Lectin affinity high-performance liquid chromatography: interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin.
We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.
Department of Pathology, Washington University Medical School, St. Louis, Missouri 63110.
E D
ED
Green
R M
RM
Brodbeck
J U
JU
Baenziger
D000373:Agglutinins / Q000032:analysis*; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000032:analysis*; D013286:Datura stramonium / Q000032:analysis; D006026:Glycoside Hydrolases / Q000378:metabolism*; D041743:Herb-Drug Interactions; D037102:Lectins / Q000032:analysis*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D010835:Phytohemagglutinins / Q000032:analysis*; D010936:Plant Extracts / Q000032:analysis; D037121:Plant Lectins; D010946:Plants, Medicinal; D010947:Plants, Toxic
D000373:Agglutinins; D037102:Lectins; D009844:Oligosaccharides; D010835:Phytohemagglutinins; D010936:Plant Extracts; D037121:Plant Lectins; D003208:Concanavalin A; D006026:Glycoside Hydrolases; C020668:glycanase