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.

Separation of bovine heart galactose lectin from endogenous glycoproteins co-purified with the lectin during affinity chromatography.

During affinity chromatographic purification of bovine heart 14 kDa galactose-binding lectin (galectin 1) on lactose-Sepharose, several high molecular weight non-lectin glycoproteins were co-purified with the lectin. Glycoprotein binding to the affinity matrix was neither hydrophobic nor ionic, but galactose-dependensti nce lactose abolished binding. Purification of galectin from the co-purified glycoproteins by affinity electrophoresis in presence of the specific sugar lactose increased agglutination activity about 65-fold, indicating that a complex containing galectin molecules bound sugar specifically to endogenous glycoproteins with sugar binding sites still available had been retained on lactose-Sepharose.

Relative affinities of bovine brain 14 kDa galactose lectin binding to individual endogenous gangliosides.

Sugar-specific binding of bovine brain 14 kDa galactose-binding lectin (BBL) to individual endogenous gangliosides (GM1, GM2, GD1a, GD1b and GT1b) was studied using affinity electrophoresis of ganglioside-lectin mixture in polyacrylamide gel at pH 8.3. Unbound (free) lectin moved ahead while ganglioside-lectin complex moved very little. Sugar-specificity of binding was confirmed by reversal of the interaction by (i) presence of lactose along with the lectin and (ii) pretreatment of gangliosides with another galactose-binding lectin, Ricinus communis agglutinin. Stoichiometry of ganglioside-BBL interaction revealed that GT1b had the highest affinity for the lectin followed by GD1b and GM2, while GM1 and GD1a had the least affinity. Results indicated that a terminal sialic acid moiety covering a galactose moiety may at times enhance BBL recognition of the latter and that changes in ganglioside pattern is a possible modulator of lectin function in vivo.

Anomer specificity of the 14 kDa galactose-binding lectin: A reappraisal.

A β-anomer preference among galactosides has been attributed to the S-type 14 kDa galactose binding lectin. Here the anomeric preference of this lectin from bovine brain (BBL) is reexamined using inhibition of lectin-mediated haemagglutination, binding of the lectin to dot-blotted glycoproteins and affinity electrophoresis of the lectin through polysaccharide-containing gels. 1·0-methyl α-D-galactoside was 8 times better inhibitor of BBL than the corresponding ß-anomer. The terminal galactose in bovine thyroglobulin (exclusively. α-linked) were also nearly 8 times more inhibitory than those in asialofetuin (exclusively ß-linked). The terminal α-galactose-containing endogenous glycoproteins of bovine brain were nearly 4 times better inhibitors of BBL than laminin. Removal of terminal α-galactose units by α-galactosidase fully abolished the BBL binding of thyroglobulin and endogenous glycoproteins. BBL was also sugar-specifically retarded by polyacrylamide gel containing guar galactommannan which bears only α-linked galactose. Data indicated that α-galactosides were sometimes better than their β-anomers in binding to BBL. The significance of this observation to the physiological role of galactose-binding lectins is discussed.

Major bovine brain stem glycoproteins sugar-specifically recognized by endogenous 14 kDa galactose-binding lectin.

In order to identify the complementary glycoproteins (receptors) that are recognized in bovine brain stem by endogenous 14 kDa galactose-binding lectin (BBL), probable glycoproteins were first selected by affinity chromatography of soluble tissue glycoproteins on Rinicus communis agglutinin (RCA)-Sepharose since this lectin had similar sugar specificity to the endogenous lectin. From Western blot of RCA-binding glycoproteins, the lectin, as its peroxidase conjugate sugar-specifically recognized chiefly an 84 kDa glycoprotein subunit and a few minor subunits. On alkaline pH PAGE of the RCA-binding brain stem glycoproteins, a prominent fast moving protein was separated which, on electroelution and dot blotting, was also recognized by BBL sugar-specifically. This glycoprotein was composed of 55 kDa and 58 kDa subunits as seen by SDS-PAGE and was also immunologically distinct from the 84 kDa subunit. Qualitative test on dot blots of the electroeluted glycoproteins using peroxidase conjugates of plant lectins of varying specificities as well as the human serum anti-alpha-galactoside antibody indicated differences in carbohydrate composition between the 84 kDa subunit and the alkaline PAGE fast moving glycoprotein. Membrane-bound brain stem glycoproteins were not recognized by BBL.

Identification of endogenous soluble glycoprotein receptors of bovine brain grey matter 14 kDa beta-galactoside-binding lectin.

The 14 kDa beta-galactoside-binding lectin from bovine brain grey matter (BBL) covalently attached to caproic acid-Sepharose by the N-hydroxy succinimide procedure was used to isolate endogenous glycoprotein receptors of this lectin. BBL-Sepharose could sugar-specifically retain several endogenous soluble glycoproteins with subunit molecular mass (in kDA) 44, 51, 60, 123 and 186. BBL, conjugated with horse radish peroxidase, could sugar-specifically recognize several glycoprotein subunits with molecular mass (in kDA) 58, 87, and 117 and 186 on Western blots. The only protein from an extract of bovine brain grey matter, that retained on Sepharose-immobilized endogenous N-linked glycoproteins and subsequently eluted with beta-galactosides was BBL as confirmed by electrophoresis and agglutination inhibition measurement. N-linked glycoproteins from bovine heart and even from human placenta were also efficient receptors of BBL. These results suggest that 14 kDa beta-galactoside-binding lectin is the major protein, if not the only one, that sugar-specifically interacts with endogenous soluble glycoproteins in bovine brain grey matter.

Sugar-specific interaction of bovine brain beta-galactoside-binding lectin with endogenous gangliosides.

Sugar-specific binding of the 14 kDa beta-galactoside-binding lectin from bovine brain grey matter to mixed endogenous gangliosides was demonstrated by affinity electrophoresis and hemagglutination inhibition. Gangliosides prepared by Folch extraction, base treatment and silica gel chromatography, when incorporated in native or desialated form in polyacrylamide gel above their critical micellar concentration, arrested the mobility of the lectin during electrophoresis at pH 8.2. This effect was sugar-specific since it was reversed if lactose, but not sucrose, was present in the gel. Also, retention of the brain lectin by ganglioside and its reversal by lactose were concentration-dependent. In presence of bovine serum albumin, at pH 7.4 native and desialylated gangliosides equally inhibited agglutination of trypsinized rabbit red cells by bovine brain lectin, but not that by the alpha-galactoside-specific antibody from human serum. Results suggested the possibility of endogenous gangliosides acting as cell surface receptors in mediation of brain lectin function.

Human brain creatine kinase binding to immobilized cibacron blue F3GA: characterization and use in purification of the enzyme.

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) from adult human brain grey matter was purified by cibacron blue F3GA-Sepharose affinity chromatography. By gel electrophoresis of the purified enzyme under non-denaturing conditions a single protein band was observed. The dye-bound enzyme was eluted using its substrate, ATP. Reversibility of the binding of purified creatine kinase to blue Sepharose by ATP in a concentration-dependent manner indicated that the cibacron blue molecule which structurally mimics nucleotides occupied the substrate binding site of the enzyme. Also the marked dependence of enzyme binding to blue Sepharose on Mg2+ concentration suggested that Mg2+ ion is capable of combining with the dye moiety to form a site-specific binding complex that is similar to the physiological substrate of creatine kinase, namely Mg(2+)-ATP or Mg(2+)-ADP.

Rapid isolation of human plasma anti-alpha-galactoside antibody using sugar-specific binding to guar galactomannan or agarose.

A method of purifying the naturally occurring antibody to alpha-galactoside moiety (anti-alpha-Gal) in human plasma by a single-step affinity chromatography on cross-linked guar galactomannan (CLGG) or agarose (Sepharose 4B) is described. IgG nature of the two preparations, as revealed by agar gel diffusion, as well as their preference for alpha-anomer of galactose, as revealed in inhibition of their agglutination of trypsinized rabbit erythrocytes by sugars, identified them with anti-alpha-Gal. The antibody binding capacity of Sepharose 4B was only a third of that of CLGG. Both gels showed similar dependence on ionic strength for binding. The pH optimum for binding of anti-alpha-Gal to CLGG was 8.0. Significantly anti-alpha-Gal binding to Sepharose was unaffected by CNBr activation and ligand coupling to the gel, thus warning that contaminating plasma could introduce artifacts in agarose-based chromatography of human tissue biomolecules.

Alpha-galactoside-binding isolectins from wild jack fruit seed (Artocarpus hirsuta): purification and properties.

Five isolectins with marked specificity for alpha-linked galactose were purified from the wild jack (Artocarpus hirsuta) seeds by affinity chromatography on cross-linked guar gum. They were composed of a glycosylated subunit A (Mr = 16 kDa) and a nonglycosylated subunit B (Mr = 11 kDa) in noncovalent association. The isolectins which eluted as a single peak of Mr 45 kDa on gel filtration in Biogel P-100 and in a TSK G-3000 SW high pressure column, were resolved into five peaks on electrophoresis at pH 4.5. Sodium dodecyl sulphate polyacrylamide gel electrophoreogram of the major isolectin band suggested that the isolectins may be the five possible tetrameric combinations of A and B subunits. The combined isolectins bound only two molecules of 4-methyl umbelliferyl alpha-D-galactoside with a binding constant of 4.75 x 10(4) M-1. The pH optimum of sugar binding was 7.0. The isolectins specifically bound to human IgG and IgA but not to IgM.

A galactomannan-hydrolysing α-galactosidase from jack fruit (Artocarpus integrifolia) seed: Affinity chromatographic purification and properties.

An acid α-galactosidase from the seeds of the jack fruit seed (Artocarpus integrifolia) has been purified to homogeneity by affinity chromatography on a matrix formed by cross-linking the soluble α-galactose-bearing guar seed galactomannan. The 35kDa enzyme was a homotetramer of 9·5kDa subunits. Its carbohydrate part (5·5%) was composed of galactose and arabinose. The Km with p-nitrophenyl α-D-galactoside as substrate was 0·35 mM. The Ki values indicated inhibition by galactose, 1-O-methyl α-galactose and melibiose in the decreasing order. Among α-galactosides, the enzyme liberated galactose from melibiose, but not from raffinose or stachyose at its pH optimum (5·2). The guar seed galactomannan was however efficiently degalactosidated; limited enzyme treatment abolished the precipitability of the polysaccharide by the α-galactose-specific jack fruit seed lectin, and complete hydrolysis yielded insoluble polysaccharide. Though similar in sugar specificity and subunit assembly, α-galactosidase and the lectin coexisting in the jack fruit seed gave no indication of immunological identity.

Physicochemical properties and binding-site amino acid residues of galactoside-binding protein of human placenta.

The galactose-binding lectin of human placenta has been purified to homogeneity by affinity chromatography on asialo-fetuin column. The protein, extractable from the tissue only with lactose is apparently membrane-bound. Molecular weight determination of native protein and subunit indicated a dimer of 13·4 kDa subunits. Inhibition of haemagglutination with various saccharides indicate that thiodigalactoside is the best inhibitor followed by lactose· However, p-nitrophenyl- and 1-O-methyl derivatives of galactose showed that α-anomers inhibited slightly better than β-anomer. Modification of amino acid residues indicated involvement of arginine, lysine and histidine residues at the saccharidebinding site. Cysteine residue modificatioin also abolished haemagglutinating activity. Amino acid composition of the lectin is also presented·

Oligosaccharide structure determination of glycoconjugates using lectins.

Lectins, the divalent or polyvalent (glyco) proteins of non-immune origin of the cells agglutinate cells or other materials, that display more than one saccharide of sufficient complementarity. Lectins considered 'identical' in terms of mono- and disaccharide specificity can be differentiated by their ability to recognise the fine differences in more complex structures. The present review discusses the interaction of lectins with various oligosaccharides and their resultant separations due to structural variations.

Binding site amino acid residues of jack fruit (Artocarpusintegrifolia) seed lectin: Chemical modification and protein difference spectral studies.

The effect of chemical modification of amino acid residues essential for sugar binding in the α-D-galactoside specific jack fruit (Artocarpus integrifolia) seed lectin and the protection of the residues by specific sugar from modification were studied. Citraconylation or maleylation of 75 % of its lysyl residues or acetylation of 70 % of the tyrosyl residues completely abolished sugar binding and agglutination without dissociation of subunits. 1-Omethyl α-D-galactoside could protect its essential lysyl and tyrosyl groups from modification. Tryptophan could not be detected in the protein. Difference absorption spectra on binding of the above sugar confirmed the role of tyrosine residues and showed an association constant Κ = 0·4 × 103 Μ-1. Data suggests that the lectin could be immobilized without any loss of sugar binding activity.

Plant lectins specific for N-acetyl-β-D-galactosamine.

N-Acetyl-D-galactosamine in β-linkage being ubiquitous in cell surface glycoproteins, their interaction with lectins specific for this sugar moiety may be a significant event in cell adhesion phenomena. This article discusses the common β-N-acetyl galactosamine-specific lectins, with particular stress on the lectin from winged beans (Psophocarpus tetragonolobus).

α -D-Galactose-specific lectin from jack fruit (A rtocarpus integra) seed.

An α-D-galactose-specific lectin from the seeds of jack fruit (Artocarpus integra) has been isolated in pure form by affinity chromatography on immobilised guar gum (a galactomannan). The lectin is shown to be a glycoprotein containing 3% carbohydrate and having a molecular weight of 39,500 as determined by gel filtration. Sodium dodecyl sulphate gel electrophoresis revealed a single polypeptide of 10,500 dalton, indicating that the native lectin is a tetrarner of identical subunits. The hemagglutinating activity of the lectin towards erythrocytes of all blood groups is found to be the same.