Reconstitution of functional L-selectin ligands on a cultured human endothelial cell line by cotransfection of alpha 1 -> 3 fucosyltransferase VII and newly cloned GlcNAc beta : 6-sulfotransferase cDNA

Recently, we proposed sialyl 6-sulfo Lewis X as a major carbohydrate-cappin g group of the L-selectin ligands on high endothelial venules in human lymp h nodes. In this study we succeeded in reconstituting functional L-selectin ligands on a cultured human endothelial cell line, ECV304, by transfecting the alpha 1-->3fucosyltranseferase VII (Fuc-T VII) and newly cloned GlcNAc beta:6-sulfotransferase (6-Sul-T) cDNAs. The ECV304 cells transfected with Fuc-T VII cDNA expressed conventional sialyl Lewis X detected with specifi c antibodies including 2H5, whereas the cells transfected with 6-Sul-T cDNA expressed sialyl 6-sulfo lactosamine as well as MECA-79-defined carbohydra te determinants, but these singly transfected cells failed to express sialy l 6-sulfo Lewis X, as detected with the antisialyl 6-sulfo Lewis X mAb G152 , Sialyl 6-sulfo Lewis X appeared only on the cells that were cotransfected with both 6-Sul-T and Fuc-T VII cDNAs, Significant adhesion of L-selectin- expressing cells was seen only to the doubly transfected ECV304 cells and w as inhibited by G152, No adhesion was observed to the cells transfected eit her with 6-Sul-T or with Fuc-T VII cDNA alone. The mRNAs of the two enzymes were expressed or were inducible upon interleukin 1 stimulation in human e ndothelial cells. These results indicate that a set of carbohydrate determi nants synthesized by the concerted action of the two enzymes, as typically represented by the sialyl 6-sulfo Lewis X-capping group, serves as an essen tial component of the ligand for L-selectin and that the reagents 2H5 and M ECA-79, utilized in earlier studies to detect L-selectin ligand on high end othelial venules, recognize two different aspects of the same set of synthe tic products.