Enzymatic analysis of cell surface lactosaminyl glycans by flow cytometry.
Cell surface expressed lactosaminyl glycans were determined on live cells by flow cytometry using a sialyltransferase mediated labeling procedure. Fluorescent CMP-sialic acid and Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase were applied to probe expression of acceptor glycans on untreated or sialidase pretreated erythrocytes. After enzymatic fluorescence labeling, erythrocytes were treated with endo-beta-galactosidase or trypsin to distinguish polylactosaminyl- and complex-type glycans. The expression of lactosaminyl sequences on cord- was 20% lower than on adult cells. After sialidase treatment fluorescence incorporation on both cell types increased twofold compared to untreated cells indicating a low sialylation extent. A recombinant alpha 2,3-sialyltransferase was preferentially labeling polylactosaminyl glycans. Taking advantage of the different fine specificity as determined here, alpha 2,6- and alpha 2,3-sialyltransferase can be applied to distinguish certain types of lactosaminyl glycans.
Bollheimer
L C
LC
Institut fũr Biochemie II, Universität Heidelberg, FRG.
Schwartz-Albiez
R
R
Sauer
A
A
Gross
H J
HJ
D000328:Adult; D000606:Amino Sugars / Q000097:blood*; D002850:Chromatography, Gel; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006026:Glycoside Hydrolases; D006801:Humans; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000378:metabolism; D011134:Polysaccharides / Q000097:blood*; D012794:Sialic Acids / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology; D001616:beta-Galactosidase / Q000494:pharmacology; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D000606:Amino Sugars; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011134:Polysaccharides; D012794:Sialic Acids; C039448:lactosaminoglycan; D012799:Sialyltransferases; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D009439:Neuraminidase; D001616:beta-Galactosidase; D014357:Trypsin; D019158:N-Acetylneuraminic Acid; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase; C000722087:ST3GAL1 protein, human