Purification and properties of the α-3/4-L-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line

• Published in: Glycoconjugate journal 1993-04, Vol.10 (2), p.152-164
• Main Authors: JOHNSON, P. H, DONALD, A. S. R, WATKINS, W. M
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbohydrate Metabolism; Carbohydrate Sequence; Carcinoma, Squamous Cell; Cell Division; Chromatography, Gel; Chromatography, Ion Exchange; Enzymes and enzyme inhibitors; Ethylmaleimide - pharmacology; Fucosyltransferases - isolation & purification; Fucosyltransferases - metabolism; Fucosyltransferases - secretion; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Hydroxymercuribenzoates - pharmacology; Molecular Sequence Data; Solubility; Substrate Specificity; Transferases; Tumor Cells, Cultured
• ISSN: 0282-0080; 1573-4986
• DOI: 10.1007/BF00737712

A soluble alpha-3/4-fucosyltransferase secreted into the growth medium of the human A431 epidermoid carcinoma cell line has been purified 700,000 fold by a series of steps involving chromatography on Phenyl Sepharose 4B, CM-Sephadex C-50 and GDP-hexanolamine Sepharose 4B. The untreated spent culture medium transferred almost ten times more fucose to the subterminal N-acetylglucosamine residue in the Type 1 (Gal beta 1-3GlcNAc) disaccharide than to the subterminal sugar in the Type 2 (Gal beta 1-4GlcNAc) disaccharide; the relative activity with these two substrates remained virtually unchanged throughout the purification procedure. At no stage was any alpha-3-fucosyltransferase species acting solely on N-acetylglucosamine residues in Type 2 chains separated from the bulk of the alpha-3/4-fucosyltransferase activity. The purified enzyme preparation showed insignificant activity with glycoprotein substrates having N-linked oligosaccharide chains with terminal Type 2 sequences but transferred fucose to a mucin-type glycoprotein with O-linked oligosaccharide chains with terminal Type 1 structures. Lactose was a poor substrate but the activity of the enzyme was influenced by the presence of substituents on the terminal beta-galactosyl residue and 2'-fucosyllactose was almost as good an acceptor as the Type 1 disaccharide. The properties of the purified enzyme with regard to specificity, divalent cation requirements, pH optimum, and M(r), closely resembled those of the Lewis-blood-group gene associated alpha-3/4-fucosyltransferase isolated from human milk.