Keyhole limpet hemocyanin (KLH) of the mollusc Megathura crenulata is known to serologically cross-react with Schistosoma mansoni glycoconjugates in a carbohydrate-dependent manner. To elucidate the structural basis for this cross-reactivity, KLH glycans were released from tryptic glycopeptides and fluorescently labeled. Cross-reacting glycans were identified using a polyclonal antiserum reacting with soluble S. mansoni egg antigens, isolated by a threedimensional fractionation scheme and analyzed by different mass spectrometric techniques as well as linkage analysis and exoglycosidase treatment. The results revealed that cross-reacting species comprise ϳ4.5% of released glycans. They all represent novel types of N-glycans with a Fuc(␣1-3)GalNAc(1-4)[Fuc(␣1-3)]GlcNAc motif, which is known to occur also in schistosomal glycoconjugates. The tetrasaccharide unit is attached to the 3-linked antenna of a trimannosyl core, which can be further decorated by galactosyl residues, a xylose residue in 2-position of the central mannose and/or a fucose at the innermost N-acetylglucosamine. This study provides for the first time detailed structural data on the KLH carbohydrate entities responsible for cross-reactivity with glycoconjugates from S. mansoni.Hemocyanins act as oxygen-transporting proteins in many arthropod and mollusc species (1). In addition to this biochemical function, the hemocyanin of the Californian giant keyhole limpet Megathura crenulata, a marine gastropod, is known to be a potent immunoactivator (for reviews, see Refs. 2 and 3). Due to these immunostimulatory properties, keyhole limpet hemocyanin (KLH) 2 is widely used in research and clinical studies. Present fields of application are, for example, immunotherapy of bladder cancer (4 -7), based on the assumed expression of Gal(1-3)GalNAc determinants as cross-reacting epitopes (8), and its use as a carrier of polysialic acid or low molecular weight haptens, such as synthetic oligosaccharides, gangliosides, or (glyco)peptides, designed for potential application in anticancer therapy (9 -14). Intriguingly, KLH has been further demonstrated to exhibit a carbohydrate-based cross-reactivity with glycoconjugates from Schistosoma mansoni (15) thus allowing the diagnosis of S. mansoni (6, 7, 16), Schistosoma hematobium (17), and Schistosoma japonicum (18) infections by enzymelinked immunosorbent assay (ELISA). Hence, KLH has also been discussed as a potential candidate for vaccination against schistosomiasis (15). On the other hand, agarose beads coated with KLH or KLH-glycopeptides have been shown to mimic in a carbohydrate-dependent manner the hepatic granuloma formation mediated by S. mansoni eggs (19). Therefore, KLH may also be regarded as a model antigen to study the immunopathological mechanisms of schistosome infections.Although it is generally accepted that the oligosaccharide constituents of KLH are of prime significance for its antigenicity and biomedical properties (2), knowledge on the carbohydrate structure of this glycoprotein is still incomp...