Carbohydrate-based probes for detection of cellular lectins

Carbohydrate (spacered saccharide residue, Glyc) probes with various tags w ere synthesized as analytical tools for study of cellular lectins, i.e., Gl yc-polyacrylamide-H-3, Glyc-PAA-biotin, Glyc-PAA-fluorescein (flu), and Gly c-PAA-digoxigenin, where PAA is a soluble polyacrylamide carrier of approxi mate to 30 kDa. Binding of all types of probes, where Glyc is the sialyl Le wis X (SiaLe(X)) tetrasaccharide or a blank saccharide, was assessed using Chinese hamster ovary (CHO) cells either transfected with the E-selectin cD NA or mock-transfected. High binding of SiaLe(X)-PAA-H-3 to E-selectin-tran sfected cells and absence of binding to control cells (both native and perm eabilized) allowed the conclusion that the polyacrylamide carrier and the s pacer arm do not contribute significantly to the binding. The biotinylated probe showed a high level of nonspecific binding in cell enzyme-linked assa ys. A similarly built digoxigenin-labeled probe was significantly better. I n flow cytometry assays, the fluorescein probe demonstrated a specific bind ing to E-selectin-transfected cells of a similar level to that given by an anti-E-selectin antibody. In addition, it could be inhibited by the anti-E- selectin antibody, further demonstrating specificity. Tumors were obtained from nude mice by injection of CHO E-selectin or mock-transfected cells. Th e fluorescent SiaLe(X)-PAA-flu probe could bind to tumor sections from E-se lectin-positive CHO cells, but not from control CHO cells. These probes can thus be used to reveal specifically complex carbohydrate-binding sites on cells either in culture or on tissue sections. (C) 1998 Academic Press.