Glycoprotein biosynthesis: The characterization of two glycoprotein: Fucosyl transferases in HeLa cells

Abstract

Two glycoprotein: fucosyl transferases that transfer fucose from GDP-fucose onto glycoprotein receptors were found in a Triton X-100 extract of HeLa cells. One enzyme, designated the fetuin:fucosyl transferase, utilizes as a receptor fetuin from which sialic acid and galactose were removed, thus leaving an available terminal N-acetylglucosamine residue. This enzyme has an optimum pH of 6.0, temperature optimum of 30 °, and requires Mg 2+. The other enzyme designated the PSM: fucosyl transferase, utilizes as a receptor PSM from which sialic acid and fucose were removed leaving an available terminal galactose residue. This enzyme has an optimum pH of 6.8, temperature of 37 °, and does not require metal ion activation. Both enzymes showed a high degree of specificity when tested with various glycoprotein receptors. It was concluded that one enzyme was involved in formation of a fucosyl- N-acetylglucosamine linkage, probably 1–4, while the other enzyme recognized only galactose with probable formation of the α- l-fucosyl-(1–2)- O- d-galactose linkage found in blood group substances and porcine submaxillary glycoprotein. The galactose residues of fetuin and α 1-glycoprotein were relatively inactive as receptors. Approximately 1.1–2.4% of the theoretical receptor sites reacted with GDP-fucose after 24-hr incubation. Both enzymes required macromolecular receptors; several mono- and disaccharides were nonfunctional as receptors. Both enzymes were were found in the smooth internal membranes containing the multienzyme group of glycosyl transferases involved in biosynthesis of the carbohydrate units of membrane glycoproteins. The fucosyl transferases are strongly bound to these membranes; dissociation with the detergent Triton X-100 solubilizes the enzymes. The detergent-solubilized enzymes were purified 11-fold by gel filtration on Sephadex G-200 followed by centrifugation at 300,000 g for 4 hr. The role of the fucosyl enzymes in the mechanism of glycoprotein biosynthesis is discussed. Of special interest in this regard is the synthesis of “artificial” glycoproteins illustrated by the replacement of the β-galactosyl-(1–4)- n-acetylglucosamine linkage present in native fetuin by the fucosyl- N-acetylglucosamine linkage formed with the fetuin:fucosyl transferase.