No abstract

Three different stages of the avian schistosome Trichobilharzia ocellata appeared to contain a novel N-acetylgalactosaminyltransferase activity. To investigate its function in the biosynthesis of schistosome glycoconjugates, the enzyme was partially purified from cercariae, a free-living stage of the parasite, by affinity chromatography on UDP-Sepharose. Acceptor specificity studies showed that the enzyme catalyses the transfer of N-acetylgalactosamine (GalNAc) from UDP-GalNAc to oligosaccharides, glycopeptides and glycoproteins carrying a terminally beta-linked N-acetylglucosamine (GlcNAc) residue, regardless of the underlying structure. Analysis of the products obtained with GlcNAc and a desialylated and degalactosylated diantennary glycopeptide by 400 MHz 1H-NMR spectroscopy revealed that a GalNAc beta 1-->4GlcNAc (N,N'-diacetyllactosediamine,lacdiNAc) unit was formed. The enzyme can therefore be described as a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminytransferase (beta 4-GalNAcT). Using specific acceptors, the enzyme could be distinguished from all other beta 4-GalNAcTs described to date, including the one from pituitary gland that is involved in the specific glycosylation of pituitary glycohormones. By contrast, the enzymatic properties of the schistosome beta 4-GalNAcT (except for the sugar-donor specificity) strongly resemble those of the beta 4-galactosyltransferase of higher animals, an enzyme which is known to control the synthesis of Gal1-->4GlcNAc (lacNAc)-type oligosaccharide chains. By analogy, the beta 4-GalNAcT is concluded to control the key step in the synthesis of lacdiNAc-type chains. LacdiNAc-type glycans are also common to the mollusc Lymnaea stagnalis, which is the intermediate host of T.ocellata. It is proposed that the schistosome beta 4-GalNAcT functions in the expression of specific carbohydrate structures that contribute to a molecular mimicry, enabling the schistosome to evade the defence system of the snail host.

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.

Both the albumen gland, one of the female accessory sex glands, and connective tissue of the freshwater snail Lymnaea stagnalis contain N-acetylgalactosaminyltransferase activity, capable of transferring GalNAc from UDP-GalNAc in Pl -4 linkage to the terminal GlcNAc residue of GlcNAcP-R. The albumin gland enzyme was partially purified by affinity chromatography on UDP-hexanolamine-Sepharose 4B. Using GlcNAcPl -2Manal-6(GlcNAcP1-2Manal-3)Man~l-4GlcNAcP1-4GlcNAc or GlcNAcPlOMe as substrates, the enzyme showed an absolute requirement for MnZ+ with an optimum concentration of 12.5-50 mM. The optimal pH was approximately pH 7.0. The enzyme activity was independent of the Triton X-100 concentration in the range 0.25-2.5%, and no activation effect was found. The more labile connective tissue microsomal enzyme, subjected to the same optimization procedure, gave comparable results. Both enzyme activities have similar substrate specificities towards GlcNAc or GlcNAcPlOMe, and towards oligosaccharides or glycopeptides with a non-reducing terminal P-GlcNAc unit, but cannot act on GlcNAcal-OMe. Saccharides with non-reducing terminal Gal or GalNAc residues, and free GalNAc, Gal or Glc residues are not acceptors. Product analysis was carried out for albumen gland N-acetylgalactosaminyltransferase and four acceptors having GlcNAcPl -R as the terminal non-reducing unit, and for connective tissue N-acetylgalactosaminyltransferase with GlcNAcPl-OMe as acceptor. In all instances, products with GalNAc PI -4-linked to GlcNAc were obtained, showing that the connective tissue and the albumen gland activities are probably from one enzyme. This enzyme activity can be identified as UDP-GalNAc :GlcNAcP-R Pl -4 N-acetylgalactosaminyltransferase, and is probably involved in the biosynthesis of N,N'-diacetyllactosediamine-containing glycoproteins, like hemocyanin, in the snail L. stugnalis.

The N,N'-diacetyllactosediamine (lacdiNAc) pathway of complex-type oligosaccharide synthesis is controlled by a UDP-GalNAc:GlcNAcf3-R j81-*4-N-acetylgalactosaminyltransferase (f34-GalNAcT) that acts analogously to the common UDP-Gal:GlcNAcj8-R 131-*4-galactosyltransferase ((34-GaiT). LacdiNAc-based chains particularly occur in invertebrates and cognate ,B4-GalNAcTs have been identified in the snail Lymnaea stagnalis, in two schistosomal species, and in several lepidopteran insect cell lines. Because of the similarity in reactions catalyzed by both enzymes, we investigated whetherL. stagnalis albumen gland (84-GalNAcT would share with mammalian f34-GaIT the property of interacting with a-lactalbumin (c-LA), a protein that only occurs in the lactating mammary gland, to form a complex in which the specificity of the enzyme is changed. It was found that, under conditions where (84-GalT forms the lactose synthase complex with a-LA, the snail .84-GalNAcT was induced by this protein to act on Glc with a > 100-fold increased efficiency, resulting in the formation of the lactose analog GalNAc,81-4Glc. This forms the second example of a glycosyltransferase, the specificity of which can be altered by a modifier protein. So far, however, no protein fraction could be isolated from L. stagnalis that could likewise interact with the 134-GalNAcT. Neither had lysozyme c, a protein that is homologous to a-LA, an effect on the specificity of the enzyme. These results raise the question of how the capability to interact with a-LA has been conserved in the snail enzyme during evolution without any apparent selective pressure. They also suggest that snail (4-GalNAcT and mammalian f84-GalT show similarity at a molecular level and allows the identification of the f34-GalNAcT as a candidate member of the f4-GaIT family.