Mucin biosynthesis enzymic properties of human-tracheal epithelial GDP-l-fucose:β-d-galactoside α-(1→2)-l-fucosyltransferase

The human-tracheal, epithelial α-(1→2)-l-fucosyltransferase that transfers l-fucose from GDP-l-fucose to an acceptor containing a β-d-galactopyranosyl group at the nonreducing terminal was characterized. Optimal enzyme activity was obtained at pH 6.5. 20-30mm MnCl₂ (or CaCl₂, and 0.05% Triton X-100 or 0.5% Tween 20. Mg²⁺ and Ba²⁺ ions moderately enhanced the enzyme activity, whereas Fe²⁺, Co²⁺, Zn²⁺, and Cd²⁺ ions were inhibitory. The enzyme activity was inhibited by N-ethylmaleimide and nucleotides of guanine, inosine, xanthine, and uridine. However, ATP and dithiothreitol did not affect the enzyme activity. The apparent Michaelis constant for GDP-l-fucose, freezing point-depressing glycoproteins (expressed as Gal→GalNAc→Thr), and phenyl β-d-galactopyranoside was 0.29, 5.70, and 25.4mm, respectively. Under alkali-borohydride conditions (0.05m NaOHM NaBH₄, 45°, 20 h), an l-[¹⁴C]fucosyltrisaccharide was released from the product obtained by use of freezing point-depressing glycoprotein as the acceptor. The α-l anomeric configuration of the fucoside was determined by the release of l-[¹⁴C]fucose from the purified trisaccharide by Turbo cornutus α-l-fucosidase. The (1→2) linkage of the l-fucosyl group to the d-galactosyl residue was established by methylation technique (m.s.-g.l.c.). The present enzyme has properties similar to those of the human milk α-(1→2)-l-fucosyltransferase which is encoded by a secretor gene.