Identification of an  3-fucosyltransferase and a novel 2 -fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fuc 1->2Fuc 1->3[Gal(NAc) 1->4]GlcNAc sequence

Abstract

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-->4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-->2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1-->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3-FucT resembles human FucT V and VI rather than other known FucTs. N-ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-->4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-->2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-->3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2-FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.

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

“…After isolation and purification of the products, the structures of the oligosaccharides were verified by 1 H-NMR spectroscopy. The spectral data (not shown) of LacdiNAc and LDN-DF were identical to those of the same compounds published by Hokke et al (1998). The chemical shifts of the structural-reporter-group protons of the monofucosylated product LDN-F were comparable to those of GalNAcβ1-4(Fucα1-3)GlcNAcβ-OCH 3 (compound GN[F]Gn in Bergwerff et al, 1993), with the exception of GlcNAc H-1 which shifted slightly due to the presence of a different aglycon in the reference compound.…”

“…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. During the incubations the progress of the reactions was monitored by HPAEC with pulsed amperometric detection on a CarboPac PA-1 pellicular anion-exchange column (0.9 × 2.5 cm, Dionex) as described previously (Joziasse et al, 1993).…”

Avian Schistosomes and Outbreaks of Cercarial Dermatitis

“…The thick glycocalyx that served as a protective layer for the free-living cercaria is shed, as its carbohydrate-rich composition is a target of the host complement cascade. In the schistosome species studied so far, the glycocalyx is markedly rich in fucose residues (154,(196)(197)(198). There is an obvious loss of saccharide moieties at the surfaces of transformed schistosomula of T. szidati and T. regenti, leading to reduced immunoreactivity and attractiveness for fucose-specific lectins (154,155,199,200).…”

LacdiNAc-Glycans Constitute a Parasite Pattern for Galectin-3-Mediated Immune Recognition

“…The structure was verified by 1 H-nuclear magnetic resonance spectroscopy as described (29). LN-(CH 2 ) 3 -NH 2 and LDN-(CH 2 ) 3 -NH 2 (5 mol each) were then covalently coupled to 1 ml HiTrap columns (Pharmacia, Peapack, NJ) according to the manufacturer's instruction.…”