Quantifiable fluorescent glycan microarrays

A glycan microarray was developed by using 2,6-diaminopyridine (DAP) as a fluorescent linker and printing of the glycan-DAP conjugates (GDAPs) on epoxy-activated glass slides. Importantly, all coupled GDAPs showed a detectable level of concentration-dependent GDAP fluorescence under blue laser excitation (495 nm) that can be used for both grid location and on-slide quantification. A glycan array including a large number of GDAP’s derived from natural and commercially available free glycans was constructed and glycan interactions with various plant lectins were investigated. In addition, binding parameters of lectins to glycans were obtained by varying both the amount of GDAPs on the array and the lectin concentration in analyses. These data demonstrate the general utility of GDAP microarrays for functional glycomic analyses and for determining binding parameters of glycan binding proteins (GBPs).     

Effects of polymer structure on the inhibition of cholera toxin by linear polypeptide-based glycopolymers

A variety of important biological events are mediated by the multivalent interaction between relevant oligosaccharides and multiple saccharide receptors on lectins, toxins, and cell surfaces; a variety of glycopolymeric materials have therefore been investigated in studies aimed at manipulating these events. The synthesis of protein- and polypeptide-based glycopolymers via protein engineering and other methods offers opportunities to control both the number and the spacing of saccharides on a scaffold, as well as the conformation of the polymer backbone, and will therefore facilitate the structure-based design of polymers for inhibition of multivalent binding events. In initial studies, we have synthesized a family of galactose-functionalized glycopolymers with a poly(L-glutamic acid) backbone, in which the density and linker length of the pendant carbohydrate moiety were varied. The composition of the glycopolymers was determined via (1)H NMR spectroscopy, and the impact of saccharide density and linker length, as well as the potential for these polypeptide-based glycopolymers to act as high-affinity inhibitors of the cholera toxin, has been indicated via competitive enzyme-linked immunosorbent assay and fluorescence titration experiments. The results of these studies suggest strategies for optimizing the binding of linear glycopolymers to bacterial toxins and will aid in the design of additional protein-based materials for studying the impact of multivalency, spacing, and backbone rigidity in a variety of biologically relevant binding events.     

Distribution and nature of saccharides of plasma membranes of rat ascites hepatoma cells as detected by ferritin-conjugated lectins and dialysed iron

Electron microscopic cytochemical studies were made on saccharides involved in the plasma membranes of rat ascites hepatoma cells (AH7974F) using ferritin-conjugated lectins and dialysed iron (DI). In the rat hepatoma cells, saccharide receptors for each of the three lectins used (concanavalin A (ConA), wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA)) were shown to be distributed homogeneously throughout the plasma membranes. When the cells were agglutinated, however, the saccharide receptors for each lectin appeared to form clusters on the plasma membranes. The cluster formation induced by one lectin was found to lead to a changed distribution of saccharide receptors for another lectin. None of the cluster formation types induced by lectins yield any noticeable effects upon the distribution of DI reactive acidic saccharides on the plasma membranes.     

Capping of saccharides on the plasma membrane of lymphocytes as studied by fluorescein-labelled lectins

The capping of saccharides on the plasma membrane of rat splenic lymphocytes was studied by means of fluorescein-labelled lectins. Treatment of unfixed splenic lymphocytes with any one of the three lectins, concanavalin A (Con A), Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) led to the formation of caps of each saccharide receptor on the plasma membrane. Treatment of unfixed lymphocytes with Con A was found to result in the formation of caps of saccharide receptors for RCA, whereas cap formations were never noted in such double treatment of the cells with all other combined uses of two lectins. These results are taken to indicate that the saccharide receptors for Con A are associated with those for RCA in the plasma membrane of rat splenic lymphocytes.     

A C-terminal carbohydrate-binding domain in the endothelial cell regulatory protein, pigpen: new function for an EWS family member

The potential for encoding information in carbohydrate (CHO) structures has long been recognized. Selective CHO-binding proteins known as lectins and the biological events they mediate are well known. However, many lectins were originally discovered for biological activities other than saccharide binding, and only subsequently was it realized that one or more of their key functions were mediated by specific CHO recognition. Our previous observations suggested that the nuclear protein pigpen had an affinity for CHO structures. This would represent a new attribute for proteins of the EWS (Ewing's sarcoma) family, of which pigpen is a member. In this study we demonstrate that a CHO-binding domain resides in the C-terminus of the molecule and can be preferentially inhibited by saccharides, most notably N-acetyl-d-galactosamine (GalNAc) and the GalNAc-containing polysaccharide, chondroitin sulfate. Ligand blotting experiments were subsequently performed with fractionated, [(3)H]galactose-labeled cells to demonstrate the presence of chondroitin sulfate-inhibitable endogenous CHO ligands for pigpen in endothelial nuclei. Finally, microinjection of polysaccharide competitor into the nucleus of cultured endothelial cells resulted in a loss of pigpen focal accumulations, suggesting that the CHO-binding activity may be instrumental in subcellular localization of the protein. In summary, our results show ligand preference and domain specificity for pigpen's CHO affinity and provide initial evidence for physiological ligands and function. They may also shed new light on the mechanisms of oncogenic transformation involving EWS proteins.     

Cell surface receptors for lymphokines. II. Studies on the carbohydrate composition of the MIF receptor on macrophages using synthetic saccharides and plant lectins.

The carbohydrate composition of the surface receptor for macrophage migration inhibitory factor (MIF) on guinea pig macrophages has been studied by examining the interaction of MIF with different saccharides and by testing the ability of plant lectins with known saccharide binding affinities to bind to macrophages and block their response to MIF. Comparison of the effectiveness of a variety of natural and synthetic mono- and disaccharides in inhibiting MIF activity in lymphocyte supernatants revealed that inhibitory activity was confined to natural 5-methylpentose sugars (l-fucose > l-rhamnose = 6-deoxy-d-glucose) and synthetic saccharides containing α-fucosyl residues. Observations on the MIF inhibitory activity of synthetic fucosyl glycosides containing fucosyl residues of defined configuration at terminal and subterminal positions indicate that MIF interacts preferentially with terminal α-l-fucopyranosyl residues and does not recognize subterminal saccharides. Studies with disaccharides containing α-(1 → 2)-, α-(1 → 3), and α-(1 → 6)-linked l-fucosyl residues failed to reveal preferential interaction of MIF with any one linkage configuration. Incubation of macrophages before exposure to MIF with lectins that bind to terminal fucosyl residues (Lotus tetragonolobus and Ulex europaeus_I, agglutinins) rendered them unresponsive to MIF but lectins which bind to nonterminal fucosyl residues and to other saccharides had no effect. The role of fucosyl residues in the binding of MIF by macrophages is discussed with reference to the possible composition of the MIF receptor and the role of fucose-containing glycolipids as receptors for this lymphokine.     

Synthesis and characterization of drug–saccharide conjugates by enzymatic strategy in organic media

A facile and efficient strategy to prepare drug derivatives with saccharides was developed, and 12 drug–saccharide conjugates were obtained by selective enzymatic synthesis methods. When the transesterification of chlorphenesin vinyl esters with glucose was chosen as a model reaction, the influence of reaction conditions was optimized. Then, we successfully prepared a series of chlorphenesin–saccharide derivatives of three monosaccharides (glucose, mannose and galactose) and three disaccharides (maltose, lactose and sucrose). In order to study the relationship between the solubility of drug–saccharide conjugates and the structure of parent drugs, five hydrophobic drugs (chlorphenesin, mephenesin, guaifenesin, propranolol and clorprenaline) were chosen as substrates to synthesize drug–saccharide conjugates. The results indicated that the aqueous solublity of drug–saccharide derivatives was greatly improved, which was changed by different saccharides and the structure of parent drugs.     

Synthesis and characterization of drug–saccharide conjugates by enzymatic strategy in organic media

A facile and efficient strategy to prepare drug derivatives with saccharides was developed, and 12 drug–saccharide conjugates were obtained by selective enzymatic synthesis methods. When the transesterification of chlorphenesin vinyl esters with glucose was chosen as a model reaction, the influence of reaction conditions was optimized. Then, we successfully prepared a series of chlorphenesin–saccharide derivatives of three monosaccharides (glucose, mannose and galactose) and three disaccharides (maltose, lactose and sucrose). In order to study the relationship between the solubility of drug–saccharide conjugates and the structure of parent drugs, five hydrophobic drugs (chlorphenesin, mephenesin, guaifenesin, propranolol and clorprenaline) were chosen as substrates to synthesize drug–saccharide conjugates. The results indicated that the aqueous solublity of drug–saccharide derivatives was greatly improved, which was changed by different saccharides and the structure of parent drugs.     

Volumetric properties for the monosaccharide (D-xylose, D-arabinose, D-glucose, D-galactose)-NaCl-water systems at 298.15 K

Densities have been measured for monosaccharide (D-xylose, D-arabinose, D-glucose and D-galactose)-NaCl-water solutions at 298.15 K. These data have been used to determine the apparent molar volumes of these saccharides and NaCl in the studied solutions. Infinite-dilution apparent molar volumes for the saccharides (V0(phi,S)) in aqueous NaCl and those for NaCl (V0(phi,E)) in aqueous saccharide solutions have been evaluated, together with the standard transfer volumes of the saccharides (delta(t) V0S) from water to aqueous NaCl and of NaCl (delta(t) V0E) from water to aqueous saccharide solutions. It is shown that the delta(t) V0S and delta (t) V0E values are positive and increase with increasing co-solute molalities. Volumetric parameters indicating the interactions of NaCl with saccharides in water have been obtained, respectively, by using transfer volumes of the saccharides and NaCl, and the resulting values are in good agreement with each other within experimental error. The interactions between saccharides and NaCl are discussed in terms of the structural interaction model and the stereochemistry of the saccharide molecules in water.     

X-ray structure of a biantennary octasaccharide-lectin complex refined at 2.3-A resolution.

The structure and flexibility of saccharides have a profound and specific influence in several biological processes such as protein protection and the maintenance of conformational integrity, and in recognition events involving viruses, enzymes, and lectins. To establish the structural bases of these phenomena, we describe herein the extensively refined 2.3-A resolution x-ray structure of a biantennary octasaccharide of the N-acetyllactosamine type, complexed to isolectin I from Lathyrus ochrus. The two octasaccharides are located in clefts at each end of the long axis of the lectin. The conformations of both the lectin and the saccharide are slightly modified upon binding. The complex is stabilized by numerous hydrogen bonds, many of them involving water molecules. It is also stabilized by van der Waals interactions, including some with aromatic residues. A more general model of a possible lectin-glycoprotein interaction is also proposed.     

Design of a covalently bonded glycosphingolipid microarray

Glycosphingolipids (GSLs) are well known ubiquitous constituents of all eukaryotic cell membranes, yet their normal biological functions are not fully understood. As with other glycoconjugates and saccharides, solid phase display on microarrays potentially provides an effective platform for in vitro study of their functional interactions. However, with few exceptions, the most widely used microarray platforms display only the glycan moiety of GSLs, which not only ignores potential modulating effects of the lipid aglycone, but inherently limits the scope of application, excluding, for example, the major classes of plant and fungal GSLs. In this work, a prototype “universal” GSL-based covalent microarray has been designed, and preliminary evaluation of its potential utility in assaying protein-GSL binding interactions investigated. An essential step in development involved the enzymatic release of the fatty acyl moiety of the ceramide aglycone of selected mammalian GSLs with sphingolipid N-deacylase (SCDase). Derivatization of the free amino group of a typical lyso-GSL, lyso-G_M1, with a prototype linker assembled from succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester and 2-mercaptoethylamine, was also tested. Underivatized or linker-derivatized lyso-GSL were then immobilized on N-hydroxysuccinimide- or epoxide-activated glass microarray slides and probed with carbohydrate binding proteins of known or partially known specificities (i.e., cholera toxin B-chain; peanut agglutinin, a monoclonal antibody to sulfatide, Sulph 1; and a polyclonal antiserum reactive to asialo-G_M2). Preliminary evaluation of the method indicated successful immobilization of the GSLs, and selective binding of test probes. The potential utility of this methodology for designing covalent microarrays that incorporate GSLs for serodiagnosis is discussed.     

Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides

Signalling by fibroblast growth factors (FGFs) through FGF receptors (FGFRs) depends on the cell-surface polysaccharide heparan sulphate (HS) [1] [2]. HS has an ordered domain structure of highly diverse saccharide motifs that present unique displays of sulphate, carboxyl and hydroxyl groups [3]. These motifs interact with many proteins, particularly growth factors. HS binds both to FGFs [4] [5] [6] and FGFRs [7], and probably activates signalling by facilitating ligand-induced receptor dimerisation [8] [9]. Nevertheless, the extent to which specific HS saccharide sequences play a regulatory role has not been established. By screening a library of structurally diverse HS decasaccharides in bioassays of FGF signalling mediated by three different FGFR isoforms, we found that saccharides showed specificity for both ligands and receptors; some saccharides selectively activated FGF signalling through different FGFR isoforms, others acted as negative regulators. We conclude that HS saccharides play critical roles in dictating the specificity of ligand-receptor interactions in FGFR signalling. Controlled alterations in HS structures [10] would provide a mechanism for regulation of cellular responsiveness to growth factors that bind HS.     

Age-correlation of protein utilization to saccharide intake

Male Wistar rats aged 30, 90, 150 and 360 days were fed ad libitum on diets with an optimum protein and fat content for their respective ages and an increasing saccharide content. Net protein utilization (NPU) was determined from the body nitrogen and protein intake values and the course of gluconeogenesis in the liver was measured by specific phosphoenolpyruvate carboxykinase (PEPCK) activity. According to the growth curve for the standard diet, animals aged 30 and 90 days have a high growth rate (3.245 g/day), 150-day-old rats grow more slowly (1.856 g/day) and 360-day-old animals put on scarcely any weight at all (277 mg/day). In 30-day-old rats, NPU attains maximum values in the presence of a 36% saccharide content in the diet, in 3- and 5-month-old animals in the presence of 51% saccharides and in one-year-old animals in the presence of 41% saccharides in their food. The course of gluconeogenesis also corresponds to these values. PEPCK activity in the youngest age group is greatest in the presence of 31% saccharides in the food, at 90 days it is stimulated in the presence of 31-46% saccharides, at 150 days the decisive concentration is 41 and 46% and at one year proteins are used for saccharide synthesis in diet with a 31 and 36% saccharide concentration. For optimum saccharide values, PEPCK activity is reduced in every age group; together with the maximum NPU values, this indicates that proteins are used for growth and building of the organism at an early age and for the renewal of tissues and organs and maintenance of the organism in adulthood.     

Clusters,bundles, arrays and lattices: novel mechanisms for lectin-saccharide-mediated cellular interactions

Multivalent protein-carbohydrate interactions regulate essential cellular events, including cell proliferation, adhesion and death. These multivalent interactions can create homogeneous complexes of lectins, such as the galectins, with their saccharide ligands. Lectin-saccharide complexes can concentrate specific glycoproteins or glycolipids within the lattice, while excluding other cell surface molecules. The formation of lectin-saccharide lattices on the cell surface can thus organize the plasma membrane into specialized domains that perform unique functions.     

Preparation and properties of metal ion derivatives of the lentil and pea lectins

Lentil lectin (LcH) and pea lectin (PSA) belong to the class of D-glucose/D-mannose binding lectins and resemble concanavalin A (Con A) closely in physicochemical, structural, and biological properties. LcH and PSA, like Con A, are Ca2+-Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. Studies of the relationship between the metal ions binding and saccharide binding activity in LcH and PSA have been difficult due to the problem of metal ion replacement in these proteins. We now report a method of metal ion replacement in both lectins that allows substitution of the Mn2+ in the native proteins with a variety of transition metal ions, as well as substitution of the Ca2+ with Cd2+ in a particular complex. The following metal ion derivatives of both LcH and PSA have been prepared: Ca2+-Zn2+, Ca2+-Co2+, Ca2+-Ni2+, and Cd2+-Cd2+. All of these derivatives are as active as the native lectins, as demonstrated by precipitation with specific polysaccharides, saccharide inhibition of precipitation, and hemagglutination assays. The yields of these derivatives are good (generally greater than 70%), and the degree of metal ion incorporation is high (generally greater than 90%). The method of preparation is quite different from that for metal ion substitution in Con A, which proceeds via the apoprotein. In contrast, the apoproteins of LcH and PSA are unstable, aggregate above pH 4.0, and cannot be remetallized once formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new kind of carbohydrate array,its use for profiling antiglycan antibodies,and the discovery of a novel human cellulose-binding antibody

In this study, we use a novel glycan array to analyze the glycan-binding antibody repertoire in a pool of affinity-purified IgG collected from a healthy human population. The glycan array used is based on mono- and oligosaccharides covalently linked to the surface via a long linker at their reducing ends. They are thus presented to the medium with a well-defined orientation and are accessible for specific binding by glycan-binding proteins, such as antibodies and lectins. A novel anticellulose antibody was detected that binds specifically to beta4-linked saccharides with a preference for glucopyranose over galactopyranose residues. We also found previously known antiglycan antibodies against mono- and oligosaccharides that are constituents of commonly occurring bacterial polysaccharides. We propose that this array can facilitate high-throughput screening of glycan-binding proteins and the search for biomarkers for personalized medicine.     

Structural analysis of Canavalia maritima and Canavalia gladiata lectins complexed with different dimannosides: new insights into the understanding of the structure-biological activity relationship in legume lectins

Plant lectins, especially those purified from species of the Leguminosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata, respectively) in the following complexes: ConM/CGL:Man(alpha1-2)Man(alpha1-O)Me, ConM/CGL:Man(alpha1-3)Man(alpha1-O)Me and ConM/CGL:Man(alpha1-4)Man(alpha1-O)Me, which crystallized in different conditions and space group from the native proteins. The structures were solved by molecular replacement, presenting satisfactory values for R(factor) and R(free). Comparisons between ConM, CGL and ConA (Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe.     

Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.     

Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.