IMMUNODETECTION OF ALPHA-1-3 FUCOSYL-TRANSFERASE (FUCT-V)

The fucosyltransferases constitute a family of glycosyltransferases in corporating fucose residues into glycoprotein or glycolipid glycans. T hey afford one of the possible termination steps of glycoconjugate bio synthesis creating the sialyl Lewis(x) or sialyl Lewis(a) determinant, which play an important role in cell-cell interaction. While cDNA, ch romosomal localization and kinetic properties of 4 number of fucosyltr ansferases are known, immunocytochemical localization and trafficking studies have been delayed because of the lack of specific antibodies d ue to the pronounced homology of alpha 1,3 fucosyltransferases III, V and VI. Here rye report development and characterization of monospecif ic polyclonal antibodies to alpha 1-3 fucosyltransferase V (FucT-V) an d their application for immunodetection in transfected cells. Antisera against FucT-V were raised in two different ways: first by producing a fusion protein beta-galactosidase-FucT-V in Escherichia coli, and by synthesizing a peptide stretch specific for FucT-V, Poly-clonal antis era were raised against each of both antigens and characterized by enz yme-linked immunosorbent assay, neutralization of activity, immunoblot ting, immunofluorescence and immunoprecipitation of metabolically labe led COS cells, transiently transfected with cDNA encoding FucT-V. Both antibodies recognized only FucT-V. No cross-reactivity to FucT-III or FucT-VI was observed. FucT-V was localized mainly to the Golgi appara tus by colocalization with beta 1,4-galactosyltransferase, and to the cell surface of COS, CHO and HeLa cells. Expression of FucT-V in COS c ells revealed three enzyme forms of 58, 53 and 50 kDa, respectively Th ese size differences arose by post-translational modifications, as sho wn by pulse-chase experiments. Our results indicate that alpha 1-3 fuc osyltransferase is a Golgi-associated enzyme and suggest its possible occurrence on the cell surface.