Multivalent Interactions of Polyamide Based Sequence-Controlled Glycomacromolecules with Concanavalin A.

Binding of mannose presenting macromolecules to the protein receptor concanavalin A (ConA) is investigated by means of single-molecule atomic force spectroscopy (SMFS) in combination with dynamic light scattering and molecular modeling. Oligomeric (Mw ≈ 1.5-2.5 kDa) and polymeric (Mw ≈ 22-30 kDa) glycomacromolecules with controlled number and positioning of mannose units along the scaffolds accessible by combining solid phase synthesis and thiol-ene coupling are used as model systems to assess the molecular mechanisms that contribute to multivalent ConA-mannose complexes. SMFS measurements show increasing dissociation force from monovalent (≈57 pN) to pentavalent oligomers (≈75 pN) suggesting subsite binding to ConA. Polymeric glycomacromolecules with larger hydrodynamic diameters compared to the binding site spacing of ConA exhibit larger dissociation forces (≈80 pN), indicating simultaneous dissociation from multiple ConA binding sites. Nevertheless, although simultaneous dissociation of multiple ligands could be expected for such multivalent systems, predominantly single dissociation events are observed. This is rationalized by strong coiling of the macromolecules' polyamide backbone due to intramolecular hydrogen bonding hindering unfolding of the coil. Therefore, this study shows that the design of glycopolymers for multivalent receptor binding and clustering must consider 3D structure and intramolecular interactions of the scaffold.

Specific adhesion of carbohydrate hydrogel particles in competition with multivalent inhibitors evaluated by AFM.

Synthetic glycooligomers have emerged as valuable analogues for multivalent glycan structures in nature. These multivalent carbohydrates bind to specific receptors and play a key role in biological processes. In this work, we investigate the specific interaction between mannose ligand presenting soft colloidal probes (SCPs) attached to an atomic force microscope (AFM) cantilever and a Concanavalin A (ConA) receptor surface in the presence of competing glycooligomer ligands. We studied the SCP-ConA adhesion energy via the JKR approach and AFM pull-off experiments in combination with optical microscopy allowing for simultaneous determination of the contact area between SCP and ConA surface. We varied the contact time, loading rate and loading force and measured the resulting mannose/ConA interaction. The average adhesion energy per mannose ligand on the probe was 5 kJ/mol, suggesting that a fraction of mannose ligands presented on the SCP bound to the receptor surface. Adhesion measurements via competitive binding of the SCP in the presence of multivalent glycooligomer ligands did not indicate an influence of their multivalency on the glycooligomer displacement from the ConA surface. The absence of this "multivalency effect" indicates that glycooligomers and ConA do not associate via chelate complexes and shows that steric shielding by the glycooligomers does not slow their displacement upon competitive binding of a ligand presenting surface. These results highlight the high reversibility of carbohydrate-surface interactions, which could be an essential feature of recognition processes on the cell surface.

Fluorescence assay for glycan expression on living cancer cells based on competitive strategy coupled with dual-functionalized nanobiocomposites.

Cell surface glycans are a class of sophisticated biomolecules related to cancer development and progression, and their analysis is of great significance for early cancer diagnosis and treatment. In this paper, we proposed a fluorescence assay to evaluate glycan expression on living cancer cells based on a competitive strategy coupled with dual-functionalized nanobiocomposites. The competitive assay was conducted between living cancer cells and thiomannosyl derivatives using concanavalin A (Con A)-modified electrode as the interaction platform. To impart fluorescence signaling ability to competitive derivatives, quantum dots (QDs) were anchored on BSA-protected Au nanoparticles, and thiomannosyl derivatives were further immobilized on the nanoparticle surface through Au-S binding. Due to the spacing between QDs and Au nanoparticles by BSA, the {QDs-Au-BSA-mannose} nanobiocomposites maintained the fluorescence of QDs and showed binding ability with the Con A-modified electrode. Au nanorods (AuNRs)-modified electrode was used as an effective substrate to immobilize Con A. This assay was successfully applied to the analysis of two cancer cells lines (A549 and QGY-7701). The method is simple and shows promise for the study of glycan expression on living cancer cells.

Ligand accessibility to receptor binding sites enhanced by movable polyrotaxanes.

Functionalized polyrotaxanes are utilized to investigate the relation to multivalent interactions between the mannose moiety and Con A immobilized surfaces. According to the results of SPR spectroscopy, the mannose-conjugated polyrotaxanes show a higher response than any other mannose conjugate on both surfaces of high- and low-density Con A. Moreover, the results of the FRET analysis suggest that the mobility of α-cyclodextrins in the polyrotaxane more efficiently contributes to their binding interactions in a multivalent manner. This well-defined polyrotaxane system provides control over ligand density, ligand mobility, and gives an efficient response to the biological interaction receptor, which has not been easy to achieve in covalently bound polymeric systems.

Cell surface lectin-binding glycoconjugates on marine planktonic protists.

Carbohydrate-protein interactions appear to play an important role in the phagocytosis of microbial prey by free-living protozoa. The present study utilizes FITC-labelled plant lectins to investigate the presence and localization of cell surface glycoconjugates on live and fixed planktonic protists (Dunaliella primolecta, Oxyrrhis marina, Goniomonas amphinema, Paraphysomonas vestita and Euplotes vannus). With live flagellate preparations, lectins primarily bound to external cell surfaces, with minimal internal staining observed. In contrast, cell fixation permeabilized cell membranes, allowing lectins to bind to internal structures, such as nuclear membranes and food vacuoles, interfering with the characterization of cell surface glycoconjugates. The method developed to label cell surface sugar moieties of live planktonic protists successfully overcomes the problems associated with fixation, and thus provides a useful protocol for future studies on protistan cell surface carbohydrate characterization.

Analysis of lectin-bound glycoproteins in snake venom from the Elapidae and Viperidae families.

This paper describes an efficient method of studying the glycoproteins found in snake venom. The glycosylation profiles of the Elapidae and Viperidae snake families were analyzed using FITC-labeled lectin glycoconjugates. The Con A-agarose affinity enrichment technique was used to fractionate glycoproteins from the N. naja kaouthia venom. The results revealed a large number of Con A binding glycoproteins, most of which have moderate to high molecular weights. To identify the proteins, the isolated glycoprotein fractions were subjected to two-dimensional electrophoresis and MALDI-TOF MS. Protein sequences were compared with published protein databases to determine for their biological functions.

Supramolecular design for multivalent interaction: maltose mobility along polyrotaxane enhanced binding with concanavalin A.

High molecular mobility of maltose-conjugated alpha-cyclodextrins (alpha-CDs) along a poly(ethylene glycol) (PEG) chain due to the mechanically locked structure of polyrotaxanes enhanced multivalent interactions between maltose and concanavalin A (Con A). When maltose groups are conjugated with alpha-CDs that were threaded onto a PEG capped with benzyloxycarbonyl l-tyrosine (polyrotaxane), Con A-induced hemagglutination was greatly inhibited by polyrotaxanes with a certain threading % of alpha-CDs. Such an inhibitory effect was significantly superior to the other type of conjugates, in which poly(acrylic acid) was used as a backbone for maltose conjugation. The spin-spin relaxation time (T2) of the maltose C(1) proton in the polyrotaxane at a typical alpha-CD threading % was significantly larger than that of any other conjugate, which was well related to the inhibitory effect. Therefore, we concluded that the high mobility of maltose groups along the polyrotaxane structure contributes to enhanced Con A recognition.

Control of multivalent interactions by binding epitope density.

Receptor clustering by multivalent ligands can activate signaling pathways. In principle, multivalent ligand features can control clustering and the downstream signals that result, but the influence of ligand structure on these processes is incompletely understood. Using a series of synthetic polymers that vary systematically, we studied the influence of multivalent ligand binding epitope density on the clustering of a model receptor, concanavalin A (Con A). We analyze three aspects of receptor clustering: the stoichiometry of the complex, rate of cluster formation, and receptor proximity. Our experiments reveal that the density of binding sites on a multivalent ligand strongly influences each of these parameters. In general, high binding epitope density results in greater numbers of receptors bound per polymer, faster rates of clustering, and reduced inter-receptor distances. Ligands with low binding epitope density, however, are the most efficient on a binding epitope basis. Our results provide insight into the design of ligands for controlling receptor-receptor interactions and can be used to illuminate mechanisms by which natural multivalent displays function.

Proton positions in the Mn(2+) binding site of concanavalin A as determined by single-crystal high-field ENDOR spectroscopy.

High-field (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) techniques have been used for the first time to determine coordinates of ligand protons of a high-spin metal center in a protein single crystal. The protein concanavalin A contains a Mn(2+) ion which is coordinated to two water molecules, a histidine residue, and three carboxylates. Single crystals of concanavalin A were grown in H(2)O and in D(2)O to distinguish the exchangeable water protons from the nonexchangeable protons of the imidazole group. Distinct EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at 4.5 K and a magnetic field of approximately 3.4 T allowed us to assign the ENDOR signals to their respective M(S) manifolds, thus providing the signs of the hyperfine couplings. Rotation patterns were acquired in the ac and ab crystallographic planes. Two distinct crystallographic sites were identified in each plane, and the hyperfine tensors of two of the imidazole protons and the four water protons were determined by simulations of the rotation patterns. All protons have axially symmetric hyperfine tensors and, by applying the point-dipole approximation, the positions of the various protons relative to the Mn(2+) ion were determined. Likewise, the water protons involved in H-bonding to neighboring residues were identified using the published, ultrahigh-resolution X-ray crystallographic coordinates of the protein (Deacon et al. J. Chem. Soc., Faraday Trans. 1997, 93(24), 4305-4312).

Influence of cholesterol crystallization effector proteins on vesicle fusion in supersaturated model bile.

BACKGROUND: In lithogenic bile, cholesterol-rich vesicles rapidly aggregate and fuse to eventually form cholesterol crystals. This process is modulated by cholesterol crystallization effector substances. In this study, we developed a method for quantitative assessment of vesicle fusion and used it to partly characterize the mechanisms of action of cholesterol crystallization effector proteins. METHODS: Cholesterol:phospholipid (1:1) liposomes were prepared and labelled with octadecyl rhodamine B chloride (R18). Fusion of these liposomes was detected by the increase of R18 fluorescence after incubation with various proteins, such as albumin, concanavalin-A bound glycoprotein, immunoglobulins, apolipoprotein A-I and apolipoprotein B (all at 100 microg/mL). RESULTS: Fusion of cholesterol/phospholipid liposomes was increased by 16 and 14% in the presence of concanavalin-A bound glycoprotein and immunoglobulins, respectively, and decreased by 21 and 9% after addition of apolipoprotein A-I and apolipoprotein B, respectively. The effect of each protein on vesicle fusion was correlated with its hydrophobicity. CONCLUSIONS: These results suggest that nucleation effector proteins modulate the stability of vesicles and, thus, affect cholesterol crystallization. Such modulation is based upon protein-vesicle association, which defines the physico-chemical metastability of vesicular cholesterol.

Quantitative assessment of comparative potencies of cholesterol-crystal-promoting factors: relation to mechanistic characterization.

The crystallization of cholesterol is affected by various factors in bile. The present study evaluated the relative importance of cholesterol-nucleation-promoting factors and partially characterized the mechanisms of their action. Model biles with an identical relative composition of cholesterol, egg-yolk phosphatidylcholine and taurocholate, except for replacing phosphatidylcholine (5-20%) with dilinoleoyl-phosphatidylcholine or taurocholate (10-30%) with taurodeoxycholate. Cholesterol crystallization was quantitatively assessed spectrophotometrically and morphologically estimated by the laser-scattering diffraction analyser and video-enhanced microscopy in the absence and presence of concanavalin A-binding glycoprotein isolated from human bile. In a series of experiments, lipid distribution among particulate species was determined after isolation by FPLC. In all experiments, cholesterol crystallization was dose-dependently enhanced with a rank order of: concanavalin A-binding glycoprotein > dilinoleoyl - phosphatidyl choline> taurodeoxycholate. No morphological alteration was evident for vesicles and crystals, but the cholesterol/phospholipid ratio in vesicles was increased significantly by replacement with dilinoleoyl-phosphatidylcholine and excess cholesterol. A high proportion of relatively hydrophilic phosphatidylcholine species such as dilinoleoyl-phosphatidylcholine and excess cholesterol in bile cause a redistribution of cholesterol to increase a vesicular cholesterol/phospholipid ratio, eventually promoting cholesterol crystallization, whereas concanavalin A-binding glycoprotein acts via differing mechanisms.

Characterization of concanavalin A-binding glycoproteins from procyclic culture forms of Trypanosoma congolense, T. simiae and T. brucei brucei.

Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) of Trypanosoma congolense, T. simiae, and T. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCF T. congolense K45/1 identified surface-accessible glycoproteins of 81.5, 59, and 38-42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high-mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies.

The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: excretion and susceptibility to proteolysis.

Biochemical properties of the concanavalin A-binding 43-kDa glycoprotein (gp43) of Paracoccidioides brasiliensis and its deglycosylated form were compared. Deglycosylation was achieved by treatment with trifluoromethanesulfonic acid, endoglycosidase H, N-glycanase, or metabolically, by growing cells with tunicamycin. The resulting antigen in all cases had Mr 38,000, and probably derived from the gp43 by loss of N-linked high-mannose oligosaccharide chains. The presence of galactopyranose units in the carbohydrate chains was suggested by antigen binding to peanut lectin. Pulse and chase experiments using [35S]methionine metabolic labeling of P. brasiliensis growing in the presence of tunicamycin showed that the N-linked chains of gp43 are not required for antigen secretion. The 38-kDa antigen was more susceptible than the native antigen to the action of papain and pronase, thus indicating a protective role of the carbohydrate moiety against proteolysis. Both forms are equally resistant to endogenous proteases at neutral pH. The gp43, itself, has a proteolytic activity at pH 5-6, but not at neutral pH. Deglycosylation with endoglycosidase H or tunicamycin preserved epitopes in the 38-kDa molecule reactive with (a) antibodies from patients with paracoccidioidomycosis, or rabbit immunized with the gp43 and (b) mouse monoclonal antibodies against the gp43 antigen. The present results provide a basis for the understanding of diagnostic reactions and fungal virulence involving the gp43 exocellular antigen of P. brasiliensis.

Isolation and incorporation into lipid vesicles of a concanavalin A receptor from human erythrocytes.

Affinity chromatography has been used to isolate a concanavalin A receptor portion of Band 3 from humen erythrocytes in the presence of the readily-dialysable detergent, dodecyltrimethylammonium bromide. Addition of phospholipids to the isolated fraction and removal of detergent by dialysis leads to formation of vesicles containing the receptor. Intramembranous particles similar in size and shape to those seen in intact erythrocytes are a characteristic of the reconstituted preparations. Vesicles containing receptor bind concanavalin A with high affinity.