Glycobiology vol. 10 no. 6 pp. 601–609, 2000 © 2000 Oxford University Press 601 Various stages of Schistosoma express Lewis x , LacdiNAc, GalNAcβ1–4 (Fucα1–3)GlcNAc and GalNAcβ1–4(Fucα1–2Fucα1–3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins Alexandra van Remoortere 1,3,4 , Cornelis H.Hokke 2,3 , Govert J.van Dam 4 , Irma van Die 3 , André M.Deelder 4 and Dirk H.van den Eijnden 3 3 Department of Medical Chemistry, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands and 4 Department of Parasitology, L4-Q, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands Received on October 5, 1999; revised on December 10, 1999; accepted on December 18, 1999 We report here that fucosylated epitopes such as Lewis x , LacdiNAc, fucosylated LacdiNAc (LDN-F) and GalNAcβ1–4(Fucα1–2Fucα1–3)GlcNAc (LDN-DF) are expressed by schistosomes throughout their life cycle. These four epitopes were enzymatically synthesized and coupled to bovine serum albumin to yield neoglycoproteins. Subsequently these neoglycoproteins were used to probe a panel of 188 monoclonal antibodies obtained from infected or immunized mice, in ELISA and surface plasmon reso- nance analysis. Of these antibodies, 25 recognized one of the fucosylated structures synthesized, indicating that these structures are immunogenic during infection. The MAbs identified could be subdivided in four different groups based on the recognition of either the Lewis x -, the LacdiNAc-, the LDN-DF-, or both the LDN-F- and LDN-DF epitope. These monoclonal antibodies were then used to investigate the localization of the fucosylated epitopes in various stages of Schistosoma mansoni using indirect immunofluorescence. Lewis x epitopes were mainly found in the gut and on the tegument of adult worms, on egg shells, and on the oral sucker of cercariae. The LacdiNAc epitope was expressed on the tegument of adult worms, on miracidia, and on the oral sucker of cercariae. In contrast, LDN-DF epitopes were mainly present in the excretory system of adult worms, on miracidia and on whole cercariae. These also stained positive with the LDN-F/LDN-DF epitope anti- bodies, while whole parenchyma reacted characteristically only with the latter antibodies. The identification of different carbohydrate structures in various stages of schistosomes may lead to a better understanding of the function of glycans in the immune response during infection. Key words: fucose/oligosaccharide/antigenicity/enzymatic synthesis/schistosomiasis Introduction Schistosomiasis is a parasitic disease, caused by blood flukes of the genus Schistosoma that affects approximately 200 million people worldwide. The life cycle of schistosomes involves a vertebrate definitive host in which sexual reproduction takes place and a fresh water snail in which asexual multiplication occurs. Since the parasites remain in their hosts for extended time periods, they have developed mechanisms to evade or resist the immune system of both hosts. In the various developmental stages, oligosaccharides at the surface of the parasite, in particular fucosylated ones, are proposed to be involved in these processes (Cummings and Nyame, 1996, 1999; Van Dam and Deelder, 1996). The structures of several schistosomal glycoconjugates have been elucidated. For example, monomeric and polymeric Lewis x (Le x ) structures have been demonstrated on both membrane-bound and secreted glycoproteins of adult worms (Ko et al., 1990; Srivatsan et al., 1992; Van Dam et al., 1994). In addition to Le x structures, the adult worms of Schistosoma mansoni also synthesize N-linked glycans containing GalNAcβ1–4GlcNAc (LacdiNAc, LDN) and GalNAcβ1– 4(Fucα1–3)GlcNAc (LDN-F) (Srivatsan et al., 1992), the LacdiNAc analog of the Le x blood group antigen. Multifuco- sylated structures have been found on O-glycans of the cercarial glycocalyx with the following terminal structure: (Fucα1→2)±Fucα1→2Fucα1→3GalNAcβ1→4((Fucα1→2) ±Fucα1→2Fucα1→3)-GlcNAcβ1→3Galα1→ (Khoo et al., 1995) and on glycolipids of S. mansoni eggs that consist of a backbone of repeating β1–4 linked GlcNAc residues substi- tuted with Fucα1–2Fucα1–3 side chains (Khoo et al., 1997). It has long been known that the humoral immune response in schistosomiasis is mainly directed against glycoconjugates (Nash et al., 1981; Aronstein et al., 1983; Omer Ali et al., 1988). More recently, several of these carbohydrates have been identi- fied. In early infections, the most pronounced antibody response (IgM) in humans is directed against the gut-associated circu- lating cathodic antigen (CCA) (Deelder et al., 1989). Upon infection with S. mansoni, humans and primates generate cyto- lytic IgM and IgG antibodies against the Le x structure (Nyame et 1 To whom correspondence should be addressed at: Department of Medical Chemistry, Faculty of Medicine, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands 2 Present address: Department of Parasitology, L4-Q, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands