Mannoside recognition and degradation by bacteria

Mannosides constitute a vast group of glycans widely distributed in nature. Produced by almost all organisms, these carbohydrates are involved in numerous cellular processes, such as cell structuration, protein maturation and signalling, mediation of protein–protein interactions and cell recognition. The ubiquitous presence of mannosides in the environment means they are a reliable source of carbon and energy for bacteria, which have developed complex strategies to harvest them. This review focuses on the various mannosides that can be found in nature and details their structure. It underlines their involvement in cellular interactions and finally describes the latest discoveries regarding the catalytic machinery and metabolic pathways that bacteria have developed to metabolize them.     

No phospholipid monolayer-sugar interactions

Studies by a number of workers using the Langmuir film balance have shown that when carbohydrates, such as sucrose or glycerol, are dissolved in a subphase on which a phospholipid is spread, film expansion occurs (Cadenhead & Demchak, 1969; Cadenhead & Bean, 1972; Maggio et al., 1976; Maggio & Lucy, 1978). Recently such effects have been observed again, particularly with the carbohydrates galactose and trehalose (Johnston et al., 1984). The origin of these film expansions was uncertain, and various suggestions have been made to explain them. One idea was that they might be due to interactions which these carbohydrates have with the water molecules close to the polar head groups of the lipids. Recent studies in our two laboratories, described here, show that the magnitude of the expansion effects is variable and that in general they arise from surfactant impurities in the sugars. These impurities are observed in carbohydrates which are reputedly of high grade; the amount of impurity present can vary from batch to batch, and sometimes they can be difficult to remove. Film balance techniques or subphase preparation can mask the detection of minor impurities. The presence of surfactant impurities in reputedly pure carbohydrates needs to be considered in other biochemical and biophysical studies of lipids and cell membranes.     

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.     

Cell surface carbohydrates in cell adhesion.

Carbohydrates are ubiquitous constituents of cell surfaces, and possess many characteristics that make them ideal candidates for recognition molecules. In many systems where cell adhesion plays a critical role, carbohydrate binding proteins have been shown to bind to cell surface carbohydrates and participate in cell-cell interactions. Such systems include fertilization, development, pathogen-host recognition and inflammation. In particular the recent discovery of the LEC-CAMs and their importance in leukocyte biology has refocused attention on lectin-mediated cell adhesion. The LEC-CAMs offer good targets for the development of therapeutics based on carbohydrate structures.     

Relevance of plant lectins in human cell biology and immunology.

Protein-carbohydrate interactions are used for intercellular communication. Mammalian cells are known to bear a variety of glycoconjugates. Lectins, first discovered in plants, are proteins which can specifically bind carbohydrates. Given the high affinity of plant lectins for carbohydrates, they have always been important as molecular tools in the identification, purification and stimulation of specific glycoproteins on human cells. Lectins have provided important clues to the repertoire of carbohydrate structures in animal cells. The discovery of plant lectins gave a great impulse to modern glycobiology. They represent important biochemical reagents for numerous applications in the biomedical field and in research. Sequence determinations and structural characterization helped to understand the mechanism of action in many biologic systems. Plant lectins have been fundamental in human immunological studies because some of them are mitogenic/activating to lymphocytes. Understanding the molecular basis of lectin-carbohydrate interactions and of the intracellular signalling evoked holds promise for the design of novel drugs for the treatment of infectious, inflammatory and malignant diseases. It may also be of help for the structural and functional investigation of glycoconjugates and their changes during physiological and pathological processes.     

The Role of Self-Assembled Monolayers of the Purine and Pyrimidine Bases in the Emergence of Life

The experimental evidence for the spontaneous formation and structure determination of two-dimensional monolayers of the purine and pyrimidine bases is examined. The plausibility of such structures forming spontaneously at the solid-liquid interface following their prebiotic synthesis suggests a functional role for them in the emergence of life. It is proposed that prebiotic interactions of enantiomorphic monolayers of mixed base composition with racemic amino acids might be implicated in a simultaneous origin of a primitive genetic coding mechanism and biomolecular homochirality. The interactions of these monolayers with carbohydrates and other derivatives is also discussed.     

A Langmuir film balance study of the interactions of ionic and polar solutes with glycolipid monolayers

Using a Langmuir film balance experiments have been conducted to discover if dissolved salts or carbohydrates interact with glycolipid monolayers. Two types of glycolipid were studied, simple glycosides made by ether linking monosaccharides to fatty alcohols and cerebrosides extracted from natural sources. It was found that salts or carbohydrates in the subphase expanded glycolipid monolayers. That is, a monolayer spread on a solution occupied a greater area at a given pressure than it would have spread on pure water. Of the carbohydrates galactose and glucose, galactose caused a markedly greater expansion of monolayers than glucose. However, the magnitude of the expansions measured for stearyl glucoside, mannoside and galactoside films on solutions of a particular sugar were not significantly different, demonstrating that this phenomenon is independent of the glycolipid sugar residue. As with carbohydrates, salts also have differing effects on glycolipid monolayers. Although the effect an individual ion has on a monolayer cannot be directly measured, comparisons between salts indicate that there is a correlation between the size of an ion and the extent of the monolayer expansion it causes. To explain these observations two different mechanisms are proposed. In the case of salts it is suggested that large ions which have a low charge density disrupt water structure in such a way that monolayers spread on the surface of their solutions are expanded. The ability of carbohydrates to expand monolayers is explained in terms of the carbohydrate replacing water molecules bound to the polar groups of the monolayer and in so doing increasing the effective area of the lipid molecules. It is suggested that the molecular mechanisms involved in the interactions of ions and carbohydrates with glycolipid monolayers may also operate in the interactions of glycolipids and glycoproteins with extracellular agents and surfaces.     

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.     

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.     

History of lectins: from hemagglutinins to biological recognition molecules

The occurrence in nature of erythrocyte-agglutinating proteins has been known since the turn of the 19th century. By the 1960s it became apparent that such proteins also agglutinate other types of cells, and that many of them are sugar-specific. These cell-agglutinating and sugar-specific proteins have been named lectins. Although shown to occur widely in plants and to some extent also in invertebrates, very few lectins had been isolated until the early 1970s, and they had attracted little attention. This attitude changed with the demonstration that lectins are extremely useful tools for the investigation of carbohydrates on cell surfaces, in particular of the changes that the latter undergo in malignancy, as well as for the isolation and characterization of glycoproteins. In subsequent years numerous lectins have been isolated from plants as well as from microorganisms and animals, and during the past two decades the structures of hundreds of them have been established. Concurrently, it was shown that lectins function as recognition molecules in cell-molecule and cell-cell interactions in a variety of biological systems. Here we present a brief account of 100-plus years of lectin research and show how these proteins have become the focus of intense interest for biologists and in particular for the glycobiologists among them.     

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.     

Selection and syntheses of tentacle type peptides as 'artificial' lectins against various cell-surface carbohydrates

Sialyl Lewis X and its derivatives are cell-surface carbohydrates that are involved in cell-cell recognition by carbohydrate-mediated interactions. Unfortunately, owing to the similarities between carbohydrates only a limited number of tools are available for their differentiation. In this study, we prepared a selected phage-displayed peptide library against LeX (2), SLN (3), or LN (4), which compared to sLeX (1) lack sialic acid, fucose, and both sialic acid and fucose from constituents, respectively. Sequences of the selected peptides, prepared as tentacle type dimeric peptides, were prepared and shown to have micromolar affinities for the cognate carbohydrates. The specificities displayed by these 'artificial' lectins overwhelm those of natural lectins. These results suggest that they can serve as useful tools to detect changes in the terminal monosaccharide of cell-surface carbohydrates.     

Thermodynamic, kinetic, and electron microscopy studies of concanavalin A and Dioclea grandiflora lectin cross-linked with synthetic divalent carbohydrates

The jack bean lectin concanavalin A (ConA) and the Dioclea grandiflora lectin (DGL) are highly homologous Man/Glc-specific members of the Diocleinae subtribe. Both lectins bind, cross-link, and precipitate with carbohydrates possessing multiple terminal nonreducing Man residues. The present study investigates the binding and cross-linking interactions of ConA and DGL with a series of synthetic divalent carbohydrates that possess spacer groups with increasing flexibility and length between terminal alpha-mannopyranoside residues. Isothermal titration microcalorimetry was used to determine the thermodynamics of binding of the two lectins to the divalent analogs, and kinetic light scattering and electron microscopy studies were used to characterize the cross-linking interactions of the lectins with the carbohydrates. The results demonstrated that divalent analogs with flexible spacer groups between the two terminal Man residues possess higher affinities for the two lectins as compared with those with inflexible spacer groups. Furthermore, despite their high degree of homology, ConA and DGL exhibit differences in their kinetics of cross-linking and precipitation with the divalent analogs. Electron microscopy shows the loss of organized cross-linked lattices of the two lectins with analogs possessing increased distance between the terminal Man residues. The loss of lattice patterns with the analogs is distinct for each lectin. These results have important implications for the interactions of lectins with multivalent carbohydrate receptors in biological systems.     

Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity.

The production of glycoproteins with carbohydrates of defined structure and minimal heterogeneity is important for functional studies of mammalian carbohydrates. To facilitate such studies, several Chinese hamster ovary mutants that carry between two and four glycosylation mutations were developed. All of the lines grew readily in culture despite the drastic simplification of their surface carbohydrates. Therefore, both endogenous glycoproteins and those introduced by transfection can be obtained with specifically tailored carbohydrates. The lectin resistance properties of the mutants showed that each line expresses a novel array of cell surface carbohydrates useful for identifying specific roles for carbohydrates in cellular interactions. In addition, they showed that the epistatic relationships among different glycosylation mutations are not entirely predictable, providing insight into the complexity of the carbohydrate structures at the Chinese hamster ovary cell surface.     

Fabrication of carbohydrate chips and their use to probe protein-carbohydrate interactions

Carbohydrate microarrays have received considerable attention as an advanced technology for the rapid analysis of carbohydrate-protein interactions. This protocol provides detailed procedures for the preparation of carbohydrate microarrays by immobilizing hydrazide-conjugated carbohydrates on epoxide-derivatized glass slides. In addition, we describe the use we make of these microarrays in glycomics research. Unlike other techniques that require large amounts of samples and long assay times, carbohydrate microarrays are used to carry out the rapid assessment of a number of carbohydrate-recognition events with tiny amounts of carbohydrate samples. Furthermore, the microarray technology is also utilized for the rapid assay of enzyme activities. We are able to routinely prepare carbohydrate microarrays within 12 h by using hydrazide-conjugated carbohydrates and apply these microarrays for the studies of glycan-protein interactions within 8 h.     

Differential accessibility of the carbohydrate moieties of Cls-Clr-Clr-Cls, the catalytic subunit of human Cl

The catalytic subunit of human Cl, Cls-Clr-Clr-Cls, is a Ca2+-dependent tetrameric association of two serine proteases, Clr and Cls, which are glycoproteins containing asparagine-linked carbohydrates. With a view to investigate the accessibility and the possible functional role of these carbohydrates, the isolated proteases and their Ca2+-dependent complexes were submitted to deglycosylation by peptide:N-glycosidase F, an endoglycosidase that specifically hydrolyzes all classes of N-linked glycans. Treatment of isolated Clr and Cls led to the removal of the carbohydrate moieties attached to their N-terminal alpha region, whereas those located in the C-terminal gamma-B catalytic domains were resistant to hydrolysis. Formation of the Ca2+-dependent Cls-Cls dimer and Cls-Clr-Clr-Cls tetramer induced specific protection of the single carbohydrate attached to the alpha region of Cls and of one of the two carbohydrates located in the corresponding region of Clr. Sequence studies indicated that the carbohydrates protected upon homologous (Cls-Cls) or heterologous (Clr-Cls) interactions are attached to asparagine residues 159 of Cls and 204 of Clr, at the C-terminal end of the EGF-like domain of both proteases. These data bring further evidence that Ca2+-dependent interactions between Clr and Cls are mediated by their N-terminal alpha regions and strongly suggest that, inside these regions, the EGF-like domains play an essential role in these interactions.     

Structural glycobiology: a game of snakes and ladders

Oligo- and polysaccharides are infamous for being extremely flexible molecules, populating a series of well-defined rotational isomeric states under physiological conditions. Characterization of this heterogeneous conformational ensemble has been a major obstacle impeding high-resolution structure determination of carbohydrates and acting as a bottleneck in the effort to understand the relationship between the carbohydrate structure and function. This challenge has compelled the field to develop and apply theoretical and experimental methods that can explore conformational ensembles by both capturing and deconvoluting the structural and dynamic properties of carbohydrates. This review focuses on computational approaches that have been successfully used in combination with experiment to detail the three-dimensional structure of carbohydrates in a solution and in a complex with proteins. In addition, emerging experimental techniques for three-dimensional structural characterization of carbohydrate-protein complexes and future challenges in the field of structural glycobiology are discussed. The review is divided into five sections: (1) The complexity and plasticity of carbohydrates, (2) Predicting carbohydrate-protein interactions, (3) Calculating relative and absolute binding free energies for carbohydrate-protein complexes, (4) Emerging and evolving techniques for experimental characterization of carbohydrate-protein structures, and (5) Current challenges in structural glycoscience.     

Thermodynamic binding studies of galectin-1, -3 and -7

The carbohydrate binding specificities of the galectin family of animal lectins has been the source of intense recent investigations. Isothermal titration microcalorimetry (ITC) provides direct determination of the thermodynamics of binding of carbohydrates to lectins, and has provided important insights into the fine carbohydrate binding specificities of a wide number of plant and animal lectins. Recent ITC studies have been performed with galectin-1, galectin-3 and galectin-7 and their interactions with sialylated and non-sialylated carbohydrates. The results show important differences in the specificities of these three galectins toward poly-N-acetyllactosamine epitopes found on the surface of cells.     

Novel functions of complex carbohydrates elucidated by the mutant mice of glycosyltransferase genes

Complex carbohydrates consist of carbohydrate moieties and protein or lipid portions, resulting in the formation of glycoproteins, proteoglycans or glycosphingolipids. The polymorphic carbohydrate structures are believed to contain profound biological implications which are important in cell-cell or cell-extracellular matrix interactions. A number of studies to delineate the roles of carbohydrates have been performed, and demonstrated definite changes in their profiles, cellular phenotypic changes or, sometimes, morphological and functional changes in tissues after modification of their structures. Recent successes in the isolation of glycosyltransferase genes and their modification enzyme genes has enabled clearer demonstrations of the roles of complex carbohydrates. In particular, genetic modification of glycosyltransferase genes in mice can elucidate the biological significances of their products in vivo. Here, we summarize recent advances in the understanding of the roles of complex carbohydrates provided from studies of gene knock-out mice of glycosyltransferase and modification enzyme genes focusing on novel functions which had not been expected.