STABLE EXPRESSION OF THE GOLGI FORM AND SECRETORY VARIANTS OF HUMAN FUCOSYL-TRANSFERASE-III FROM BHK-21-CELLS - PURIFICATION AND CHARACTERIZATION OF AN ENGINEERED TRUNCATED FORM FROM THE CULTURE-MEDIUM

Stable BHK-21 cell Lines were constructed expressing the Golgi membran e bound form and two secretory forms of the human alpha 1,3/4-fucosylt ransferase (amino acids 35-361 and 46-361). It was found that 40% of t he enzyme activity synthesized by cells transfected with the Golgi for m of the fucosyltransferase was constitutively secreted into the mediu m. The corresponding enzyme detected by Western blot had an apparent m olecular mass similar to those of the truncated secretory forms. The s ecretory variant (amino acids 46-361) was purified by a single affinit y-chromatography step on GDP-Fractogel resin with a 20% final recovery . The purified enzyme had a unique NH2 terminus and contained N-linked endo H sensitive carbohydrate chains at its two glycosylation sites. The fucosyltransferase transferred fucose to the O-4 position of GlcNA c in small oligosaccharides, glycolipids, glycopeptides, and glycoprot eins containing the type I Gal beta 1-3GlcNAc motif. The acceptor olig osaccharide in bovine asialofetuin was identified as the Man-3 branche d triantennary isomer with one Gal beta 1-3GlcNAc. The type II motif G al beta 1-4GlcNAc in bi-, tri-, or tetraantennary neutral or alpha 2-3 /alpha 2-6 sialylated oligosaccharides with or without N-acetyllactosa mine repeats and in native glycoproteins were not modified. The solubl e forms of fucosyltransferase III secreted by stably transfected cells may be used for in vitro synthesis of the Lewis(a) determinant on car bohydrates and glycoproteins, whereas Lewis(x) and sialyl-Lewis(x) str uctures cannot be synthesized.