ENZYMATIC ANALYSIS OF CELL-SURFACE LACTOSAMINYL GLYCANS BY FLOW-CYTOMETRY

Cell surface expressed lactosaminyl glycans were determined on live ce lls by flow cytometry using a sialyltransferase mediated labeling proc edure. Fluorescent CMP-sialic acid and Gal beta 1,4GlcNAc alpha 2,6-si alyltransferase were applied to probe expression of acceptor glycans o n untreated or sialidase pretreated erythrocytes. After enzymatic fluo rescence labeling, erythrocytes were treated with endo-beta-galactosid ase or trypsin to distinguish polylactosaminyl- and complex-type glyca ns. The expression of lactosaminyl sequences on cord- was 20% lower th an on adult cells, After sialidase treatment fluorescence incorporatio n on both cell types increased twofold compared to untreated cells ind icating a low sialylation extent. A recombinant alpha 2,3-sialyltransf erase was preferentially labeling polylactosaminyl glycans. Taking adv antage of the different fine specificity as determined here, alpha 2,6 - and alpha 2,3-sialyltransferase can be applied to distinguish certai n types of lactosaminyl glycans. (C) 1995 Academic Press, Inc.