Sequential biosynthesis of sulfated and/or sialylated Lewis x determinants by transferases of the human bronchial mucosa

The structural determination of sulfated carbohydrate chains from a cystic fibrosis patient respiratory mucins has shown that sulfation may occur either on the C-3 of the terminal Gal, or on the C-6 of the GlcNAc residue of a terminal N-acetyllactosamine unit. The two enzymes responsible for the transfer of sulfate from PAPS to the C-3 of Gal or to the C-6 of GlcNAc residues have been characterized in human respiratory mucosa. These two enzymes, in conjunction with fucosyl- and sialyltransferases, allow the synthesis of different sulfated epitopes such as 3-sulfo Lewis x (with a 3-O-sulfated Gal), 6-sulfo Lewis x and 6-sulfo-sialyl Lewis x (with a 6-O-sulfated GlcNAc). In the present study, the sequential biosynthesis of these epitopes has been investigated using microsomal fractions from human respiratory mucosa incubated with radiolabeled nucleotide-sugars or PAPS, and oligosaccharide acceptors, mostly prepared from human respiratory mucins. The structures of the radiolabeled products have been determined by their coelution in HPAEC with known oligosaccharidic standards. In the biosynthesis of 6-O-sulfated carbohydrate chains by the human respiratory mucosa, the 6-O-sulfation of a terminal nonreducing GlcNAc residue precedes β1–4-galactosylation, α2–3-sialylation (to generate 6-sulfo-sialyl-N-acetyllactosamine), and α1–3-fucosylation (to generate the 6-sulfo-sialyl Lewis x determinant). The 3-O-sulfation of a terminal N-acetyllactosamine may occur if this carbohydrate unit is not substituted. Once an N-acetyllactosamine unit is synthesized, α1–3-fucosylation of the GlcNAc residue to generate a Lewis x structure blocks any further substitution. Therefore, the present study defines the pathways for the biosynthesis of Lewis x, sialyl Lewis x, sulfo Lewis x, and 6-sulfo-sialyl Lewis x determinants in the human bronchial mucosa.