Fluorescent carbohydrate probes for cell lectins

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytic al tools for the study of cellular lectins, i.e. SiaLe(x)-PAA-flu, Sia(2)-P AA-flu, GlcNAc(2)-PAA-flu, LacNAc-PAA-flu and a number of similar ones, wit h PAA a soluble polyacrylamide carrier. The binding of SiaLe(x)-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of S ia(2)-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe(x)-PAA-H-3 to the E-selectin transfec ted cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding, Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells, The fluorescent SiaLe(x)-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal sp ecifically the carbohydrate binding sites on cells in culture as well as ce lls in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins i n different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a cons tituent part of the confocal spectral imaging technique) enabled interferin g signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local conc entration to be measured. (C) 2001 Elsevier Science B.V. All rights reserve d.