Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents.
A rapid and convenient method was established for analysis of the N-linked carbohydrate chains of glycoproteins on nitrocellulose sheets. Proteins were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, reacted with peroxidase-coupled lectins, and detected by color development of the enzyme reaction. Four glycoproteins having N-linked oligosaccharide chains were used as test materials: Taka-amylase A (which has a high-mannose-type chain), ovalbumin (high-mannose-type chains and hybrid-type chains), transferrin (biantennary chains of complex type), and fetuin (triantennary chains of complex type and O-linked-type chains). Concanavalin A interacted with Taka-amylase A, transferrin, and ovalbumin but barely interacted with fetuin. After treatment of the glycoproteins on a nitrocellulose sheet with endo-beta-N-acetylglucosaminidase H, transferrin reacted with concanavalin A but Taka-amylase A and ovalbumin did not. Wheat germ agglutinin interacted with Taka-amylase A but not ovalbumin; therefore, they were distinguishable from each other. Fetuin and transferrin were detected by Ricinus communis agglutinin or peanut agglutinin after removal of sialic acid by treatment with neuraminidase or by weak-acid hydrolysis. Erythroagglutinating Phaseolus vulgaris agglutinin detected fetuin and transferrin. Thus, the combined use of these procedures distinguished the four different types of N-linked glycoproteins. This method was also applied to the analysis of membrane glycoproteins from sheep red blood cells. The terminally positioned sugars of sialic acid, alpha-fucose, alpha-galactose, and alpha-N-acetylgalactosamine were also detected with lectins from Limulus polyphemus, Lotus tetragonolobus, Maclura pomifera, and Dolichos biflorus, respectively.
S
S
Kijimoto-Ochiai
Y U
YU
Katagiri
H
H
Ochiai
D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D003101:Collodion; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis* / Q000145:classification; D006735:Horseradish Peroxidase; D007202:Indicators and Reagents; D037102:Lectins; D008358:Mannose; D009844:Oligosaccharides / Q000032:analysis*; D010835:Phytohemagglutinins; D012794:Sialic Acids; D013194:Staining and Labeling
D006023:Glycoproteins; D007202:Indicators and Reagents; D037102:Lectins; D009844:Oligosaccharides; D010835:Phytohemagglutinins; D012794:Sialic Acids; D003208:Concanavalin A; D003101:Collodion; D006735:Horseradish Peroxidase; D008358:Mannose