ABSTRACT
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.
Subject(s)
Animals , Binding, Competitive , Brain/metabolism , Cattle , Electrophoresis, Polyacrylamide Gel , Galectins , Gangliosides/chemistry , Hemagglutinins/metabolism , Lactose/metabolism , Lectins/metabolism , N-Acetylneuraminic Acid/metabolism , Plant LectinsABSTRACT
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.
Subject(s)
Animals , Brain Stem/metabolism , Carrier Proteins/metabolism , Cattle , Galectins , Glycoproteins/metabolism , Hemagglutinins/metabolism , Molecular WeightABSTRACT
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.