Enzymatic characterization of human alpha1,3-fucosyltransferase Fuc-TVII synthesized in a B cell lymphoma cell line.

The human alpha1,3-fucosyltransferase, Fuc-TVII, a key enzyme in the biosynthesis of selectin ligands, was expressed as a soluble protein-A chimeric form in a human B cell lymphoma cell line, Namalwa KJM-1, and purified using IgG-Sepharose. The enzymatic properties of recombinant soluble Fuc-TVII were then examined. Its enzyme activity was highest at pH 7.5, and the presence of 25 mM Mn2+ was required for full activity. Fuc-TVII exhibits an acceptor specificity restricted to alpha2,3-sialylated type 2 oligosaccharides, and the apparent Km values for alpha2,3-sialyl lacto-N-neotetraose and GDP-fucose were 3.08 mM and 16.4 microM, respectively. The inhibitory effects of various nucleotides on the activity of Fuc-TVII reflected its donor specificity for the nucleotide portion of GDP. Fuc-TVII was demonstrated to be useful for the synthesis of a sialyl Lewis x hexasaccharide from lacto-N-neotetraose in combination with an alpha2, 3-sialyltransferase, ST3Gal IV. Polyethylene glycols enhanced the thermal stability of Fuc-TVII, leading to increased formation of the reaction product.