Acceptor specificity of the human leukocyte α3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The α3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcα(2–3)Galβ(1–4)[Fucα(1–3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte α3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLex and sialyl-dimeric-Lex structures in vitro, from 3′-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3′-sulfated acceptors. Studies using fucosylated NeuAcα(2–3)-(Galβ(1–4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and trifucosylated structures from mono-fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucTVII is capable of generating complex selectin ligands, in vitro, however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.