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),-EtPhNHCOCF,] 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),-EtPhNHCOCF, hexasaccharide, mono-, di-, or trifucosylated oligosaccharide species were formed, containing the LewisX determinant (Gal~l+4[Fucal+3]GlcNAcPl-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-P-galactosidase treatment by HPLC and reverse-phase chromatography. In addition, the fucosylated (LacNAc),-EtPhNHCOCF, structures were characterized by HPLC and were identified by 400-MHz 'H-NMR spectroscopy. There is a highly preferred order in which the fucosyl residues are attached to (LacNAc),-EtPhNHCOCF,. 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 a-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.

Conversion of GalNAcβ(1–4)GlcNAcβ‐OMe into GalNAcβ(1–4)‐[Fucα(1–3)]GlcNAcβ‐OMe using human milk α3/4‐fucosyltransferase synthesis of a novel terminal element in glycoprotein glycans

“…Functionally purified preparations of human milk ~3/4-fucosyltransferase have been used in the enzyme assisted synthesis of ~(1 3)-fucosylated oligosaccharides, including Le x [7][8][9]12]. Here it is demonstrated that the enzyme can also be used to produce the GalNAc-containing variant of Le x in high yield.…”

“…GDP-[3H]Fuc (7 Ci/mmol) was purchased from DuPont NEN (Boston, MA), and was diluted with unlabelled GDP-Fuc. Partially purified human milk c~3/4-fucosyltransferase, kindly provided by Mrs. T. De Vries, was isolated from pooled human milk as described in [11] with minor modifications [12]. The resulting preparation contained the enzyme in a 225-fold purified form (1.4 mU/ml) in a solution of 50 mM sodium cacodylate buffer, pH 7.2, containing 50% (by vol.)…”

Various stages of Schistosoma express Lewisx, LacdiNAc, GalNAc 1-4 (Fuc 1-3)GlcNAc and GalNAc 1-4(Fuc 1-2Fuc 1-3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins

“…Five micromoles of GlcNAc-R was incubated with 6 µmol of UDP-[ 3 H]GalNAc (0.09 Ci/mol) and partially purified UDP-GalNAc:GlcNAcβ β1→4-N-acetylgalactosaminyltransferase (β4-GalNAcT) from the albumen gland of Lymnea stagnalis (Mulder et al, 1995) in an incubation mixture containing 100 mM sodium cacodylate pH 6.5, 20 mM MnCl 2 , 100 mM NaCl, 600 mM GlcNAc, 4 mM ATP, and 0.5% (v/v) Triton X-100 for 18 h at 37°C. Three micromoles of the produced LacdiNAc-R was fucosylated with 4 µmol of GDPFuc and partially purified GDP-Fuc:[Galβ1→4]GlcNAcβ α1→3-fucosyltransferase (α3-FucT) (1.4 mU) from human milk (DeVries et al, 1993) in an incubation mixture containing 100 mM sodium cacodylate pH 7.0, 100 mM NaCl, 20 mM MnCl 2 , 4 mM ATP and 0.5% (v/v) Triton X-100, for 48 h at 37°C (Bergwerff et al, 1993). Two µmol of LDN-F was further fucosylated with partially purified GDP-Fuc:Fucα α1→2-fucosyltransferase (α2-FucT) from T.ocellata (Hokke et al, 1998) under the same conditions as described previously, for 72 h at 24°C.…”

Isolation and characterization of linear polylactosamines containing one and two site-specifically positioned Lewis x determinants: WGA agarose chromatography in fractionation of mixtures generated by random, partial enzymatic alpha3-fucosylation of pure polylactosamines

“…For instance, endo-β-galactosidase of B.fragilis, cleaving internal β-galactosidic bonds of primary polylactosamine backbones, does not hydrolyze these bonds in LacNAc units that are α1, 3-fucosylated (de Vries et al, 1993), β1,6′-N-acetylglucosaminylated (Scudder et al, 1984), or 6′-sulfated (Scudder et al, 1983). Analogous examples also include α1,3-fucosyltransferases, which transfer to all LacNAc units of linear polylactosamine chains (de Vries et al, 1993;Niemelä et al, 1998), but work poorly at branch-bearing LacNAc units .…”