The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: Carbohydrate–protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate–protein interactions. We investigated whether carbohydrate–lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp–GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.     

Biochemistry of carbohydrate-protein interaction.

Recognition of glycoconjugates is an important event in biological systems, and is frequently in the form of carbohydrate-protein interactions. To thoroughly understand these interactions, well-defined carbohydrate ligands must be available. Naturally derived glycoconjugates can be highly purified, and their structures (including conformational structures) can be elucidated to provide such ligands. This requires highly effective methods of separation, such as various forms of high-performance liquid chromatography. Alternatively, structurally well-defined glycoconjugates can be synthesized for this purpose. These include conjugates of carbohydrate derivatives to proteins, lipids, and nonbiological carriers and polymers. The efficacy of these conjugates is amply demonstrated in the studies of carbohydrate-binding proteins from animals. Hepatic carbohydrate receptors, requiring calcium for binding, recognize only the terminal sugar residues. Although different sugar specificities are manifested by different species, there is some commonality in the requirement of the substituents of the sugar rings. Clustering of the target sugars in proper geometric arrangement greatly enhances the binding by these proteins. Some other animal carbohydrate-binding proteins, however, may require penultimate sugars for optimal binding.     

Effects of clustered epitopes in multivalent ligand-receptor interactions

Many biological ligands are composed of clustered binding epitopes. However, the effects of clustered epitopes on the affinity of ligand-receptor interactions in many cases are not well understood. Clustered carbohydrate epitopes are present in naturally occurring multivalent carbohydrates and glycoproteins, which are receptors on the surface of cells. Recent studies have provided evidence that the enhanced affinities of lectins, which are carbohydrate binding proteins, for multivalent carbohydrates and glycoproteins are due to internal diffusion of lectin molecules from epitope to epitope in these multivalent ligands before dissociation. Indeed, binding of lectins to mucins, which are large linear glycoproteins, appears to be similar to the internal diffusion mechanism(s) of protein ligands binding to DNA, which have been termed the "bind and slide" or "bind and hop" mechanisms. The observed increasing negative cooperativity and gradient of decreasing microaffinity constants of a lectin binding to multivalent carbohydrates and glycoproteins result in an initial fraction of lectin molecules that bind with very high affinity and dynamic motion. These findings have important implications for the mechanisms of binding of lectins to mucins, and for other ligand-biopolymer interactions and clustered ligand-receptor systems in general.     

Facile and rapid direct gold surface immobilization with controlled orientation for carbohydrates

Effective surface immobilization is a prerequisite for numerous carbohydrate-related studies including carbohydrate-biomolecule interactions. In the present work, we report a simple and rapid modification technique for diverse carbohydrate types in which direct oriented immobilization onto a gold surface is accomplished by coupling the amine group of a thiol group-bearing aminophenyl disulfide as a new coupling reagent with an aldehyde group of the terminal reducing sugar in the carbohydrate. To demonstrate the generality of this proposed reductive amination method, we examined its use for three types of carbohydrates: glucose (monosaccharide), lactose (disaccharide), and GM1 pentasaccharide. Through successful mass identifications of the modified carbohydrates, direct binding assays on gold surface using surface plasmon resonance and electrochemical methods, and a terminal galactose-binding lectin assay using atomic force microscopy, we confirmed several advantages including direct and rapid one-step immobilization onto a gold surface and exposure of functional carbohydrate moieties through oriented modification of the terminal reducing sugar. Therefore, this facile modification and immobilization method can be successfully used for diverse biomimetic studies of carbohydrates, including carbohydrate-biomolecule interactions and carbohydrate sensor or array development for diagnosis and screening.     

Glycoconjugates for the recognition of Bacillus spores

Carbohydrates act as ligands in many biological processes, including the folding and secretion of proteins, cell-cell recognition, adhesion, and sporulation in the Bacillus genus. Fluorescent-labeled disaccharide glycoconjugates have been applied to evaluate binding to bacterial spores assuming that the spore surface is covered with carbohydrates. This study has shown that specific recognition of bacterial spores is based on interactions between disaccharide glycoconjugates acting as ligands and monosaccharide units expressed on the exterior of bacterial spores. Using fluorophore-assisted carbohydrate electrophoresis (FACE), carbohydrates that are expressed on the exterior of the spores were enumerated. The findings have an impact on how to improve ligand selection, essential for sensor development. In addition, the findings provide new information for inhibition of bacterial spores, and in general, demonstrate how carbohydrates function as recognition signals in nature.     

NMR and MD Investigations of Human Galectin-1/Oligosaccharide Complexes

The specific recognition of carbohydrates by lectins plays a major role in many cellular processes. Galectin-1 belongs to a family of 15 structurally related β-galactoside binding proteins that are able to control a variety of cellular events, including cell cycle regulation, adhesion, proliferation, and apoptosis. The three-dimensional structure of galectin-1 has been solved by x-ray crystallography in the free form and in complex with various carbohydrate ligands. In this work, we used a combination of two-dimensional NMR titration experiments and molecular-dynamics simulations with explicit solvent to study the mode of interaction between human galectin-1 and five galactose-containing ligands. Isothermal titration calorimetry measurements were performed to determine their affinities for galectin-1. The contribution of the different hexopyranose units in the protein-carbohydrate interaction was given particular consideration. Although the galactose moiety of each oligosaccharide is necessary for binding, it is not sufficient by itself. The nature of both the reducing sugar in the disaccharide and the interglycosidic linkage play essential roles in the binding to human galectin-1.     

Oligosaccharide microarrays fabricated on aminooxyacetyl functionalized glass surface for characterization of carbohydrate-protein interaction

Carbohydrate-protein interactions play important biological roles in biological processes. But there is a lack of high-throughput methods to elucidate recognition events between carbohydrates and proteins. This paper reported a convenient and efficient method for preparing oligosaccharide microarrays, wherein the underivatized oligosaccharide probes were efficiently immobilized on aminooxyacetyl functionalized glass surface by formation of oxime bonding with the carbonyl group at the reducing end of the suitable carbohydrates via irreversible condensation. Prototypes of carbohydrate microarrays containing 10 oligosaccharides were fabricated on aminooxyacetyl functionalized glass by robotic arrayer. Utilization of the prepared carbohydrate microarrays for the characterization of carbohydrate-protein interaction reveals that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. The limit of detection (LOD) for lectin ConA on the fabricated carbohydrate microarrays was determined to be approximately 0.008 microg/mL. Inhibition experiment with soluble carbohydrates also demonstrated that the binding affinities of lectins to different carbohydrates could be analyzed quantitatively by determining IC(50) values of the soluble carbohydrates with the carbohydrate microarrays. This work provides a simple procedure to prepare carbohydrate microarray for high-throughput parallel characterization of carbohydrate-protein interaction.     

Proteins that bind high-mannose sugars of the HIV envelope

A broad range of proteins bind high-mannose carbohydrates found on the surface of the envelope protein gp120 of the human immunodeficiency virus and thus interfere with the viral life cycle, providing a potential new way of controlling HIV infection. These proteins interact with the carbohydrate moieties in different ways. A group of them interacts as typical C-type lectins via a Ca2+ ion. Another group interacts with specific single, terminal sugars, without the help of a metal cation. A third group is involved in more intimate interactions, with multiple carbohydrate rings and no metal ion. Finally, there is a group of lectins for which the interaction mode has not yet been elucidated. This review summarizes, principally from a structural point of view, the current state of knowledge about these high-mannose binding proteins and their mode of sugar binding.     

Theoretical studies on the modes of binding of some of the β-glycosidically linked glucobioses to concanavalin A

The probable modes of binding for methyl-α-d-sophoroside, methyl-β-d-sophoroside, laminariboise and cellobiose to concanavalin A have been determined using theoretical methods. Methyl-d-sophorosides can bind to concanavalin A in two modes, i.e. by placing their reducing as well as non-reducing sugar units in the carbohydrate specific binding site, whereas laminaribiose and cellobiose can reach the binding site only with their non-reducing glucose units. However, the probability for methyl-α-d-sophoroside to bind to concanavalin A with its reducing sugar residue as the occupant of the binding site is much higher than it is with its non-reducing sugar residue as the occupant of the sugar binding site. A few of the probable conformers of methyl-β-d-sophoroside can bind to concanavalin A with either the reducing or non-reducing glucose unit. Higher energy conformers of cellobiose or laminaribiose can reach the binding site with their non-reducing residues alone. The relative differences in the binding affinities of these disaccharides are mainly due to the differences in the availability of proper conformers which can reach the binding site and to non-covalent interactions between the sugar and the protein. This study also suggests that though the sugar binding site of concanavalin A accommodates a single sugar residue, the residue outwards from the binding site also interacts with concanavalin A, indicating the existence of extended concanavalin A carbohydrate interactions.     

Glycolipids of cell surfaces: Isolation of specific sugar sequences using carbohydrate-binding proteins

Proteins that bind carbohydrates can be used to isolate specific sugar sequences from complex mixtures. Free sialyloligosaccharides or sialyloligosaccharides released from gangliosides by ozonolysis and alkaline fragmentation are labeled at their reducing ends by reduction with NaB[³H]₄. After partial separation by column chromatography, oligosaccharide fractions are tested for binding to anti-sialyloligosaccharide antibodies [Smith, D. F., and Ginsburg, V. (1980) J. Biol. Chem.255, 55–59] and cholera toxin by a nitrocellulose filter assay. Oligosaccharides bound by the proteins can be eluted from the filters and further characterized. The method can be used to isolate and identify carbohydrate ligands of cell surfaces.     

Design,synthesis and characterisation of affinity ligands for glycoproteins

The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein–carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar‐binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine‐based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself. Copyright © 1999 John Wiley & Sons, Ltd.     

伴刀豆球蛋白A-糖复合物的模拟分析

主要分析ConA与不同的糖特异性结合时其活性位点构象变化的特征。模拟分析了ConA糖结合活性中心氨基酸残基结构特征,同时对相应残基原子可及性表面进行了计算和分析。结果表明：（1）ConA在和不同的糖结合时,存在不同的结合方式;（2）不管ConA和什么糖结合,主要的作用是由活性中心的Tyr12、Asn14、Asp208和Arg228提供的;（3）无论是结合单糖还是寡糖,活性中心总是与第一个糖环起主要的结合作用。     

Modeling protein recognition of carbohydrates

We have a limited understanding of the details of molecular recognition of carbohydrates by proteins, which is critical to a multitude of biological processes. Furthermore, carbohydrate-modifying proteins such as glycosyl hydrolases and phosphorylases are of growing importance as potential drug targets. Interactions between proteins and carbohydrates have complex thermodynamics, and in general the specific positioning of only a few hydroxyl groups determines their binding affinities. A thorough understanding of both carbohydrate and protein structures is thus essential to predict these interactions. An atomic-level view of carbohydrate recognition through structures of carbohydrate-active enzymes complexed with transition-state inhibitors reveals some of the distinctive molecular features unique to protein-carbohydrate complexes. However, the inherent flexibility of carbohydrates and their often water-mediated hydrogen bonding to proteins makes simulation of their complexes difficult. Nonetheless, recent developments such as the parameterization of specific force fields and docking scoring functions have greatly improved our ability to predict protein-carbohydrate interactions. We review protein-carbohydrate complexes having defined molecular requirements for specific carbohydrate recognition by proteins, providing an overview of the different computational techniques available to model them.     

Capillary affinity electrophoresis for the screening of post-translational modification of proteins with carbohydrates

Glycosylation is one of the most important post-translational events for proteins, affecting their functions in health and disease, and plays significant roles in various information traffics for intracellular and intercellular biological events (Hancock, W. S. J. Proteome Res. 2002, 1, 297). We have attempted to obtain the information on the numbers and amounts of carbohydrate chains. Interaction between carbohydrate chains and proteins that recognize them is a target to understand the biological roles of glycosylation. To date, there have been a few strategies for simultaneous analysis of the interactions between complex mixtures of carbohydrates and proteins. Here, we report an approach to categorize carbohydrate chains using a few glycoprotein samples as models for the studies on the analysis of post-translational modification of proteins with carbohydrates. A combination of some specific lectins was used as carbohydrate-binding proteins. The method is based on high-resolution separation of fluorescent-labeled carbohydrates by capillary electrophoresis with laser-induced fluorescent detection in the presence of carbohydrate-binding proteins at different concentrations. The present technique affords (1) simultaneous determination of carbohydrate chains, (2) binding specificity of the constituent carbohydrate chains to specific proteins, and (3) kinetic data such as the association constant of each carbohydrate. We found that the lectins employed in the present study could discriminate subtle difference in linkages and resolved the carbohydrate mixtures. The results will be useful, for example, to understand the biological events expressed with carbohydrate changes on the cell surface.     

The molecular mechanism of sulfated carbohydrate recognition by the cysteine-rich domain of mannose receptor

The mannose receptor (MR) binds foreign and host ligands through interactions with their carbohydrates. Two portions of MR have distinct carbohydrate recognition properties. One is conferred by the amino-terminal cysteine-rich domain (Cys-MR), which plays a critical role in binding sulfated glycoproteins including pituitary hormones. The other is achieved by tandemly arranged C-type lectin domains that facilitate carbohydrate-dependent uptake of infectious microorganisms. This dual carbohydrate binding specificity enables MR to bind ligands by interacting with both sulfated and non-sulfated polysaccharide chains. We previously determined crystal structures of Cys-MR complexed with 4-SO(4)-N-acetylglucosamine and with an unidentified ligand resembling Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]). In continued efforts to elucidate the mechanism of sulfated carbohydrate recognition by Cys-MR, we characterized the binding affinities between Cys-MR and potential carbohydrate ligands using a fluorescence-based assay. We find that Cys-MR binds sulfated carbohydrates with relatively high affinities (K(D)=0.1 mM to 1.0 mM) compared to the affinities of other lectins. Cys-MR also binds Hepes with a K(D) value of 3.9 mM, consistent with the suggestion that the ligand in the original Cys-MR crystal structure is Hepes. We also determined crystal structures of Cys-MR complexed with 3-SO(4)-Lewis(x), 3-SO(4)-Lewis(a), and 6-SO(4)-N-acetylglucosamine at 1.9 A, 2.2 A, and 2.5 A resolution, respectively, and the 2.0 A structure of Cys-MR that had been treated to remove Hepes. The conformation of the Cys-MR binding site is virtually identical in all Cys-MR crystal structures, suggesting that Cys-MR does not undergo conformational changes upon ligand binding. The structures are used to rationalize the binding affinities derived from the biochemical studies and to elucidate the molecular mechanism of sulfated carbohydrate recognition by Cys-MR.     

Spatial differences of endogenous lectin expression within the cellular organization of the human heart: a glycohistochemical, immunohistochemical, and glycobiochemical study

Protein-carbohydrate recognition may be involved in an array of molecular interactions on the cellular and subcellular levels. To gain insight into the role of proteins in this type of interaction, surgically removed specimens of human endomyocardial tissue were processed for histochemical and biochemical analysis. The inherent capacity of these sections to bind individual sugar moieties, which are constituents of the carbohydrate part of cellular glycoconjugates, was assessed using a panel of biotinylated neoglycoproteins according to a standardized procedure. Together with appropriate controls, it primarily allowed localization of endogenous lectins. Differences in lectin expression were observed between layers of endocardial tissue, myocardial cell constituents, connective-tissue elements, and vascular structures. The endocardium proved to be positive with beta-galactoside-bearing probes; with neoglycoproteins carrying beta-xylosides, alpha-fucosides, and galactose-6-phosphate moieties; and with probes containing a carboxyl group within the carbohydrate structure, namely sialic acid and glucuronic acid. In contrast, only fucose-and maltose-specific receptors were apparent in the elastic layers of the endocardium. Aside from ascertaining the specificity of the protein-carbohydrate interaction by controls, i.e., lack of binding of the probe in the presence of the unlabelled neoglycoprotein and lack of binding of the labelled sugar-free carrier protein, respective sugar receptors were isolated from heart extracts by using histochemically effective carbohydrates as immobilized affinity ligand. Moreover, affinity chromatography using immobilized lactose as affinity ligand as well as the use of polyclonal antibodies against the predominant beta-galactoside-specific lectin of heart demonstrated that the lactose-specific neoglycoprotein binding was due to this lectin. Remarkably, the labelled endogenous lectin, preferred to plant lectins for detecting ligands of the endogenous lectin, localized ligands in tissue parts where the lectin itself was detected glycohistochemically as well as immunohistologically. This demonstration of receptor-ligand presence in the same system is a further step toward functional assignment of the recorded protein-carbohydrate interaction. Overall, the observed patterns of lectin expression may serve as a guideline to elucidate the precise physiological relevance of lectins and to analyze pathological conditions comparatively.     

A new lectin-gold complex for ultrastructural localization of galacturonic acids

We report the development of a cytochemical affinity technique for detection of galacturonic acids at the ultrastructural level. The highly purified gonad lectin from Aplysia depilans (AGL) was tagged with colloidal gold particles and used for labeling carbohydrates in resin-embedded sections of various plant and fungal tissues. Patterns of AGL binding sites were compared to those obtained with a D-galactose-specific lectin, Ricinus communis agglutinin I. Differences in labeling patterns were noted, indicating that the lectins exhibited differential carbohydrate binding. In addition, the considerable loss of labeling over isolated wheat coleoptile walls treated for removal of pectin, after incubation with the AGL-gold complex, strongly suggested an affinity of AGL for pectic substances. A series of cytochemical controls, including sugar inhibition tests, has proven the specificity of the technique and the high affinity of AGL towards galacturonic acids. The potential value of this new lectin for ultrastructural studies on cell wall pectic substances in plant biology and pathology is demonstrated.     

O-linked protein glycosylation structure and function

There has been a recent resurgence of interest in the post-translational modification of serine and threonine hydroxyl groups by glycosylation, because the resulting O-linked oligosaccharide chains tend to be clustered over short stretches of peptide and hence they can present multivalent carbohydrate antigenic or functional determinants for antibody recognition, mammalian cell adhesion and microorganism binding. Co-operativity can greatly increase the affinity of interactions with antibodies or carbohydrate binding proteins. Thus, in addition to their known importance in bearing tumour associated antigens in the gastrointestinal and respiratory tracts, glycoproteins with O-linked chains have been implicated as ligands or co-receptors for selectins (mammalian carbohydrate binding proteins). Microorganisms may have adopted similar mechanisms for interactions with mammalian cells in infection, by having relatively low affinity ligands (adhesins) for carbohydrate binding, which may bind with higher affinity due to the multivalency of the host ligand and which are complemented by other virulence factors such as interactions with integrin-type molecules. In addition to specific adhesion signals from O-linked carbohydrate chains, multivalent O-glycosylation is involved in determining protein conformation and forming conjugate oligosaccharide-protein antigenic, and possible functional determinants.     

Affinity enhancement by multivalent lectin–carbohydrate interaction

The binding of simple carbohydrate ligands by proteins often requires affinity enhancement to attain biologically relevant strength. This is especially true for endocytotic receptors and the molecules that engage in the first-line of defense. For such purposes, nature often utilizes a mode of affinity enhancement that arises from multiple interactions between the binding proteins and the carbohydrate ligands, which we term glycoside cluster effect. In this review article we give a number of examples and describe important factors in the multi-valent interactions that govern the degree of affinity enhancement.     

Carbohydrate accumulation and partitioning in Pinus banksiana seedlings and seedling cuttings

Tests were conducted to identify possible relations between carbohydrates and callusing-rooting of Pinus banksiana Lamb, cuttings. Terminals, upper stems, and basal (1 cm) stems of 90-day-old untreated seedlings and seedling cuttings were analyzed for sucrose, total soluble reducing sugar, starch and total non-structural carbohydrate during propagation. Seedlings were evaluated in order to determine whether data for cuttings alone properly described carbohydrate-callusing-rooting relations under conditions where stock plants and cuttings were propagated in different environments. Results indicated that seedling terminals and upper stems, but not basal stems, accumulated the measured carbohydrates much like cuttings, though to lesser concentrations. Thus, carbohydrate accumulation by cutting terminals and upper stems would have been overestimated, based on cutting data alone. In terms of rooting, results indicated that: 1) Total carbohydrate accumulation in cutting basal stems was related to callusing-rooting, but a cause-effect relation was not established; 2) The positive relation between callusing-rooting and total carbohydrate accumulation was primarily due to accumulation of reducing sugar and starch, with reducing sugar predominant. 3) Reducing sugar/starch concentration ratios were the most sensitive and convenient indicators of specific carbohydrate differences within and between seedlings and cuttings.