Although Galβ1–4GlcNAc (LacNAc) moieties are the most common constituents of N-linked glycans on vertebrate proteins, GalNAcβ1–4GlcNAc (LacdiNAc, LDN)-containing glycans are widespread in invertebrates, such as helminths. We postulated that LDN might be a molecular pattern for recognition of helminth parasites by the immune system. Using LDN-based affinity chromatography and mass spectrometry, we have identified galectin-3 as the major LDN-binding protein in macrophages. By contrast, LDN binding was not observed with galectin-1. Surface plasmon resonance (SPR) analysis and a solid phase binding assay demonstrated that galectin-3 binds directly to neoglycoconjugates carrying LDN glycans. In addition, galectin-3 bound to Schistosoma mansoni soluble egg Ags and a mAb against the LDN glycan inhibited this binding, suggesting that LDN glycans within S. mansoni soluble egg Ags contribute to galectin-3 binding. Immunocytochemistry demonstrated high levels of galectin-3 in liver granulomas of S. mansoni-infected hamsters, and a colocalization of galectin-3 and LDN glycans was observed on the parasite eggshells. Finally, we demonstrate that galectin-3 can mediate recognition and phagocytosis of LDN-coated particles by macrophages. These findings provide evidence that LDN-glycans constitute a parasite pattern for galectin-3-mediated immune recognition.

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigen Lewis x (Lex) in a polymeric form. Lex is β-d-galactose-(1-4)-[α-l-fucose-(1-3)]-β-d-acetylglucosamine. Schematically the LPS structure is (Lex) n -core-lipid A. In this report, we show that Lex expression is not a stable trait but that LPS displays a high frequency (0.2 to 0.5%) of phase variation, resulting in the presence of several LPS variants in one bacterial cell population. One type of phase variation implied the loss of α1,3-linked fucose, resulting in variants that expressed nonsubstituted polylactosamines (also called the i antigen), i.e., Lex minus fucose; LPS: (lactosamine) n -core-lipid A. The switch of Lex to i antigen was reversible. A second group of variants arose by loss of polymeric main chain which resulted in expression of monomeric Ley; LPS: (Ley)-core-lipid A. A third group of variants arose by acquisition of α1,2-linked fucose which hence expressed Lex plus Ley; LPS: (Ley)(Lex) n -core-lipid A. The second and third group of variants switched back to the parental phenotype [(Lex) n -core-lipid A] in lower frequencies. Part of the variation can be ascribed to altered expression levels of glycosyltransferase levels as assessed by assaying the activities of galactosyl-, fucosyl-, andN-acetylglucosaminyltransferases. Clearly phase variation increases the heterogeneity of H. pylori, and this process may be involved in generating the very closely related yet genetically slightly different strains that have been isolated from one patient.

In order to assess the function of the different human UDP-Gal:GlcNAc L L4-galactosyltransferases, the cDNAs of two of them, L L4-GalT I and L L4-GalT V, were expressed in the baculovirus/insect cell expression system. The soluble recombinant enzymes produced were purified from the medium and used to determine their in vitro substrate specificities. The specific activity of the recombinant L L4-GalT V was more than 15 times lower than that of L L4-GalT I, using GlcNAcL L-S-pNP as an acceptor. Whereas L L4-GalT I efficiently acts on all substrates having a terminal L L-linked GlcNAc, L L4-GalT V appeared to be far more restricted in acceptor usage. L L4-GalT V acts with high preference on acceptors that contain the GlcNAcL L1C C6GalNAc structural element, as found in O-linked core 2-, 4-and 6-based glycans, but not on substrates related to N-linked or blood group I-active oligosaccharides. These results suggest that L L4-GalT V may function in the synthesis of lacNAc units on O-linked chains, particularly in tissues which do not express L L4-GalT I, such as brain.z 1999 Federation of European Biochemical Societies.

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Three putative K K1C C3/4-fucosyltransferase (K K1C C3/ 4-FucT) genes have been detected in the Arabidopsis thaliana genome. The products of two of these genes have been identified in vivo as core K K1C C3-FucTs involved in N-glycosylation. An orthologue of the third gene was isolated from a Beta vulgaris cDNA library. The encoded enzyme efficiently fucosylates GalL L1C C3GlcNAcL L1C C3GalL L1C C4Glc. Analysis of the product by 400 MHz 1 H-nuclear magnetic resonance spectroscopy showed that the product is K K1C C4-fucosylated at the Nacetylglucosamine residue. In vitro, the recombinant B. vulgaris K K1C C4-FucT acts efficiently only on neutral type 1 chain-based glycan structures. In plants the enzyme is expected to be involved in Lewis a formation on N-linked glycans. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.