Purification and properties of the alpha-3/4-L-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line.
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.
Johnson
P H
PH
Division of Immunochemical Genetics, MRC Clinical Research Centre, Middlesex, UK.
Donald
A S
AS
Watkins
W M
WM
D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002294:Carcinoma, Squamous Cell; D002455:Cell Division; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005033:Ethylmaleimide / Q000494:pharmacology; D005647:Fucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D008969:Molecular Sequence Data; D012995:Solubility; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase; D005033:Ethylmaleimide