Sugar Interaction Research Articles

Reconciliation of opposing views on membrane–sugar interactions

It is well established that small sugars exert different types of stabilization of biomembranes both in vivo and in vitro. However, the essential question of whether sugars are bound to or expelled from membrane surfaces, i.e., the sign and size of the free energy of the interaction, remains unresolved, and this prevents a molecular understanding of the stabilizing mechanism. We have used small-angle neutron scattering and thermodynamic measurements to show that sugars may be either bound or expelled depending on the concentration of sugar. At low concentration, small sugars bind quite strongly to a lipid bilayer, and the accumulation of sugar at the interface makes the membrane thinner and laterally expanded. Above ∼0.2 M the sugars gradually become expelled from the membrane surface, and this repulsive mode of interaction counteracts membrane thinning. The dual nature of sugar-membrane interactions offers a reconciliation of conflicting views in earlier reports on sugar-induced modulations of membrane properties.

Self-assembly of trehalose molecules on a lysozyme surface: the broken glass hypothesis

To help understand how sugar interactions with proteins stabilise biomolecular structures, we compare the three main hypotheses for the phenomenon with the results of long molecular dynamics simulations on lysozyme in aqueous trehalose solution (0.75 M). We show that the water replacement and water entrapment hypotheses need not be mutually exclusive, because the trehalose molecules assemble in distinctive clusters on the surface of the protein. The flexibility of the protein backbone is reduced under the sugar patches supporting earlier findings that link reduced flexibility of the protein with its higher stability. The results explain the apparent contradiction between different experimental and theoretical results for trehalose effects on proteins.

Interaction of glycated human serum albumin with endothelial cells in a hemodynamic environment: structural and functional correlates

Advanced glycation end products (AGEs) are known to be involved in the pathogenesis of several diseases, in particular diabetes, via signaling through their receptor. Numerous studies have been carried out on protein-sugar interactions at very high concentrations of the latter. The objective of this investigation was to determine the effects of nonenzymatic glycation induced by reducing sugars on the secondary structure of human serum albumin (HSA) under different physiological conditions and to correlate that with expression of RAGE (receptor for advanced glycation end products) on HUVECs (human umbilical vein endothelial cells) in a controlled hemodynamic environment. Our results indicate that RAGE expression is shear stress modulated and that glycated HSA enhances the expression further. The secondary structure of AGE-HSA derived from glucose at 20 mM contains higher α-helical content and elicits maximum expression of the receptor. The effect of shear stress at 10 dynes cm(-2) is independent of AGE-HSA.

Purification and characterization of an Anti-proliferative and mitogenic plant lectin from tubers of Arisaema speciosum

A novel lectin having specificity for N-acetyllactosamine was purified from the tubers of Arisaema speciosum Martius (family: Araceae) by affinity chromatography on asialofetuin-linked amino activated silica beads. The lectin have four identical subunits of 13.5 kDa each and native molecular mass of 51.2 kDa. ASL agglutinated rabbit, rat, sheep and guinea pig erythrocytes but human ABO blood group erythrocytes were not even after neuraminidase treatment. ASL gave as single band on native PAGE, pH 4.5 and multiple bands in isoelectric focusing and at PAGE 8.3. ASL is a glycoprotein with 0.7% carbohydrate content, stable up to 50°C and at pH 2 to 10. The lectin had no requirement for divalent cations and was stable up to 4M Urea, 3M thiourea and 3M Guanidine-HCl. ASL was rich in acidic amino acids but cysteine was completely absent. Amino acid modification revealed the involvement of tryptophan and tyrosine residues in lectin sugar interaction. The secondary structure estimated in far UV CD spectrum was 38% α-helix, 27% β-sheet and 35% random contributions. ASL showed mitogenic potential towards human peripheral blood mononuclear cell. ASL produced more that 50% growth inhibition of seven human cancer cell lines viz. SW-620, IMR-32, PC-3, SK-N-SH, Colo-205, HCT-15 and HT-29 at 100 μg/ml out of eleven cell lines used.

Sugar signalling and antioxidant network connections in plant cells

Sugars play important roles as both nutrients and regulatory molecules throughout plant life. Sugar metabolism and signalling function in an intricate network with numerous hormones and reactive oxygen species (ROS) production, signalling and scavenging systems. Although hexokinase is well known to fulfil a crucial role in glucose sensing processes, a scenario is emerging in which the catalytic activity of mitochondria-associated hexokinase regulates glucose-6-phosphate and ROS levels, stimulating antioxidant defence mechanisms and the synthesis of phenolic compounds. As a new concept, it can be hypothesized that the synergistic interaction of sugars (or sugar-like compounds) and phenolic compounds forms part of an integrated redox system, quenching ROS and contributing to stress tolerance, especially in tissues or organelles with high soluble sugar concentrations.

Impact of sucrose level on storage stability of proteins in freeze-dried solids: I. correlation of protein–sugar interaction with native structure preservation

The purpose of this study is to investigate protein–sugar interactions in dried protein solids as a function of sucrose level using water sorption isotherm data and secondary structure information from Fourier transform infrared (FTIR) spectroscopy. Three IgG1 fusion proteins and two cytokines were freeze-dried with sucrose at different sucrose/protein mass ratios. The water monolayer of the colyophilized sucrose/protein samples, as determined by BET analysis of water sorption data, was found to be lower than that expected based on additive contributions of pure protein and pure sucrose. This negative deviation suggests the presence of a solid-state interaction between protein and sucrose that reduces the availability of total water-binding sites. The difference in water monolayer between colyophilized and a physical mixture of protein and sucrose reached a maximum value at sucrose/protein mass ratio of 1/1 for these proteins, suggesting saturation of the protein–sugar interaction at this ratio. In addition, for four proteins studied, the normalized peak height of the major band in the FTIR spectra reached a plateau at about a 1/1 mass ratio. Therefore, it appears that there is a coupling between the preservation of protein secondary structure and the protein–sugar interaction as measured by water sorption isotherms. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3131–3144, 2009

Structure of the Murine Unglycosylated IgG1 Fc Fragment

A prototypic IgG antibody can be divided into two major structural units: the antigen-binding fragment (Fab) and the Fc fragment that mediates effector functions. The IgG Fc fragment is a homodimer of the two C-terminal domains (CH2 and CH3) of the heavy chains. Characteristic of the Fc part is the presence of a sugar moiety at the inner face of the CH2 domains. The structure of this complex branched oligosaccharide is generally resolved in crystal structures of Fc fragments due to numerous well-defined sugar–protein interactions and a small number of sugar–sugar interactions. This suggested that sugars play an important role in the structure of the Fc fragment. To address this question directly, we determined the crystal structure of the unglycosylated Fc fragment of the murine IgG1 MAK33. The structures of the CH3 domains of the unglycosylated Fc fragment superimpose perfectly with the structure of the isolated MAK33 CH3 domain. The unglycosylated CH2 domains, in contrast, approach each other much more closely compared to known structures of partly deglycosylated Fc fragments with rigid-body motions between 10 and 14 Å, leading to a strongly “closed” conformation of the unglycosylated Fc fragment. The glycosylation sites in the C′E loop and the BC and FG loops are well defined in the unglycosylated CH2 domain, however, with increased mobility and with a significant displacement of about 4.9 Å for the unglycosylated Asn residue compared to the glycosylated structure. Thus, glycosylation both stabilizes the C′E-loop conformation within the CH2 domain and also helps to ensure an “open” conformation, as seen upon Fc receptor binding. These structural data provide a rationale for the observation that deglycosylation of antibodies often compromises their ability to bind and activate Fcγ receptors.

The effect of MgCl2 on the kinetics of the Maillard reaction in both aqueous and dehydrated systems

Abstract The modification of Maillard reaction kinetics induced by MgCl 2 was evaluated in both liquid and dehydrated model systems with special emphasis on the interactions of the salt with water and/or the sugars. In liquid trehalose systems, browning is accelerated by the presence of MgCl 2 due to the increased sugar hydrolysis and to the reduction of water mobility caused by the salt (shown by the decrease in 1 H NMR relaxation times T 2 ), counteracting the inhibitory effect of water on the reaction. In water restricted trehalose systems, MgCl 2 inhibited the Maillard reaction. The salt–sugar interactions, manifested by the delayed sugar crystallization, decreased the reaction rate by affecting the reactivity of reducing sugars. Molecular and supramolecular effects in the presence of MgCl 2 have been observed in the present work, and must be taken into account considering high technological interest in finding strategies to either inhibit or enhance the Maillard reaction depending on the application.

NMR studies on the interaction of sugars with the C‐terminal domain of an R‐type lectin from the earthworm Lumbricus terrestris

The R-type lectin EW29, isolated from the earthworm Lumbricus terrestris, consists of two homologous domains (14,500 Da) showing 27% identity with each other. The C-terminal domain (Ch; C-half) of EW29 (EW29Ch) has two sugar-binding sites in subdomains alpha and gamma, and the protein uses these sugar-binding sites for its function as a single-domain-type hemagglutinin. In order to determine the sugar-binding ability and specificity for each of the two sugar-binding sites in EW29Ch, ligand-induced chemical-shift changes in EW29Ch were monitored using (1)H-(15)N HSQC spectra as a function of increasing concentrations of lactose, melibiose, D-galactose, methyl alpha-D-galactopyranoside and methyl beta-D-galactopyranoside. Shift perturbation patterns for well-resolved resonances confirmed that all of these sugars associated independently with the two sugar-binding sites of EW29Ch. NMR titration experiments showed that the sugar-binding site in subdomain alpha had a slow or intermediate exchange regime on the chemical-shift timescale (K(d) = 10(-2) to 10(-1) mM), whereas that in subdomain gamma had a fast exchange regime for these sugars (K(d) = 2-6 mM). Thus, our results suggest that the two sugar-binding sites of EW29Ch in the same molecule retain its hemagglutinating activity, but this activity is 10-fold lower than that of the whole protein because EW29Ch has two sugar-binding sites in the same molecule, one of which has a weak binding mode.

Molecular interaction of imino sugars with human α-galactosidase: Insight into the mechanism of complex formation and pharmacological chaperone action in Fabry disease

Enzyme enhancement therapy (EET) for Fabry disease involving imino sugars has been developed and attracted interest. It is thought that imino sugars act as pharmacological chaperones for wild-type and mutant α-galactosidases (GLAs) in cells, but the mechanisms underlying the molecular interactions between the imino sugars and the enzyme have not been clarified yet. We examined various kinds of imino sugars and found that galactostatin bisulfite (GBS) inhibited GLA in vitro and increased the enzyme activity in cultured Fabry fibroblasts as in the case of 1-deoxygalactonojirimycin (DGJ). Then, we analyzed the molecular interactions between the imino sugars and recombinant human GLA by means of isothermal titration calorimetry and surface plasmon resonance biosensor assays, and first determined the thermodynamic and binding-kinetics parameters of imino sugar and GLA complex formation. The results revealed that DGJ bound to the enzyme more strongly than GBS, the binding of DGJ to the enzyme protein being enthalpy-driven. In the case of GBS, the reaction was mainly enthalpy-driven, but there was a possibility that entropy-driven factors were involved in the binding. Structural analysis in silico revealed that both the chemicals fit into the active-site pocket and undergo hydrogen bonding with residues comprising the active-site pocket including the catalytic ones. The side chain of GBS was oriented towards the entrance of the active-site pocket, and thus it could be in contact with residues comprising the wall of the active-site pocket. Thermodynamic, kinetic and structural studies should provide us with a lot of information for improving EET for Fabry disease.

Preparation of Cellobiose-conjugated Polyacrylamide and its Interaction with a Cellulose Matrix for Papermaking Application

Cellobiose, a structural subunit of cellulose, was utilized as a retention promoter of polymer additives to a cellulose matrix in an aqueous papermaking system. beta-D-Cellobiosylamine was conjugated with anionic polyacrylamide (A-PAM) through a condensation reaction using water-soluble carbodiimide. Glyco-modified A-PAM containing cellobiose residues (CB-A-PAM), which was added to a pulp suspension using tap water, had good retention efficiency and enhanced the strength of paper sheets. Surface plasmon resonance analysis gave useful information on the affinity of CB-A-PAM for a cellulose matrix. Cellobiose and A-PAM had negligible interaction with cellulose, but CB-A-PAM was significantly adsorbed on the cellulose surface. It was presumed that the many cellobiose moieties in one CB-APAM molecule cooperatively enhanced its affinity for cellulose. Such sugar−sugar interaction is a novel concept for the retention system of papermaking additives and the functional design of carbohydrate-based materials.

C-terminal Loop 13 of Na+/Glucose Cotransporter 1 Contains Both Stereospecific and Non-stereospecific Sugar Interaction Sites

To investigate whether the C-terminal loop 13 of rabbit sodium/glucose cotransporter SGLT1 is involved in the recognition of the substrate d-glucose, isolated loop 13 (amino acids (aa) 541-638) was immobilized to a lipid bilayer. Interactions were investigated by surface plasmon resonance spectroscopy using an antibody directed against the late part of the loop (aa 606-631) or the glucoside transport inhibitor phlorizin. Specific binding of the antibody to the loop could be detected. The number of bound antibodies decreased upon the addition of d-glucose but not upon the addition of l-glucose. Phlorizin also significantly lowered the number of bound antibodies. Binding of phlorizin to the loop could also be demonstrated directly. Binding of phlorizin was, however, reduced to a similar extent upon the addition of either d-glucose or l-glucose, indicating their unspecific competition with the inhibitor's sugar moiety. Thus, the presence of a stereospecific glucose interaction site in the late part of the loop and a second, but non-stereospecific, sugar binding site on the same loop was assumed. To investigate whether the early part of loop 13 contains this non-stereospecific sugar binding site, peptides containing aa 541-598 were expressed in Escherichia coli and purified. Both d-glucose and l-glucose quenched the peptides tryptophan fluorescence and reduced the Trp accessibility to acrylamide to a similar degree. In view of the recently proposed transmembrane orientation of loop 13, the two binding sites may be part of the extracellular (stereospecific) and intracellular (non-stereospecific) sugar interaction sites of SGLT1.

Interaction of Volatiles, Sugars, and Acids on Perception of Tomato Aroma and Flavor Descriptors

To better understand the effect of sugars and acid levels on perception of aroma volatiles, intensity of tomato earthy/medicinal/musty, green/grassy/viney, and fruity/floral aroma and flavor descriptors were evaluated using coarsely chopped partially deodorized tomato puree. This puree was spiked with 1.5% to 3% sugar (glucose/fructose combinations), 0.1% to 0.2% acid (citric/malic acid combinations), or water and 2 levels of 12 individual food-grade volatiles reported to contribute to tomato flavor. A panel consisting of 6 to 8 trained members rated 9 aroma, 8 taste, and 1 aftertaste descriptors of the spiked and nonspiked purees. The panelists detected significant differences (P < or = 0.1) for various individual aroma compound/sugar/acid combinations for a range of descriptors. Adding 0.2% acids alone to bland tomato puree decreased green and floral aromas as well as sweet taste. Adding 3% sugars alone increased green and musty aromas and decreased floral aroma as well as sour, citrus, and bitter tastes. Principal component analysis (PCA) explained 56.5% of the variation in the first 3 principal components (PCs) for added acids and volatiles to bland tomato puree. The effect of added acids with the various aroma compounds generally increased perception of overall and ripe tomato taste and aroma, tropical aroma, and sour taste, and decreased sweet, fruity, and bitter tastes. PCA for added sugars with volatiles explained 67.8% of the variation in first 3 PCs, and sugars generally decreased perception of sour, bitter, and citrus tastes and green aroma, while enhancing perception of flavors associated with ripe, tropical, and aromatic tomatoes. Adding sugars, acids, and volatiles together had a similar effect to addition of sugars alone.

Electrochemical evaluation of lectin–sugar interaction on gold electrode modified with colloidal gold and polyvinyl butyral

In this work, ConA and CramoLL lectins were immobilized on gold nanoparticles (AuNp) with polyvinyl butyral (PVB), and adsorbed on the surface of gold (Au) electrodes. Electrochemical impedance spectroscopy (EIS), in the frequency range from 100 mHz to 100 KHz, and cyclic voltammetry (CV), from −0.2 to 0.7 V, were performed on these electrodes, in phosphate buffer (PBS) solution containing 10 mM K 3[Fe(CN) 6]/K 4[Fe(CN) 6] (1:1) mixture as a redox probe. EIS and CV measurements showed that redox probe reactions on the modified Au electrodes were partially blocked due to the adsorption of AuNp-ConA-PVB and AuNp-CramoLL-PVB. SEM images showed the presence of aggregates of AuNp-ConA on PVB spherules in a tridimensional structure on the surface of the Au electrode. Bovine serum albumin (BSA) was adsorbed on the AuNp-Lectin-PVB modified electrode in order to block the remaining free gold sites. Both EIS and CV techniques yielded results that confirm positive responses of the lectins to ovalbumin agglutination. These results indicate an improvement of the sensitivity for detection of sugars that can be applicable to construction of a biosensor sensitive to glycoproteins in solution.

Boronate-containing polymers form affinity complexes with mucin and enable tight and reversible occlusion of mucosal lumen by poly(vinyl alcohol) gel

Copolymers of N-acryloyl- m-aminophenylboronic acid (NAAPBA) with N, N-dimethylacrylamide (DMAA) formed insoluble interpolymer complexes with mucin from porcine stomach at pH 9.0. The complex formation based on boronate–sugar interactions took place between the similarly charged macromolecules and resulted in coacervate particles formation, which depended both on pH and ionic strength of the solution. The coacervation rate displayed a maximum at the intermediate DMAA-NAAPBA copolymer: mucin weight ratio, that is a pattern typical of interpolymer complex formation. The effective hydrodynamic particle diameter of the coacervates monotonously grew from 155 ± 20 nm up to 730 ± 120 nm in 2 days in 0.1 M sodium bicarbonate buffer solution, pH 9.0. Electrophoretic mobility of the resultant nanoparticles was intermediate between those of individual polymers, whereas the particles zeta-potential was −7.5 ± 0.4 mV in the above buffer solution. Pre-treatment of the inner mucosal epithelium of excised male pig urethras with 5% (w/v) solutions of acrylamide-NAAPBA or DMAA-NAAPBA copolymers at pH 8.8 allowed for tight occlusion of the lumen by poly(vinyl alcohol) – borax gel injected via a two-way catheter. Leakage of 0.15 M NaCl solution through the thus occluded organs could be prevented, while the leakage through the organs occluded by the gel without the pre-treatment was unavoidable. The gel plug could be quickly dissolved on demand after injection of 5% (w/v) aqueous fructose solution into the lumen. The described technique may be useful for temporal occlusion of mucosal lumens in living organisms. In contrast to the conventional mucoadhesive polymers like polyacrylic acid or chitosan, the boronate-containing copolymers display their mucoadhesivity at weakly alkaline pH of 8–9 and physiological ionic strength.

Kinetic hybrid i-motifs: Intercepting DNA with RNA to form a DNA 2–RNA 2 i-motif

We have constructed and characterized a long-lived hybrid DNA 2–RNA 2 i-motif that is kinetically formed by mixing equivalent amount of C-rich RNA ( R) and C-rich DNA ( D). Circular dichroism shows that these hybrids are distinct from their parent DNA 4 or RNA 4 i-motif. pH dependent CD and UV thermal melting experiments showed that the complexes were maximally stable at pH 4.5, the p K a of cytosine, consistent with the complex being held by CH +–C base pairs. Fluorescence studies confirmed their tetrameric nature and established the relative strand polarities of the RNA and DNA strands in the complex. These showed that in a hybrid D 2 R 2 i-motif two DNA strands occupy one narrow groove and the two RNA strands occupy the other. This suggests that even the sugar–sugar interactions are highly specific. Interestingly, this hybrid slowly disproportionates into DNA 4 i-motifs and ssRNA which would be valuable to study intermediates in DNA 4 i-motif formation.

Inihbition of Protein Glycation with Varying Concentrations of Lysine

The causes of the complications of diabetes mellitus have been explored in ongoing studies in our laboratory using an In Vitro system. This system is used to screen compounds for their ability to inhibit the glycation of proteins by sugars. Vascular damage seen in diabetic patients is impacted by such protein sugar interaction. During this study it was found that lysine is an effective inhibitor of glycation. The inhibition of glycation was studied using a spectrophotometric assay for glycoproteins. Studies indicated lysine as most effective as an inhibitor of glycation at a concentration of 100 μg/ml. Lower levels of inhibition were observed when a concentration of 500 μg/ml was used. When the concentration was lowered to 50 μg/ml and 200 μg/ml the results showed the inhibition of glycation was roughly equal at each respective concentration. Further research is being performed to find the actual concentration at which the lysine inhibition no longer performs efficiently. Preliminary results indicated that further investigation is needed to determine the mechanism of inhibition and the possible use of lysine in the treatment of diabetic patients.This research project was funded by the Smith‐Glynn‐Callaway Medical Foundation.

Atomic Mapping of the Sugar Interactions in One-Site and Two-Site Mutants of Cyanovirin-N by NMR Spectroscopy

The details of the interaction between two mutants of Cyanovirin-N (CV-N), an HIV inactivating protein, and di- and trimannosides, substructures of Man-9, were investigated by STD NMR spectroscopy. One mutant, CV-N (mutDB), contains only one carbohydrate-binding site on domain A, whereas in CV-N (mutDA), the specificity of domain A for trimannose was changed while the site in domain B was kept intact, allowing for a dissection of the overall binding. Results of the STD NMR experiments revealed close contact between the protein binding site on domain A and H2, H3, and H4 of the nonreducing terminal mannose unit for Manalpha(1-2)Manalpha OMe, Manalpha(1-2)Manalpha(1-3)Manalpha OMe, and Manalpha(1-2)Manalpha(1-6)Manalpha OMe. The Manalpha(1-2)Manalpha(1-2)Manalpha OMe trisaccharide interacted with CV-N with the highest affinity. Further dissection of the interaction was achieved by NMR experiments with synthetic 2'-, 3'-, 4'-, and 6'-deoxy analogues of the disaccharide Manalpha(1-2)Manalpha OMe. STD and (1)H- (15)N HSQC NMR spectroscopy revealed that the 2'- and 6'-deoxy dimannosides were recognized by CV-N, whereas no binding was detected for the 3'- and 4'-deoxy sugars. These results demonstrate that the 3'- and 4'-hydroxyl groups on the terminal residue are engaged in key polar interactions with the protein and are required for high-affinity binding.

Binding parameters and thermodynamics of the interaction of imino sugars with a recombinant human acid α-glucosidase (alglucosidase alfa): Insight into the complex formation mechanism

BackgroundRecently, enzyme enhancement therapy (EET) for Pompe disease involving imino sugars, which act as potential inhibitors of acid α-glucosidases in vitro, to improve the stability and/or transportation of mutant acid α-glucosidases in cells was studied and attracted interest. However, the mechanism underlying the molecular interaction between the imino sugars and the enzyme has not been clarified yet. MethodsWe examined the inhibitory and binding effects of four imino sugars on a recombinant human acid α-glucosidase, alglucosidase alfa, by means of inhibition assaying and isothermal titration calorimetry (ITC). Furthermore, we built structural models of complexes of the catalytic domain of the enzyme with the imino sugars bound to its active site by homology modeling, and examined the molecular interaction between them. ResultsAll of the imino sugars examined exhibited a competitive inhibitory action against the enzyme, 1-deoxynojirimycin (DNJ) exhibiting the strongest action among them. ITC revealed that one compound molecule binds to one enzyme molecule and that DNJ most strongly binds to the enzyme among them. Structural analysis revealed that the active site of the enzyme is almost completely occupied by DNJ. ConclusionThese biochemical and structural analyses increased our understanding of the molecular interaction between a human acid α-glucosidase and imino sugars.

The Crystal Structure of the Complex of Bla g 2 with an Antibody Fragment Reveals Cation-pi and Sugar Interactions

The Crystal Structure of the Complex of Bla g 2 with an Antibody Fragment Reveals Cation-pi and Sugar Interactions