Carbohydrate microarrays reveal sulphation as a modulator of siglec binding.

Siglecs are receptors on cells of the immune, haemopoietic, and nervous systems that recognize sialyl-glycans with differing preferences for sialic acid linkage and oligosaccharide backbone sequence. We investigate here siglec binding using microarrays of Lewis(x) (Le(x))- and 3'-sialyl-Le(x)-related probes with different sulphation patterns. These include sulphation at position 3 of the terminal galactose of Le(x), position 6 of the galactose of Le(x) and sialyl-Le(x), position 6 of N-acetylglucosamine of Le(x) and sialyl-Le(x), or both positions of sialyl-Le(x). Recombinant soluble forms of five siglecs have been investigated: human Siglec-7, -8, -9, and murine Siglec-F and CD22 (Siglec-2). Each siglec has a different binding pattern. Unlike two C-type lectins of leukocytes, L-selectin and Langerin, which also bind to sulphated analogues of sialyl-Le(x), the siglecs do not give detectable binding signals with sulphated analogues that are lacking sialic acid. The sulphate groups modulate, however, positively or negatively the siglec binding intensities to the sialyl-Le(x) sequence.

High and low affinity carbohydrate ligands revealed for murine SIGN-R1 by carbohydrate array and cell binding approaches, and differing specificities for SIGN-R3 and langerin.

The number of receptors of the 'C-type' lectin family is greater than previously thought with a considerable proportion on cells (dendritic cells and macrophages) critical for innate immunity. Establishing that they bind carbohydrates, unravelling and comparing details of their ligands is crucial for understanding the molecular basis of the cell-cell and cell-pathogen interactions that they mediate. Here we use carbohydrate arrays as a new approach to discovering the ligands of three recently described C-type lectin-type receptors on antigen-presenting cells: murine SIGN-R1, SIGN-R3 and langerin. The arrays encompass an extensive panel including polysaccharides, glycoproteins, oligosaccharides and monosaccharides. These are probed with soluble forms of the receptors (IgG-Fc chimeras). The dominant specificities found for SIGN-R1 and SIGN-R3 are mannose- and fucose-related, as expressed on high mannose type N-glycans and Lewis(a/b)/Lewis(x/y)-type sequences, respectively, with subtle differences between the receptors. The dominant specificity for langerin is unique so far: a Lewisx-related sequence with sulfate at position 6 of the terminal galactose. The polysaccharide dextran, known from classical studies to elicit a T-independent response, and whose cellular uptake has been shown recently to be mediated by membrane-associated SIGN-R1, gave no binding signals with the soluble form of the protein. We highlight here the additional need for cell-based assays for detecting biologically relevant low affinity ligands, for we show with SIGN-R1-transfected cells that dextran is such a low affinity ligand for SIGN-R1 that binding is detectable only with the cell membrane-associated receptor. But there is a close relationship between dextran recognition and mannose/fucose recognition, with dextran- and mannose-conjugates co-localizing in intracellular compartments.

Biophysical characterization of triacyl monosaccharide lipid a partial structures in relation to bioactivity.

Synthetic triacyl glucosamine monosaccharide lipid A part structures corresponding to the non-reducing moiety of enterobacterial lipid A with an acyloxyacyl chain linked to position 3 of the glucosamine and an unbranched chain linked to position 2 (group 1) and vice versa (group 2) were analyzed biophysically: Fourier-transform infrared spectroscopy was performed to characterize the gel-to-liquid crystalline phase transition, the phosphate band contour, and the orientation of the glucosamine with respect to the membrane surface. Small-angle x-ray diffraction was applied for the elucidation of the supramolecular aggregate structure and, with that, of the molecular shape. With fluorescence resonance energy transfer the lipopolysaccharide-binding protein (LBP)-mediated intercalation of the lipid A partial structures into phospholipid liposomes was monitored. The physical data clearly exhibit a classification of the synthetic compounds into two groups: group 1 compounds have sharp phase transitions, indicating dense acyl chain packing and an inclination of the glucosamine backbone with respect to the membrane surface of 30 degrees with the phosphate buried in the membrane. Group 2 compounds have a very broad phase transition, indicating poorly packed acyl chains, and an inclination of -30 degrees with the phosphate group sticking outward. For the first group unilamellar phases are observed superimposed by a non-lamellar structure, and for the second one only multilamellar aggregate structures. The cytokine-inducing capacity in human mononuclear cells is relatively high for the first group and low or absent for the second group. Based on these data a model of the intra and intermolecular conformations is proposed which also extends the concept of "endotoxic conformation."