THE USE OF HUMAN-MILK FUCOSYL-TRANSFERASE IN THE SYNTHESIS OF TUMOR-ASSOCIATED TRIMERIC-X DETERMINANTS

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 ty pe-2 structures, whether sialylated or not. When fucose residues were added enzymically to the (LacNAc)3-EtPhNHCOCF, hexasaccharide, mono-, di-, or trifucosylated oligosaccharide species were formed, containing the Lewis(x) determinant (Galbeta1-->4[Fucalpha1-->3]GlcNAcbeta1-->3) . With excess GDP-fucose and prolonged reaction times, the trifucosyla ted product was formed in almost quantitative yield. Kinetic analysis of the fucosylation reaction indicated that there is a significant dif ference in the rate of transfer of the first, second and third fucose residues onto the acceptor molecule. The location of the fucose residu es in the monofucosylated and difucosylated intermediate products was assessed by analyzing the digests obtained after endo-beta-galactosida se treatment by HPLC and reverse-phase chromatography. In addition, th e fucosylated (LacNAc)3-EtPhNHCOCF, structures were characterized by H PLC and were identified by 400-MHz H-1-NMR spectroscopy. There is a hi ghly preferred order in which the fucosyl residues are attached to (La cN-Ac)3-EtPhNHCOCF3. In the major pathway, the first two fucose residu es are transferred with equal preference to the medial (GN3) and proxi mal (GN1) GlcNAc residues, whereas the third fucose is attached to the distal (GN5) GlcNAc residue. These results are of relevance in unders tanding the role of alpha-3-fucosyltransferase in the biosynthesis of Lewis(X)-related cell-surface carbohydrate structures, that function a s ligands for selectin-type cell-adhesion molecules and may play a rol e in the invasion and metastasis of several carcinoma.