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Amino acid replacement mutations in certain chronic lymphocytic leukemia (CLL) stereotyped B cell receptor (BCR) immunoglobulins (IGs) at defined positions within antigen-binding sites strongly imply antigen selection. Prime examples of this are CLL subset 4 BCR IGs using IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements. Conspicuously, and unlike most CLL IGs, subset 4 IGs do not bind apoptotic cells. By testing the (auto)antigenic reactivities of subset 4 IGs toward viable lymphoid-lineage cells and specific autoantigens typically bound by IGHV4-34+ IGs, we found that IGs from both subset 4 and non-subset 4 IGHV4-34-expressing CLL cases bound naïve B cells. However, only subset 4 IGs reacted with memory B cells. Furthermore, subset 4 IGs did not bind DNA nor i or I carbohydrate antigens that are common targets of IGHV4-34-utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively. Notably, we found that subset 4 IG binding to memory B lymphocytes depends on an aspartic acid at position 66 of FR3 in the rearranged IGKV2-30 gene; this amino acid residue is acquired by somatic mutation. Our findings illustrate the importance of positive and negative selection criteria for structural elements in CLL IGs and suggest that autoantigens driving normal B cells to become subset 4 CLL cells differ from those driving IGHV4-34+ B cells in other diseases.

Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.

We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are obtained from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewis(x) sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewis(x) sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-gamma (IFN-gamma) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewis(x), and sulfo-Lewis(a), in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.

The closing years of the second millennium have been uplifting for carbohydrate biology. Optimism that oligosaccharide sequences are bearers of crucial biological information has been borne out by the constellation of efforts of carbohydrate chemists, biochemists, immunochemists, and cell- and molecular biologists. The direct involvement of specific oligosaccharide sequences in protein targeting and folding, and in mechanisms of infection, inflammation and immunity is now unquestioned. With the emergence of families of proteins with carbohydrate-binding activities, assignments of information content for defined oligosaccharide sequences will become more common, but the pinpointing and elucidation of the bioactive domains on oligosaccharides will continue to pose challenges even to the most experienced carbohydrate biologists. The neoglycolipid technology incorporates some of the key requirements for this challenge: namely the resolution of complex glycan mixtures, and ligand binding coupled with sequence determination by mass spectrometry.

The interaction between glycosphingolipids and recombinant human GM2-activator was studied in a microwell binding assay. A-series gangliosides like GM3, GM2 and GM1 were strongly bound by the recombinant human GM2 activator. A weak binding was observed to GD1b and sulfatide, while neutral glycolipids were not bound. Optimal binding occurred at pH 4.2 and was inhibited by increasing concentrations of citrate buffer and NaCl. In contrast with these in vitro results the recombinant human GM2-activator is able to restore the degradation of GA2 in fibroblasts from patients with the AB variant of GM2 gangliosidosis in vivo.

Galectins are beta-galactoside-binding proteins that occur intra- and extracellularly in many animal tissues. They have been proposed to form networks of glycoconjugates on the cell surface, where they may modulate various cell response pathways such as growth, activation and adhesion. The high resolution X-ray crystallographic analyses of three crystal forms of bovine galectin-1 in complex with biantennary saccharides of N-acetyllactosamine type reveal infinite chains of lectin dimers cross-linked through N-acetyllactosamine units located at the end of the oligosaccharide antenna. The oligosaccharide adopts a different low energy conformation in each of the three crystal forms.

We wish to retract this Article owing to an inability to reproduce the results. This retraction has not been signed by K.B. and A.F., and M.P. is deceased (J.O’B. cannot be traced).

The cellular biosynthetic machinery can be harnessed to incorporate traceable markers into cell-surface glycans in vivo.

Corrigendum: Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray

Nat. Biotechnol. 22, 1244–1245 (2004) On Page 1245, col. 2, paragraph 2, last line, “others3” should have been “others2”.

The hope that hybridoma antibodies would reveal unique cell surface antigens during embryogenesis, differentiation and oncogenesis has been replaced by the realization that such antigens are mainly carbohydrate structures of glycoproteins and glycolipids occurring in many cell types. These findings either may reflect limitations in the methods of selection of hybridoma antibodies or may point to important roles for the diverse carbohydrate structures as receptors for regulators of cell growth and differentiation.

There is much interest in developmentally regulated molecules which may have function in cell interactions and sorting during embryogenesis and differentiation. Numerous antisera have been raised which detect antigens that are expressed in early embryonic cells and become restricted during differentiation, being expressed in only a minority of adult cells (reviewed in refs 1–3). The precise antigenic determinants recognized by such antisera have not been defined. However, studies using a hybri-doma antibody against mouse spleen cells4 and monoclonal autoantibodies of patients with cold agglutinin disease5 have shown that two defined carbohydrate antigen systems, the Forssman and the li antigens, have stage-specific expression in early mouse embryos. We now describe evidence that the stage-specific embryonic antigen SSEA-1 (ref. 6) involves the carbohydrate sequence Galβ1 → 4GlcNAc ↑1,3 Fucα This determinant is formed by α 1 → 3 fucosylation of blood group I or i antigens which are branched or linear oligosaccharides, respectively7–9, built of Galβ1 →4GlcNAc units and known as type 2 precursor chains10 of the major blood group antigens. Thus, we introduce the concept of simple glycosylation changes as a basis for stage-specific expression of embryonic antigens.