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T Feizi

Publications and source records attributed to T Feizi.

At least 19 recordsLinked to original sources
PubMed2001

NMR studies of mannitol-terminating oligosaccharides derived by reductive alkaline hydrolysis from brain glycoproteins.

Interest in the characterisation of O-mannosyl glycan structures has been stimulated following the identification of mannitol-terminating oligosaccharides among the chains released from mammalian proteins in nervous and muscle tissues, and by the discovery of a putative human O-mannosyl transferase. Several mass spectrometry methods have been applied to structure elucidation particularly when low amounts of oligosaccharide are available for analysis. However, when sufficient amounts are available, a combination of through-bond homo- and heteronuclear, and of through-space homonuclear NMR experiments permit the complete identification of these oligosaccharide sequences. We describe here the assignment of 1H and 13C NMR chemical shifts from such experiments for four mannitol-terminating oligosaccharide alditols, GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)[Fucalpha-(1-->3)]GlcNAcbeta-(1-->2)Manol and NeuAcalpha-(2-->3)Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, that were released from brain glycopeptides by alkaline borohydride treatment.

Animals↗
PubMed2001

10E4 antigen of Scrapie lesions contains an unusual nonsulfated heparan motif.

The carbohydrate antigen on heparan sulfate recognized by monoclonal antibody 10E4 is uniquely codistributed with the abnormal prion protein, PrP(Sc), even in the earliest detectable brain lesions of scrapie-infected mice. Determining the chemical structure of 10E4 antigen is, therefore, an important aspect of structure elucidation of scrapie lesions, and a prerequisite for designing experiments to understand its role in scrapie pathogenesis. Toward this aim, we have examined preparations of heparan sulfate, with differing sulfate contents, for binding by 10E4 antibody. The highest antigenicity was observed in a preparation (HS-1) with the lowest sulfate content. HS-1 was partially depolymerized with heparin lyase III, and oligosaccharide fragments examined for 10E4 antigen expression by the neoglycolipid technology. An antigen-positive and two antigen-negative tetrasaccharides were isolated and examined by electrospray mass spectrometry. The antigen-positive tetrasaccharide sequence on heparan sulfate was thus deduced to contain a unique unsulfated motif that includes an N-unsubstituted glucosamine in the sequence, UA-GlcN-UA-GlcNAc. Antibody binding experiments with neoglycolipids prepared from a series of heparin/heparan sulfate disaccharides, and the trisaccharide derived from the antigen-positive tetrasaccharide after removal of the terminal hexuronic acid, show that both the penultimate glucosamine and the outer nonsulfated hexuronic acid are important for 10E4 antigenicity.

Animals↗
PubMed2001

New structural insights into lectin-type proteins of the immune system.

New structural data have emerged for the ligand-binding sites of C-type lectin domains and C-type lectin-like domains of receptors of the immune system. These include binding sites for oligosaccharide or polypeptide ligands, or both oligosaccharide and polypeptide ligands. The structural basis for the binding of a lectin domain of the beta-trefoil family to different sulfooligosaccharide sequences has been revealed. Lectin activity has been documented for a beta/alpha TIM barrel fold that does not have the chitinase activity of the prototype enzyme with this fold.

Animals↗
PubMed2001

A monoclonal antibody, MIN/3/60, that recognizes the sulpho-Lewis(x) and sulpho-Lewis(a) sequences detects a sub-population of epithelial glycans in the crypts of human colonic epithelium.

Monoclonal antibodies (MAbs) directed to Lewis(x) (Le(x)) and related carbohydrate sequences have been invaluable in anticipating biological roles for these oligosaccharides by detecting the remarkable changes that occur in their expression from the earliest stages of embryogenesis, through development and sequential stages of cell differentiation and maturation. A notable impact has been in the molecular dissection of ligand-receptor interactions in key cell adhesion events at the initial stages of leukocyte recruitment in inflammation, and almost certainly in the metastasis of epithelial tumours. Antibodies that recognise Le(x) and the 3'-sialyl forms were observed to identify leukocyte subsets; these were subsequently found to match those recognized by the leukocyte-endothelium adhesion molecules, the E- and P-selectins. We now describe a MAb (rat hybridoma MIN/3/60) raised to 3'-sulpho-Le(x), a carbohydrate sequence which, in vitro, is bound not only by the E-, L-, and P-selectins, but also by the cysteine-rich domain of the macrophage endocytosis receptor. We observe that MIN/3/60 is bispecific, however; it binds 3'-sulpho-Le(a) as well as 3'-sulpho-Le(x). Nevertheless, our exploratory studies reveal that it may be a useful histochemical reagent when used in conjunction with a monospecific antibody to 3'-sulpho-Le(a). The MIN/3/60 antibody reveals a sub-population of epithelial glycans in the crypts of Lieberkühn in normal human colon.

Animals↗
PubMed2001

Carrier protein-modulated presentation and recognition of an N-glycan: observations on the interactions of Man(8) glycoform of ribonuclease B with conglutinin.

Conglutinin is a serum lectin of the innate immune system, which binds high mannose N-glycans when these are appropriately presented on proteins. Here we use the conglutinin-ribonuclease B (RNaseB)-recognition system as a model to investigate the structural basis of selective recognition of protein-bound oligosaccharides by this carbohydrate-binding receptor. Conglutinin shows little binding to the isolated RNaseB-Man(8 )glycoform, and no binding to Man(5-6) glycoforms. In contrast, when the protein moiety is reduced and denatured we observe that conglutinin binds strongly to the isolated RNaseB-Man(8) glycoform and weakly to the Man(5-6) glycoforms. These results are in accord with observations on the binding to the N-glycans in the absence of carrier protein. NMR analyses of native RNaseB-Man(8) and -Man(5-6) glycoforms reveal that the three-dimensional structure of the protein moiety is essentially identical to that of non-glycosylated RNase (RNaseA). Thus there are no perceptible differences between the RNase protein forms that could account for differential availability of the N-glycan for conglutinin-binding. After reduction and denaturation, the NMR spectrum became typical of a non-structured polypeptide, although the conformational preferences of the N-glycosidic linkage were unchanged, and most importantly, the Man(8 )oligosaccharide retained the average conformational behavior of the free oligosaccharide irrespective of the carrier protein fold. This conformational freedom is clearly not translated into full availability of the oligosaccharide for the carbohydrate-recognition protein. We propose, therefore, that the differing bioactivity of the N-glycan is a reflection of the existence of different geometries of presentation of the carbohydrate determinant in relation to the protein surface within the glycan:carrier protein ensemble.

Carbohydrate Conformation↗
PubMed2000

Conformational studies of the Man8 oligosaccharide on native ribonuclease B and on the reduced and denatured protein.

Site-specific presentation of oligosaccharides in the context of carrier proteins can influence markedly their recognition by carbohydrate-binding proteins. On RNaseB, the Man5-9 N-glycans at Asn-34 are bound by the serum lectin conglutinin when the glycoprotein is reduced and denatured, but there is no binding to the N-glycans on the native form of RNaseB. The RNaseB Man8, which is a glycoform preferentially bound by conglutinin, is the subject of the present study. The conformational behavior of the protein-linked oligosaccharide Man8 is investigated on the native and on the reduced and denatured RNaseB, using a combination of NMR and theoretical calculations. Quantitative data on the NOESY crosspeaks have been obtained, thereby allowing the comparison of mobilities of homologous linkages within the glycan chain. Oligosaccharide conformations compatible with the NMR data have been explored by molecular modeling of the free oligosaccharide, using two different force fields (AMBER and SYBYL). There are some differences between the results produced by the two force fields, the AMBER simulations providing a better agreement with the experimental data. The results indicate that both on the native and on the reduced heat-denatured glycoprotein, the RNase Man8 oligosaccharide exhibits a conformational behavior very similar to that of the free oligosaccharide. However, this conformational freedom of the N-glcyan does not amount to full availability for carbohydrate-recognition proteins and enzymes.

Magnetic Resonance Spectroscopy↗
PubMed2000

Crystal structure of the cysteine-rich domain of mannose receptor complexed with a sulfated carbohydrate ligand.

The macrophage and epithelial cell mannose receptor (MR) binds carbohydrates on foreign and host molecules. Two portions of MR recognize carbohydrates: tandemly arranged C-type lectin domains facilitate carbohydrate-dependent macrophage uptake of infectious organisms, and the NH(2)-terminal cysteine-rich domain (Cys-MR) binds to sulfated glycoproteins including pituitary hormones. To elucidate the mechanism of sulfated carbohydrate recognition, we determined crystal structures of Cys-MR alone and complexed with 4-sulfated-N-acetylgalactosamine at 1.7 and 2.2 A resolution, respectively. Cys-MR folds into an approximately three-fold symmetric beta-trefoil shape resembling fibroblast growth factor. The sulfate portions of 4-sulfated-N-acetylgalactosamine and an unidentified ligand found in the native crystals bind in a neutral pocket in the third lobe. We use the structures to rationalize the carbohydrate binding specificities of Cys-MR and compare the recognition properties of Cys-MR with other beta-trefoil proteins.

Acetylgalactosamine↗
PubMed2000

The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated N-glycans of lutropin.

The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells.

Acetylgalactosamine↗
PubMed2000

Expression in Escherichia coli, folding in vitro, and characterization of the carbohydrate recognition domain of the natural killer cell receptor NKR-P1A.

NKR-P1A is a homodimeric type II transmembrane protein of the C-type lectin family found on natural killer (NK) cells and NK-like T cells and is an activator of cytotoxicity. Toward structure determination by NMR, the recombinant carbohydrate-recognition domain (CRD) of NKR-P1A has been expressed in high-yield in Escherichia coli and folded in vitro. The purified protein behaves as a monomer in size-exclusion chromatography and is bound by the conformation-sensitive antibody, 3.2.3, indicating a folded structure. A polypeptide tag at the N-terminus is selectively cleaved from the CRD after limited trypsin digestion in further support of a compact folded structure. The disulfide bonds have been identified by peptide mapping and electrospray mass spectrometry. These are characteristic of a long form CRD. The 1D NMR spectrum of the unlabeled CRD and the 2D HSQC spectrum of the (15)N-labeled CRD are those of a folded protein. Chemical shifts of H(alpha) and NH protons indicate a considerable amount of beta-strand structure. Successful folding in the absence of Ca(2+), coupled with the lack of chemical shift changes upon addition of Ca(2+), suggests that the NKR-P1A-CRD may not be a Ca(2+)-binding protein.

Amino Acid Sequence↗
PubMed2000

Carbohydrate-mediated recognition systems in innate immunity.

There is growing interest in carbohydrate-recognizing receptors of the innate immune system. Among them are members of the C-type lectin family, which include the collectins and the selectins and which operate by ligating exogenous (microbial) or endogenous carbohydrates. De novo assignments of the sequences of ligands for carbohydrate-recognizing receptors are among the most challenging topics in cell biology. This is because of the heterogeneity of oligosaccharides on proteins and lipids, and their availability only in limited amounts. To address the need for a microprocedure for direct binding studies with oligosaccharides derived from glycoproteins, we introduced the neoglycolipid technology for generating solid phase oligosaccharide probes for binding experiments. The technology has enabled assignments of unsuspected oligosaccharide ligands for the selectins and given valuable insights into those for the collectins. The ligands so far identified appear not to be unique for a given receptor system; there are considerable cross-reactions. Specificity can be created, however, through different modes of oligosaccharide presentation on macromolecular carriers, or the expression of a particular oligosaccharide sequence on a selected cell type in a given body compartment, and the regulated expression of the receptor protein at the desired location. The existence of unique ligand structures is not ruled out, however. Co-ligation of a receptor may also occur to a second carbohydrate or even to a non-carbohydrate ligand to create a unique assembly. A further group of C-type lectin-like proteins occurs on natural killer (NK) cells and NK T cells, and is associated with activation or inhibition of the cell effector functions. An important challenge is to determine whether carbohydrates are among physiological ligands for this important group of receptors.

Animals↗
PubMed2000

Fluorescent neoglycolipids. Improved probes for oligosaccharide ligand discovery.

A second generation of lipid-linked oligosaccharide probes, fluorescent neoglycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), which has been used extensively to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N-aminoacetyl-N-(9-anthracenylmethyl)-1, 2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHP-neoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequence-specific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl-Lea- and sialyl-Lex-active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary-ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno-detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state-of-the-art technologies for carbohydrate analysis.

Amination↗
PubMed1999

Recombinant soluble human CD69 dimer produced in Escherichia coli: reevaluation of saccharide binding.

We reevaluate here an earlier report of monosaccharide binding by the C-type lectin-like, leukocyte surface protein CD69 in the form of a recombinant soluble dimer, and we examine polysaccharide binding by the protein. We have expressed in Escherichia coli a new construct of the extracellular part (Q(65)-K(199)) of human CD69. We describe the folding in vitro to produce, in good yield, the protein in a soluble, disulphide-linked, dimeric form, and the results of binding experiments with monosaccharides: glucose, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine, linked to bovine serum albumin. Monosaccharide-binding signals are not detectable. Among the polysaccharides, heparin, chondroitin sulphates A, B, and C, fucoidan, and dextran sulphate, CD69 dimer gives a weak binding signal with fucoidan.

Amino Acid Sequence↗
PubMed1999

Influence of oligosaccharide presentation on the interactions of carbohydrate sequence-specific antibodies and the selectins. Observations with biotinylated oligosaccharides.

This study was aimed at investigating the efficacy of presentation of biotinylated oligosaccharides on streptavidin-coated microwells for interactions with (a) three monoclonal antibodies directed at sialyl-Lewisa (Le(a)) or sulfo-Le(a)-related sequences, and (b) the endothelium-leukocyte adhesion molecules, the E-, L- and P-selectins which recognize both the sulfo- and sialyl-Le(a) series. With the antibodies it was observed that if the biotinylated oligosaccharide incorporated the entire antigenic determinant, and additional saccharide length was not included, the biotinyl tag spacer length was a critical factor in the strength of the binding signal. If oligosaccharide chain beyond the determinant was included, the biotinyl tag spacer length was less important. The E-selectin binding data with the biotinylated sialyl- and sulfo-oligosaccharides were in overall accord with previous knowledge. With the L- and P-selectins, however, unexpectedly low binding signals were elicited by biotinyl sulfo-Le(a) sequences relative to those with the sialyl-analogs. This suppression was more pronounced with the rodent than the human L-selectin. Such differential availabilities of oligosaccharides displayed on streptavidin may relate to biological situations, such as the differential reactivities of the three selectins with a given oligosaccharide ligand presented on different carrier proteins, or on different O-glycan cores on mucin-type glycoproteins.

Animals↗
PubMed1999

L-selectin interactions with novel mono- and multisulfated Lewisx sequences in comparison with the potent ligand 3'-sulfated Lewisa.

The cell adhesion molecule L-selectin binds to 3'-sialyl-Lewis (Le)x and -Lea and to 3'-sulfo-Lex and -Lea sequences. The binding to 3'-sialyl-Lex is strongly affected by the presence of 6-O-sulfate as found on oligosaccharides of the counter receptor, GlyCAM-1; 6-O-sulfate on the N-acetylglucosamine (6-sulfation) enhances, whereas 6-O-sulfate on the galactose (6'-sulfation) virtually abolishes binding. To extend knowledge on the specificity of L-selectin, we have investigated interactions with novel sulfo-oligosaccharides based on the Lex pentasaccharide sequence. We observe that, also with 3'-sulfo-Lex, the 6-sulfation enhances and 6'-sulfation suppresses L-selectin binding. The 6'-sulfation without 3'-sialyl or 3'-sulfate gives no binding signal with L-selectin. Where the 6-sulfo,3'-sialyl-Lex is on an extended di-N-acetyllactosamine backbone, additional 6-O-sulfates on the inner galactose and inner N-acetylglucosamine do not influence the binding. Although binding to the 6,3'-sulfo-Lex and 6-sulfo, 3'-sialyl-Lex sequences is comparable, the former is a more effective inhibitor of L-selectin binding. This difference is most apparent when L-selectin is in paucivalent form (predominantly di- and tetramer) rather than multivalent. Indeed, as inhibitors of the paucivalent L-selectin, the 3'-sulfo-Lex series are more potent than the corresponding 3'-sialyl-Lex series. Thus, for synthetic strategies to design therapeutic oligosaccharide analogs as antagonists of L-selectin binding, those based on the simpler 3'-sulfo-Lex (and also the 3'-sulfo-Lea) would seem most appropriate.

Carbohydrate Sequence↗
PubMed1999

Core-branching pattern and sequence analysis of mannitol-terminating oligosaccharides by neoglycolipid technology.

The occurrence of mannitol-terminating oligosaccharides (2-substituted or 2,6-disubstituted) among the O-glycans released by alkaline borohydride treatment from glycoproteins of the nervous system has prompted the development of a microscale method to analyze the core-branching pattern and sequence by the neoglycolipid (NGL) technology, analogous to a method previously described for GalNAcol-terminating oligosaccharides (M. S. Stoll, E. F. Hounsell, A. M. Lawson, W. Chai, and T. Feizi, Eur. J. Biochem. 189, 499-507, 1990). The approach involves the selective cleavage at the core mannitol by mild periodate treatment and analysis of the reaction products as NGLs by in situ TLC/liquid secondary ion mass spectrometry. Oxidation conditions have been optimized using as reference compounds 2-, 3-, 4-, or 6-monosubstituted mannobi-itols, 3,6-disubstituted mannitol-terminating pentasaccharides, and 2-mono- and 2,6-disubstituted mannitol-terminating neutral and sialylated oligosaccharides isolated from brain glycopeptides. When a 2:1 molar ratio of periodate to alditol is used, the core mannitol is cleaved at the C3-C4 threo-diol bond and in the absence of a threo-diol cleavage occurs to a lesser extent at erythro-diols. Saccharide ring diols are not cleaved under these conditions, and it is also shown that the side chain of sialic acid on the oligosaccharide is largely unaffected. Substituents at 2- and 6-positions of the core mannitol can be identified, and the method is applicable to neutral and sialylated oligosaccharide alditols. Typically, the starting material is 5 nmol of oligosaccharide and 0.5-1 nmol of derivatives is applied for analysis. By this strategy, the core-branching pattern and position of sialic acid of two branched monosialylated mannitol-terminating oligosaccharide isomers have been determined.

Animals↗