition profile of Bauhinia purpurea agglutinin (BPA)

Bauhinia purpurea agglutinin (BPA) is a Galbeta1-3GalNAc (T) specific leguminous lectin that has been widely used in multifarious cytochemical and immunological studies of cells and tissues under pathological or malignant conditions. Despite these diverse applications, knowledge of its carbohydrate specificity was mainly limited to molecular or submolecular T disaccharides. Thus, the requirement of high density polyvalent or multi-antennary carbohydrate structural units for BPA binding and an updated affinity profile were further evaluated by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. Among the glycoproteins (gps) tested and expressed as 50% nanogram inhibition, the high density polyvalent GalNAcalpha1-Ser/Thr (Tn) and Galbeta1-3/4GlcNAc (I/II) glycotopes present on macromolecules generated a great enhancement of binding affinity for BPA as compared to their monomers. The most potent inhibitors were a Tn-containing gp (asialo OSM) and a I/II containing gp (human blood group precursor gp), which were up to 1.7 x 10(4) and 2.3 x 10(3) times more potent than monovalent Gal and GalNAc, respectively. However, multi-antennary glycopeptides, such as tri-antennary Galbeta1-4GlcNAc, which was slightly more active than II or Gal, gave only a minor contribution. Regarding the carbohydrate structural units studied by the inhibition assay, blood group GalNAcbeta1-3/4Gal (P/S) active glycotopes were active ligands. The overall binding profile of BPA was: high density polyvalent T/Tn and II clusters > Tn-glycopeptides (M.W. <3.0 x 10(3))/Talpha monomer > monovalent P/S > Tn monomer and GalNAc > tri-antennary II > Gal > Man and Glc (inactive). These findings give evidence for the binding of this lectin to dense cell surface T, Tn and I/II glycoconjugates and should facilitate future usage of this lectin in biotechnological and medical applications.     

Isolation and characterization of an N-acetylgalactosamine specific lectin from Salvia sclarea seeds

Crude extracts from Salvia sclarea seeds were known to contain a lectin which specifically agglutinates Tn erythrocytes (Bird, G. W. G., and Wingham, G. (1974) Vox Sang. 26, 163-166). We have purified the lectin to homogeneity by ion-exchange chromatography and affinity chromatography. The agglutinin was found to be a glycoprotein of Mr = 50,000, composed of two identical subunits of Mr = 35,000 linked together by disulfide bonds. The purified lectin agglutinates specifically Tn erythrocytes and, at higher concentrations, also Cad erythrocytes. Native A, B, or O red blood cells are not agglutinated by the lectin and, even after treatment with sialidase or papain, these cells are not recognized. Tn red cells present 1.45 X 10(6) accessible sites to the lectin which binds to these erythrocytes with an association constant of 1.8 X 10(6) M-1. On Cad red cells, 1.73 X 10(6) sites are accessible to the lectin which binds with an association constant of 1.0 X 10(6) M-1. The carbohydrate specificity of the S. sclarea lectin has been determined in detail, using well defined monosaccharide, oligosaccharide, and glycopeptide structures. The lectin was found to be specific for terminal N-acetylgalactosamine (GalNAc) residues. It binds preferentially alpha GalNAc determinants either linked to Ser or Thr (as in Tn structures) or linked in 1-3 to a beta GalNAc or to an unsubstituted beta Gal. Although more weakly, the lectin binds beta GalNAc residues linked in 1-4 to a beta Gal (as in Cad structures). It does not recognize beta GalNAc determinants linked in 1-3 to a Gal (as in globoside) or the alpha GalNAc residues of blood group A structures.     

Lectinochemical characterization of a GalNAc and multi-Galbeta1-->4GlcNAc reactive lectin from Wistaria sinensis seeds.

An agglutinin that has high affinity for GalNAcbeta1-->, was isolated from seeds of Wistaria sinensis by adsorption to immobilized mild acid-treated hog gastric mucin on Sepharose 4B matrix and elution with aqueous 0.2 M lactose. The binding property of this lectin was characterized by quantitative precipitin assay (QPA) and by inhibition of biotinylated lectin-glycan interaction. Of the 37 glycoforms tested by QPA, this agglutinin reacted best with a GalNAcbeta1-->4 containing glycoprotein (GP) [Tamm-Horsfall Sd(a+) GP]; a Galbeta1-->4GlcNAc containing GP (human blood group precursor glycoprotein from ovarian cyst fluid and asialo human alpha1-acid GP) and a GalNAcalpha1-->3GalNAc containing GP (asialo bird nest GP), but poorly or not at all with most sialic acid containing glycoproteins. Among the oligosaccharides tested, GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc (Fp) was the most active ligand. It was as active as GalNAc and two to 11 times more active than Tn cluster mixtures, Galbeta1--> 3/4GlcNAc (I/II), GalNAcalpha1-->3(L-Fucalpha1-->2)Gal (Ah), Galbeta1-->4Glc (L), Galbeta1-->3GalNAc (T) and Galalpha1--> 3Galalpha-->methyl (B). Of the monosaccharides and their glycosides tested, p-nitrophenyl betaGalNAc was the best inhibitor; it was approximately 1.7 and 2.5 times more potent than its corresponding alpha anomer and GalNAc (or Fp), respectively. GalNAc was 53.3 times more active than Gal. From the present observations, it can be concluded that the Wistaria agglutinin (WSA) binds to the C-3, C-4 and C-6 positions of GalNAc and Gal residues; the N-acetyl group at C-2 enhances its binding dramatically. The combining site of WSA for GalNAc related ligands is most likely of a shallow type, able to recognize both alpha and beta anomers of GalNAc. Gal ligands must be Galbeta1-->3/4GlcNAc related, in which subterminal beta1-->3/4 GlcNAc contributes significantly to binding; hydrophobicity is important for binding of the beta anomer of Gal. The decreasing order of the affinity of WSA for mammalian structural carbohydrate units is Fp >/= multi-II > monomeric II >/= Tn, I and Ah >/= E and L > T > Gal.     

The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2-6)Gal/GalNAc sequence

Carbohydrate binding properties of a new plant lectin isolated from elderberry (Sambucus nigra L.) (SNA) bark were studied using the techniques of quantitative precipitation, hapten inhibition, and equilibrium dialysis. Purified SNA precipitates highly sialylated glycoproteins such as fetuin, orosomucoid, and ovine submaxillary mucin, but not their asialo derivatives. Hapten inhibition experiments showed that both D-Gal and D-GalNAc are weak inhibitors of SNA-glycophorin precipitation, but neither New5Ac nor Neu5Gc is an inhibitor. A series of oligosaccharides which contain the terminal Neu5Ac(alpha 2-6)Gal sequence showed an extremely high inhibitory potency (1,600-10,000 times more inhibitory than Gal). On the other hand, oligosaccharides with the Neu5Ac(alpha 2-3)Gal linkage were only 30-80 times more inhibitory than Gal, thus showing a marked preference for the 2,6-linked isomer. Hapten inhibition with Gal and its epimers suggested that the equatorial OH at C-3 and the axial OH at C-4 of the D-pyranose ring are strict requirements for binding. Conversion of the Neu5Ac residue to its 7-carbon analogue by selective periodate oxidation of its glyceryl side chain, followed by NaBH4 reduction, completely destroyed the ability of fetuin and orosomucoid to precipitate with SNA. Moreover, the same treatment of Neu5Ac(alpha 2-3)lactitol also abolished its ability to inhibit the precipitation reaction, suggesting that the glyceryl side chain of NBu5Ac (especially the C-8 and/or C-9 portion) is an important determinant for SNA. The increased inhibitory potency of various glycosides with beta-linked nonpolar aglycons suggested the presence of a hydrophibic interacting region adjacent to the carbohydrate binding site. The results of equilibrium dialysis using [3H] Neu5Ac(alpha 2-6)lactitol as ligand showed the presence of two equivalent, noninteracting carbohydrate binding sites in this tetrameric glycoprotein lectin (Ka = 3.9 X 10(5) M-1).     

Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin

The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.     

Catfish (Clarias batrachus) serum lectin recognizes polyvalent Tn [alpha-D-GalpNAc1-Ser/Thr], Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc1-Ser/Thr], and II [beta-D-Galp(1-->4)-beta-D-GlcpNAc1-] mammalian glycotopes

A new calcium dependent GalNAc/Gal specific lectin was isolated from the serum of Indian catfish, Clarias batrachus and designated as C. batrachus lectin (CBL). It is a disulfide-linked homodecameric lectin of 74.65kDa subunits and the oligomeric form is essential for its activity. Binding specificity of CBL was investigated by enzyme-linked lectin-sorbent assay using a series of simple sugars, polysaccharides, and glycoproteins. GalNAc was more potent inhibitor than Gal; and alpha glycosides of both were more inhibitory than their beta counterparts. CBL showed maximum affinity for human tumor-associated Tn-antigens (GalNAcalpha1-Ser/Thr) at the molecular level and was 3.5 times higher than GalNAc. CBL interacted strongly with polyvalent Tn and Talpha (Galbeta1,3GalNAcalpha1-) as well as multivalent-II (Galbeta1,4GlcNAcbeta1-) antigens containing glycoproteins and intensity of inhibition was 10(3)-10(5) times more than monovalent ones. The overall specificity of CBL lies in the order of polyvalent Tn, Talpha and II>monovalent TnMe-alphaGalNAc>monovalent Talpha> Me-betaGalNAc>Me-alphaGal>monovalent T>GalNAc>monovalent F>monovalent II>Me-betaGal>Gal.     

Isolation and characterization of amaranthin, a lectin present in the seeds of Amaranthus caudatus, that recognizes the T- (or cryptic T)-antigen

A lectin (Amaranthin) present in the seeds of Amaranthus caudatus has been isolated by fractionation on DEAE-cellulose followed by affinity chromatography on Synsorb-T beads (Gal beta 1,3GalNAc alpha-O-R-Synsorb). The lectin appeared homogeneous by gel electrophoresis at pH 4.3 and gave a single protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr = 33,000-36,000. A native Mr = 54,000 was determined by gel filtration suggesting that amaranthin exists as a homodimer. Compositional analysis revealed high amounts of acidic and hydroxyamino acids and relatively large amounts of lysine, methionine, and tryptophan for a plant protein. Amaranthin formed a precipitate with asialo-bovine submaxillary mucin, asialo-ovine submaxillary, porcine submaxillary mucin, asialo-fetuin and asialoglycophorin. Hapten inhibition of precipitate formation between amaranthin and asialo-ovine submaxillary indicated that the T-disaccharide and its alpha-linked glycosides (Gal beta 1,3GalNAc alpha-O-R; R = OH, methyl, -(CH2)8-COOCH3, allyl, o-nitrophenyl, or benzyl) were the best inhibitors. N-Acetylgalactosamine, the only monosaccharide which inhibited precipitation, was 350-fold less effective than Gal beta 1,3GalNAc alpha-O-R. Hapten inhibition with derivatives of the T-disaccharide suggested that the C'-4 axial hydroxyl group of the galactosyl moiety, and the C-4 axial hydroxyl group, and the C-2 acetamido group of the GalNAc unit are the most important loci for lectin interaction. NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-(CH2)8CO2CH3 was as potent an inhibitor as Gal beta 1,3GalNAc alpha-O-(CH2)8CO2-CH3, and amaranthin was precipitated by NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-BSA (where BSA is bovine serum albumin), indicating that the amaranthin-combining site tolerates substitutions at the C'-3 hydroxyl group. Amaranthin was precipitated by a Gal beta 1,3GalNAc alpha-O-BSA glycoconjugate but not by the anomeric Gal beta 1,3GalNAc beta-O-BSA glycoconjugate illustrating that the disaccharide must be linked alpha in order to interact with the lectin. Metal ions do not appear to be required for lectin activity. A study of pH dependence showed significant precipitate formation between pH 4 to 9 with a maximum at pH 5. Hapten inhibition and glycoconjugate precipitation assays were also conducted for peanut (Arachis hypogaea) agglutinin. A comparison between the carbohydrate-binding specificities of amaranthin and peanut (Arachis hypogaea) agglutinin is discussed.     

The binding of fucose-containing glycoproteins by hepatic lectins. Re-examination of the clearance from blood and the binding to membrane receptors and pure lectins

The nature of the hepatic receptors that bind glycoproteins through fucose at the non-reducing termini of oligosaccharides in glycoproteins has been examined by three different approaches. First, the clearance from blood of intravenously injected glycoproteins was examined in mice with the aid of neoglycoproteins of bovine serum albumin (BSA). The clearance of fucosyl-BSA was rapid and was not strongly inhibited by glycoproteins that inhibit clearance mediated by the galactose or the mannose/N-acetylglucosamine receptors of liver. The clearance of Fuc alpha 1,3(Gal beta 1,4)GlcNAc-BSA (where Fuc is fucose) was inhibited weakly by either Fuc-BSA or Gal beta 1,4GlcNAc-BSA but strongly by a mixture of the two neoglycoproteins, suggesting that its clearance was mediated by hepatic galactose receptors as well as by a fucose-binding receptor. Second, the binding of neoglycoproteins to a membrane fraction of mouse liver was examined. Fuc-BSA binding to membranes was Ca2+ dependent but was not inhibited by glycoproteins that would inhibit the galactose or the mannose/N-acetylglucosamine receptors. In addition, the binding of Fuc-BSA and Gal beta 1,4GlcNAc-BSA differed as a function of pH, in accord with binding of Fuc-BSA through fucose-specific hepatic receptors. Finally, the binding of neoglycoproteins to the pure galactose lectin from rat liver was examined. Neither Fuc-BSA nor Fuc alpha 1,2Gal beta 1,4GlcNAc-BSA bound the galactose lectin, although Fuc alpha 1,3(Gal beta-1,4) GlcNAc-BSA bound avidly. Taken together, these studies suggest that a fucose-binding receptor that differs from the galactose and the mannose/N-acetylglucosamine receptors may exist in rat and mouse liver.     

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Carbohydrate binding specificity of the Tn-antigen binding lectin from Vicia villosa seeds (VVLB4).

2-Dansylamino-2-deoxy-D-galactose (GalNDns) is a useful fluorescent probe to study the interaction of non-fluorescent sugars with the B4 lectin from Vicia villosa seeds (VVLB4). Binding of the lectin to GalNDns leads to a 5.2-fold increase in Dansyl fluorescence with a concomitant 10 nm blue shift in its emission maximum. The strong binding of GalNDns (Ka = 7.33 x 10(4) M-1 at 20 degrees C) is due to a favourable entropic contribution to the association process. Among the other sugars studied, GalNAc alpha 1-O-Ser followed by Me alpha GalNAc are the best ligands. 2-Deoxygalactose, galactosamine and galactose are 2013, 469 and 130 times weaker ligands, respectively, as compared to GalNAc, whereas GalNDns is about 2.44 times more potent than GalNAc, indicating that substitutions at the C-2 position of GalNAc have a considerable influence on the binding affinities. Equatorial orientation of the hydroxyl group at C-3 and axial orientation at C-4 as in galactose are important for the interaction with VVLB4. The C-6 hydroxyl group is not indispensable. The binding site of the lectin is directed exclusively towards monosaccharides alone. Interestingly enough, despite its preference for Me alpha GalNAc over Me beta GalNAc, in oligosaccharides, the lectin prefers terminal beta-linked GalNAc as compared to the alpha-linked one.     

Carbohydrate specificity of a lectin isolated from the fungus Sclerotium rolfsii

In order to investigate the functional roles of a phytopathogenic fungal lectin (SRL) isolated from the bodies of Sclerotium rolfsii, the binding properties of SRL were studied by enzyme linked lectinosorbent assay and by inhibition of SRL-glycan interaction. Among glycoproteins (gp) tested for binding, SRL reacted strongly with GalNAc alpha1-->4Ser/Thr (Tn) and/or Gal beta1-->3GalNAc alpha1-->(T(alpha)) containing gps: human T(alpha) and Tn glycophorin, asialo salivary gps, and asialofetuin, but its reactivity toward sialylated glycoproteins was reduced significantly. Of the sugar ligands tested for inhibition of SRL-asialofetuin binding, Thomsen-Friedenreich residue (T(alpha)) was the best, being 22.4 and 2.24 x 10(3) more active than GalNAc and Gal beta1--> residues, respectively. Other ligands tested were inactive. When the glycans used as inhibitors, T(alpha), and/or Tn containing gps, especially asialo PSM, asialo BSM, asialo OSM, active antifreeze gp, asialo glycophorin and Tn-glycophorin were very active, and 1.0 x 10(4) times more potent than GalNAc. From these results, it is clear that the combining site of SRL should be of a cavity type and recognizes only Tn and T(alpha) residues of glycans; it is suggested that T(alpha) and Tn glycotopes, which are present only in abnormal carbohydrate sequences of higher orders of mammal, are the most likely sites for phytopathogenic fungal attachment as an initial step of infection. The affinity of SRL for ligands can be ranked in decreasing order as follows: multivalent T(alpha) and Tn > monomeric T(alpha) and Tn > GalNAc > II (Gal beta1-->4GlcNAc), L (Gal beta1-->4Glc), and Gal.     

Comparison of the carbohydrate-binding specificities of seven N-acetyl-D-galactosamine-recognizing lectins

Seven plant lectins, Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin (GSA, isolectin A4), Helix pomatia agglutinin (HPA), soybean (Glycine max) agglutinin (SBA), Salvia sclarea agglutinin (SSA), Vicia villosa agglutinin (VVA, isolectin B4) and Wistaria floribunda agglutinin (WFA), known to be specific for N-acetyl-D-galactosamine-(GalNAc) bearing glycoconjugates, have been compared by the binding of their radiolabelled derivatives, to eight well-characterized synthetic oligosaccharides immobilized via a spacer on an inert silica matrix (Synsorb). The eight oligosaccharides included the Forssman, the blood group A and the T antigens, as well as alpha GalNAc coupled directly to the support (Tn antigen) and also structures with GalNAc linked alpha or beta to positions 3 or 4 of an unsubstituted Gal. The binding studies clearly distinguished the lectins into alpha GalNAc-specific agglutinins like DBA, GSA and SSA, and lectins which recognize alpha- as well as beta-linked GalNAc residues like HPA, VVA, WFA and SBA. HPA was the only lectin which bound to the beta Gal1----3 alpha GalNAc-Synsorb adsorbent (T antigen) indicating that it also recognizes internal GalNAc residues. Among the alpha GalNAc-specific lectins, DBA strongly recognized blood group A structures while GSA displayed weaker recognition, and SSA bound only slightly to this affinity matrix. In addition, DBA and SSA were able to distinguish between GalNAc linked alpha 1----3 and GalNAc linked alpha 1----4, to the support, the latter being a much weaker ligand. These results were corroborated by the binding of the lectins to biological substrates as determined by their hemagglutination titers with native and enzyme-treated red blood cells carrying known GalNAc determinants, e.g. blood group A, and the Cad and Tn antigens. For SSA, the binding to the alpha GalNAc matrix was inhibited by a number of glycopeptides and glycoproteins confirming the strong preference of this lectin for alpha GalNAc-Ser/Thr-bearing glycoproteins.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.