The use of streptavidin-biotinylglycans as a tool for characterization of oligosaccharide-binding specificity of lectin.
A new rapid and sensitive method for characterizing lectin specificity using streptavidin-biotinylglycans as a tool is presented. This assay is analogous to enzyme immunoassay and takes advantage of the strong, irreversible adsorption of streptavidin to the wells of the chambers of titer plates. A series of streptavidin-biotinylglycans was first coated on a microtiter plate, and then one of six lectins, concanavalin A, wheat germ agglutinin, Phaseolus vulgaris (red kidney bean) erythro-agglutinin, Lens culinaris (lentil) agglutinin, Datura stramoniun agglutinin, or Sambucus nigra (elderberry bark) agglutinin coupled to horseradish peroxidase, was added. After incubation and thorough washing, only the lectin bound to a complementary glycan remained and could be detected and quantified by the peroxidase reaction. It was established that the lectins retained their oligosaccharide-binding specificities after coupling to the peroxidase, that the binding was inhibited by addition of the corresponding sugar inhibitors, and that the color intensity produced by the enzyme reaction is proportional to the amount of lectin-peroxidase bound to biotinylglycan complexed with streptavidin immobilized on the plate. As an example, it was found that the peroxidase-D. stramoniun agglutinin conjugate strongly bound biotinylglycans, GlcNAc3-Man5-R, GalGlcNAc3Man5-R, and GlcNAc3-4Man3-R (R = GlcNAc2-[6-(biotinamido)hexanoyl]-Asn). As little as 10 pmol/ml of lectin was detected. With the growing availability of biotinylglycans, the method should represent a reliable and simple procedure for screening lectin-oligosaccharide recognition qualitatively and quantitatively.
Shao
M C
MC
Department of Biochemistry, Shanghai Medical University, People's Republic of China.
D001426:Bacterial Proteins; D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D002240:Carbohydrate Sequence; D006735:Horseradish Peroxidase; D037102:Lectins / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry; D019809:Streptavidin; D013379:Substrate Specificity
D001426:Bacterial Proteins; D037102:Lectins; D009844:Oligosaccharides; D011134:Polysaccharides; D001710:Biotin; D019809:Streptavidin; D006735:Horseradish Peroxidase