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Ten Feizi

Publications and source records attributed to Ten Feizi.

At least 19 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2006

Preparation of neoglycolipids with ring-closed cores via chemoselective oxime-ligation for microarray analysis of carbohydrate-protein interactions.

Affinities of most oligosaccharide-protein interactions are so low that multivalent forms of ligand and protein are required for detecting interactions. The neoglycolipid (NGL) technology was designed to address the need for microscale presentation of oligosaccharides in a multivalent form for studying carbohydrate-protein interactions, and this is now the basis of a state-of-the-art carbohydrate microarray system. NGL technology involves conjugating oligosaccharides by reductive amination to the aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE). Other than ring-opening of the monosaccharide residues at reducing ends, oligosaccharides remain intact, and the NGLs derived from trisaccharides or larger oligosaccharides have performed well for the majority of carbohydrate-recognition systems that have the peripheral or backbone regions of oligosaccharides as recognition motifs. However, ring-opening of reducing end monosaccharides limits applicability to very short oligosaccharides (di- and trisaccharides) and, potentially, to N-glycans recognized by proteins such as Pisum sativum agglutinin (pea lectin) that require both intact core and backbone regions for strong binding. This chapter describes a method for preparing NGLs (designated AO-NGLs) from reducing oligosaccharides by chemoselective oxime-ligation to a new lipid reagent, N-aminooxyacetyl-DHPE. Microarray analyses of the AO-NGL derived from Lewis x (Le(x)) trisaccharide probed with anti-Le(x) antibodies indicate that a significant proportion of the core monosaccharide linked to lipid is in ring-closed form. Thus, AO-NGLs have broadened the applicability of NGLs as probes in studies of carbohydrate-protein interactions.

Carbohydrate Conformation↗
PubMed2005

Ligands for the beta-glucan receptor, Dectin-1, assigned using "designer" microarrays of oligosaccharide probes (neoglycolipids) generated from glucan polysaccharides.

Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate beta-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in beta1,3- or both beta1,3- and beta1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate "designer" microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular beta-glucans and compete with zymosan binding and triggering of tumor necrosis factor-alpha secretion by a Dectin-1-expressing macrophage cell line.

Animals↗
PubMed2004

Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor.

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.

Amino Acid Sequence↗
PubMed2004

Relative susceptibilities of the glucosamine-glucuronic acid and N-acetylglucosamine-glucuronic acid linkages to heparin lyase III.

Heparin lyases are valuable tools for generating oligosaccharide fragments and in sequence determination of heparan sulfate (HS). Heparin lyase III is known to cleave the linkages between N-acetylglucosamine (GlcNAc) or N-sulfated glucosamine (GlcNS) and glucuronic acid (GlcA) as the primary sites and the linkages between GlcNAc, GlcNAc(6S), or GlcNS and iduronic acid as secondary sites. N-Unsubstituted glucosamine (GlcN) occurs as a minor component in HS, and it has been associated with various bioactivities. Here we investigate the specificity of heparin lyase III toward the GlcN-GlcA linkage using a recombinant enzyme of high purity and as substrates the partially de-N-acetylated polysaccharide of Escherichia coli K5 strain and derived hexasaccharides. The specificity of lyase III toward the GlcN-GlcA linkage is deduced by sequencing of the oligosaccharide products using electrospray mass spectrometry with collision-induced dissociation and MS/MS scanning. The results demonstrate that under controlled conditions for partial digestion, lyase III does not act at the GlcN-GlcA linkage, whereas GlcNAc-GlcA is cleaved. Even under forced conditions for exhaustive digestion, the GlcN-GlcA linkage is only partly cleaved. It is this property of lyase III that has enabled the isolation of a unique, nonsulfated antigenic determinant DeltaUA-GlcN-UA-GlcNAc from HS and from partially de-N-acetylated K5 polysaccharide. It was unexpected that pentasaccharide fragments were also detected among the digestion products of the K5 polysaccharide used. It is possible that these are products of an additional glycosidase activity of lyase III, although other mechanisms cannot be completely ruled out.

Acetylation↗
PubMed2004

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.

Animals↗
PubMed2003

Interactions of the gastrotropic bacterium Helicobacter pylori with the leukocyte-endothelium adhesion molecules, the selectins--a preliminary report.

The deleterious effects of Helicobacter pylori infection of the stomach are largely the result of a vigorous chronic inflammatory response, and include chronic gastritis, peptic ulceration and gastric cancer. We are exploring the possibility that carbohydrate components on H. pylori contribute to the persistent inflammation through interactions with leukocyte-endothelial adhesion molecules of the host. Lipopolysaccharides of most H. pylori strains contain sequences related to the Lewis (Le(x) or Le(a)) antigens. Carbohydrate sequences of this family encompass ligands for the leukocyte-endothelium adhesion molecules of the host, namely, the E- and P-selectins, which are expressed on inflamed endothelia, and L-selectin, which is constitutively expressed on leukocytes. Here we investigate H. pylori isolates from patients with chronic gastritis, duodenal ulcer and gastric cancer for their interactions with the selectins. Our results provide unequivocal evidence of interactions of isolates from each of the diagnostic groups with E- and L-selectins.

Adult↗
PubMed2003

Carbohydrate microarrays - a new set of technologies at the frontiers of glycomics.

Carbohydrate microarray technologies are new developments at the frontiers of glycomics. Results of 'proof of concept' experiments with carbohydrate-binding proteins of the immune system - antibodies, selectins, a cytokine and a chemokine - and several plant lectins indicate that microarrays of carbohydrates (glycoconjugates, oligosaccharides and monosaccharides) will greatly facilitate not only surveys of proteins for carbohydrate-binding activities but also elucidation of their ligands. It is predicted that both naturally occurring and synthetic carbohydrates will be required for the fabrication of microarrays that are sufficiently comprehensive and representative of entire glycomes. New leads to biological pathways that involve carbohydrate-protein interactions and new therapeutic targets are among biomedically important outcomes anticipated from applications of carbohydrate microarrays.

Carbohydrates↗
PubMed2002

Synthesis and selectin-binding activity of N-deacetylsialyl Lewis X ganglioside.

A novel analogue of sialyl Lewis X ganglioside, N-deacetylsialyl Lewis X ganglioside, was synthesized. Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-alpha-D-galacto-2-nonulopyranosylonate-(2 --> 3)-2,4,6-tri-O-benzoyl-D-galactopyranosyl trichloroacetimidate was coupled with 2-(trimethylsilyl)ethyl [2-acetamido-6-O-benzyl-2-deoxy-3-O-(4-methoxybenzyl)-beta-D-glucopyranosyl]-(1 --> 3)-[2,4,6-tri-O-benzyl-beta-D-galactopyranosyl]-(1 --> 4)-2,3,6-tri-O-benzyl-beta-D-galactopyranoside to give the desired pentasaccharide in high yield. The glycosylation of the pentasaccharide acceptor, which was derived from its precursor by removal of the 3-methoxybenzyl group, with the phenyl 1-thioglycoside derivative of L-fucose using N-iodosuccinimide-trifluoromethanesulfonic acid as promoter, produced the hexasaccharide. Proper manipulation of the protecting groups of the hexasaccharide afforded the corresponding glycosyl imidate, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol. Selective reduction of the azido group, N-acylation with octadecanoic acid, and the complete removal of the protecting groups gave the desired N-deacetylsialyl Lewis X ganglioside. L-Selectin bound more strongly to N-deacetylsialyl Lewis X ganglioside than to the sialyl Lewis X ganglioside, whereas E- and P-selectins bound equally well to the two gangliosides.

E-Selectin↗
PubMed2002

Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions.

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.

Animals↗
PubMed2002

Chemically synthesized solid phase oligosaccharide probes for carbohydrate-binding receptors. Interactions of the E-, L- and P-selectins with sialyl-Le(x) and O-sulphated forms linked to biotin or to polyacrylamide.

There is a growing interest in chemically defined oligosaccharide reagents for identifying proteins that bind carbohydrates and determining the specificities of carbohydrate-binding proteins. Here, we compare three sets of chemically synthesized commercially available oligosaccharide conjugates as immobilized probes, for the binding signals that they elicit with known carbohydrate-binding receptors of the immune system, the E-, P- and L-selectins. The first set of conjugates is of oligosaccharides linked to biotin via a nine-carbon spacer. The second and third sets are multivalent derivatives in which the oligosaccharides are linked, via a three-carbon spacer to poly[N-(2-hydroxyethyl)acrylamide] (PAA) or to biotinylated PAA with an average of 20% substitution of the hydroxyethyl-amide groups by carbohydrate. The conjugates were immobilized on streptavidin-coated microwells if biotinylated, otherwise by drying in uncoated wells. The most robust binding curves, overall, were with the biotinylated PAA derivatives of the ligands immobilized on streptavidin wells. These reagents have permitted a reevaluation of selectin binding signals elicited by sialyl-Lewis(x) (SLe(x)) analogues having sulphate at position 6 of the galactose (6'SuSLe(x)) or of the N-acetylglucosamine (6SuSLe(x)). The results clarify the role of 6SuSLe(x), rather then 6'SuSLe(x), as a ligand for the selectins.

Acrylic Resins↗
PubMed2002

Mannose receptor-mediated regulation of serum glycoprotein homeostasis.

Carbohydrates are thought to function as tags that mark circulatory glycoproteins for rapid clearance. To examine the role of the mannose receptor (MR) in glycoprotein clearance, we generated mice genetically deficient in MR. MR-/- mice were defective in clearing proteins bearing accessible mannose and N-acetylglucosamine residues and had elevated levels of eight different lysosomal hydrolases. Proteomic analysis of MR-/- and control mouse sera showed that an additional 4 out of 52 proteins identified were elevated in MR-/- serum. Each of these is up-regulated during inflammation and wound healing. Thus, MR appears to operate as an essential regulator of serum glycoprotein homeostasis.

Animals↗
PubMed2002

Synergistic interactions of the two classes of ligand, sialyl-Lewis(a/x) fuco-oligosaccharides and short sulpho-motifs, with the P- and L-selectins: implications for therapeutic inhibitor designs.

The E-, L- and P-selectins are carbohydrate-recognizing cell-adhesion molecules mediating selective leucocyte recruitment in inflammation. The 3'-sialyl- and 3'-sulpho-oligosaccharides of Lewis(x) (Le(x)) and Lewis(a) (Le(a)) series are bound by them, but for high-avidity binding of P- and L-selectins to the glycoprotein counter-receptor known as P-selectin glycoprotein ligand, PSGL-1, there is a requirement for sulpho-tyrosines neighbouring a sialyl-Le(x) glycan. The two selectins can also bind 3-O- or 6-O-sulphated galacto-lipids (sulphatides). Here we compare some features of the interactions of P- and L-selectins with a novel lipid-linked sulpho-tyrosine probe, and with the sulphatides and neoglycolipids of sialyl- and sulpho-Le(x)/Le(a) fuco-oligosaccharides. The sulpho-tyrosine probe is bound by both selectins. There are close similarities in the interactions of the two selectins with sulpho-tyrosine and the sulphatides; the binding is relatively resistant to chelation of calcium ions, in contrast to the absolute requirement of calcium ions with the long fuco-oligosaccharides, including 6-sulpho-sialyl-Le(x). With both selectins, there is striking synergy in binding signals elicited by the two ligand types when presented as equimolar mixtures on a matrix. Thus, there are two operationally distinct binding sites on both L- and P-selectin; and the binding sites for sulphate groups in the two ligand types are probably distinct. When sulpho-tyrosine and sialyl-Le(x) are presented on liposomes, a potent inhibitory activity is generated toward the binding of P-selectin to HL60 cells, with 50% inhibitory concentration (IC(50)) values in the nanomolar range. These features of the lipid-linked ligand analogues, and the simple approach for their display on liposomes, may have applications in designs and screening of selectin inhibitors as anti-inflammatory compounds.

Binding Sites↗