The use of human milk fucosyltransferase in the synthesis of tumor-associated trimeric X determinants

Abstract

We have studied the fucosylation of a chemically synthesized trimer of N-acetyllactosamine [(LacNAc)3-EtPhNHCOCF3] with a fucosyltransferase preparation from normal human milk, which utilizes both type-1 and type-2 structures, whether sialylated or not. When fucose residues were added enzymically to the (LacNAc)3-EtPhNHCOCF3 hexasaccharide, mono-, di-, or trifucosylated oligosaccharide species were formed, containing the Lewisx determinant (Gal beta 1-->4[Fuc alpha 1-->3]Glc-NAc beta 1-->3). With excess GDP-fucose and prolonged reaction times, the trifucosylated product was formed in almost quantitative yield. Kinetic analysis of the fucosylation reaction indicated that there is a significant difference in the rate of transfer of the first, second and third fucose residues onto the acceptor molecule. The location of the fucose residues in the monofucosylated and difucosylated intermediate products was assessed by analyzing the digests obtained after endo-beta-galactosidase treatment by HPLC and reverse-phase chromatography. In addition, the fucosylated (LacNAc)3-EtPhNHCOCF3 structures were characterized by HPLC and were identified by 400-MHz 1H-NMR spectroscopy. There is a highly preferred order in which the fucosyl residues are attached to (LacN-Ac)3-EtPhNHCOCF3. In the major pathway, the first two fucose residues are transferred with equal preference to the medial (GN3) and proximal (GN1) GlcNAc residues, whereas the third fucose is attached to the distal (GN5) GlcNAc residue. These results are of relevance in understanding the role of alpha-3-fucosyltransferase in the biosynthesis of Lewisx-related cell-surface carbohydrate structures, that function as ligands for selectin-type cell-adhesion molecules and may play a role in the invasion and metastasis of several carcinoma.