Signaling pathways for transduction of the initial message of the glycocode into cellular responses

The sugar units of glycan structures store information and establish an alphabet of life. The language of the oligosaccharide coding units is deciphered by receptors such as lectins and the decoded message can be transduced by multiple signaling pathways. Similar to glycoconjugates, these receptors can exhibit pronounced changes in quantitative and qualitative aspects of expression, as attested by a wealth of lectin and immunohistochemical studies. Since histochemistry provides a static picture, it is essential to shed light on the mechanisms of how a recognitive protein-carbohydrate interplay can be transduced into cellular responses. Their consequences for example for cell morphology will then be visible to the histochemist. Therefore, basic signaling routes will be graphically outlined and their trigger potential will be explained by selected examples from the realm of glycosciences

The Third Dimension of Reading the Sugar Code by Lectins

Coding of biological information is not confined to nucleic acids and proteins. Endowed with the highest level of structural versatility among biomolecules, the glycan chains of cellular glycoconjugates are well-suited to generate molecular messages/signals in a minimum of space. The sequence and shape of oligosaccharides as well as spatial aspects of multivalent presentation are assumed to underlie the natural specificity/selectivity that cellular glycans have for endogenous lectins. In order to eventually unravel structure-activity profiles cyclic scaffolds have been used as platforms to produce glycoclusters and afford valuable tools. Using adhesion/growth-regulatory galectins and the pan-galectin ligand lactose as a model, emerging insights into the potential of cyclodextrins, cyclic peptides, calixarenes and glycophanes for this purpose are presented herein. The systematic testing of lectin panels with spatially defined ligand presentations can be considered as a biomimetic means to help clarify the mechanisms, which lead to the exquisite accuracy at which endogenous lectins select their physiological counterreceptors from the complexity of the cellular glycome

Detection of inflammation- and neoplasia-associated alterations in human large intestine using plant/invertebrate lectins, galectin-1 and neoglycoproteins

Commonly, plant and invertebrate lectins are accepted glycohistochemical tools for the analysis of normal and altered structures of glycans in histology and pathology. Mammalian lectins and neoglycoproteins are recent additions to this panel for the detection of lectin-reactive carbohydrate epitopes and glycoligand-binding sites. The binding profiles of these three types of probes were comparatively analyzed in normal, inflamed and neoplastic large intestine. In normal colonic mucosa the intracellular distribution of glycoconjugates and carbohydrate ligand-binding sites in enterocytes reveals a differential binding of lectins with different specificity and of neoglycoproteins to the Golgi apparatus, the rough and smooth endoplasmic reticulum and the apical cell surface. The accessible glycoligand-binding sites and the lectin-reactive carbohydrate epitopes detected by galectin-1 show the same pattern of intracellular location excluding the apical cell surface. Lectin-reactive carbohydrate epitopes detected by plant lectins of identical monosaccharide specificity as the endogenous lectin {[}Ricinus communis agglutinin-I (RCA-I), Viscum album agglutinin (VAA)], however, clearly differ with respect to their intracellular distribution. Maturation-associated differences and heterogeneity in glycohistochemical properties of epithelial cells and non-epithelial cells (macrophages, dendritic cells, lymphocytes) are found. Dissimilarities in the fine structural Ligand recognition of lectins with nominal specificity to the same monosaccharide have been demonstrated for the galactoside-specific lectins RCA-I, VAA and galectin-1 as well as the N-acetylgalactosamine (GalNAc)-specific lectins Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) and Helix pomatia agglutinin in normal mucosa and in acute appendicitis. Acute inflammation of the intestinal mucosa found in acute phlegmonous appendicitis is associated with selective changes of glycosylation of mucin in goblet cells mainly of lower and middle crypt segments resulting in an increase of DBA- and SEA-binding sites in the goblet cell population. Appendicitis causes no detectable alteration of neoglycoprotein binding. In contrast, tumorigenesis of colonic adenoma is characterized by increases in lectin-reactive galactose (Gal; Gal-beta 1,3-GalNAc), fucose and N-acetylglucosamine moieties and by enhanced presentation of respective carbohydrate ligand-binding capacity. This work reveals that endogenous lectins and neoglycoproteins are valuable glycohistochemical tools supplementing the well-known analytic capacities of plant lectins in the fields of gastrointestinal anatomy and gastroenteropathology

Introduction to glycopathology: the concept, the tools and the perspectives

Analyzing the flow of biological information is a fundamental challenge for basic sciences. The emerging results will then lend themselves to the development of new approaches for medical applications. Toward this end, the products of protein/lipid glycosylation deserve special attention. The covalent attachment of sugars to these carriers means much more than just a change of the carriers' physicochemical properties. In principle, the ubiquitous presence of glycoconjugates and the close inspection of the particular structural `talents' of carbohydrates provide suggestive evidence for information coding by sugars. In fact, the theoretical number of 'words' (oligomers) formed by 'letters' (monosaccharides) is by far higher than by using nucleotides or amino acids. In other words, glycans harbor an unsurpassed coding capacity. The cyto- and histochemical detection of dynamic changes in the profile of cellular glycans (glycome, the equivalent of the proteome) by sugar receptors such as antibodies used as tools underscores the suitability of carbohydrates for such a task. The resulting staining patterns can be likened to a molecular fingerprint. By acting as ligand (counterreceptor) for endogenous receptors (tissue lectins), glycan epitopes become partners in a specific recognition pair, and the sugar-encoded information can then be translated into effects, e.g. in growth regulation. Of note, expression of both sides of such a pair, i.e. lectin and cognate glycan, can physiologically be orchestrated for optimal efficiency. Indeed, examples how to prevent autoimmune diseases by regulatory T cells and restrict carcinoma growth by a tumor suppressor attest occurrence of co-regulation. In consequence, these glycans have potential to establish a new class of functional biomarkers, and mapping presence of their receptors is warranted. In this review, the cyto- and histochemical methods, which contribute to explore information storage and transfer within the sugar code, are described. This introduction to the toolbox is flanked by illustrating the application of each type of tool in histopathology, with focus on adhesion/growth-regulating galectins. Together with an introduction to fundamental principles of the sugar code, the review is designed to guide into this field and to inspire respective research efforts. Virtual slides: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1670639891114983

From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals (sugar coding). Here, we test the hypothesis that the common N-glycan modifications by core fucosylation and introduction of the bisecting N-acetylglucosamine moiety have long-range effects with functional consequences. Molecular dynamics simulations indicate a shift in conformational equilibria between linear extension or backfolding of the glycan antennae upon substitution. We also present a new fingerprint-like mode of presentation for this multi-parameter system. In order to delineate definite structure-function relationships, we strategically combined chemoenzymatic synthesis with bioassaying cell binding and the distribution of radioiodinated neoglycoproteins in vivo. Of clinical relevance, tailoring the core region affects serum clearance markedly, e. g., prolonging circulation time for the neoglycoprotein presenting the N-glycan with both substitutions. alpha 2,3-Sialylation is another means toward this end, similarly seen for type II branching in triantennary N-glycans. This discovery signifies that rational glycoengineering along the given lines is an attractive perspective to optimize pharmacokinetic behavior of glycosylated pharmaproteins. Of general importance for the concept of the sugar code, the presented results teach the fundamental lesson that N-glycan core substitutions convey distinct characteristics to the concerned oligosaccharide relevant for cis and trans biorecognition processes. These modifications are thus molecular switches

Diffuse pulmonary hemosiderosis after exposure to pesticides - A case report

This report describes the clinical, radiological, microscopical and ligandohistochemical findings in a 17-year-old woman who suffered from an acute onset of pulmonary hemosiderosis after inhalation of pesticides used for the cultivation of strawberries. She complained of headache, dyspnea, rhinitis, weakness and recurrent severe hemoptysis. Chest radiographs revealed bilateral patchy infiltrates, predominantly in the lower parts of both lungs. The consecutive severe anemia was treated by multiple blood transfusions which were repeated every 4-5 days. Open lung biopsies displayed signs of diffuse hemorrhage with hemosiderin-loaded macrophages, some hyaline membranes, focal fibroid deposits with intermingled histiocytes, mild interstitial fibrosis and focal intra-alveolar calcified bodies surrounded by foreign body giant cells. Analysis of endogenous lectins failed to demonstrate expression of binding capacities for maltose, fucose, mannose; lactose and sialic acid. Neither binding capacities for the macrophage-migration-inhibitory factor nor its presence, as analyzed by labeled sarcolectin, could be detected histochemically. The light microscopical findings are consistent with a longer-lasting diffuse pulmonary hemosiderosis; the presence of calcified bodies and foreign body giant cells (including the ligandohistochemical data) argues for a causal role of inhaled substances. The patient's clinical course improved after cyclophosphamide treatment, which restored her ability to work and released her from the need for recurrent blood transfusions

Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy

Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence, robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2, altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited, no labeling is needed. Owing to tryptophan’s presence in carbohydrate-binding sites, also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases, glycosidases), this assay procedure can have relevance beyond galectins

Inhibition of the adenylylation of liver plasma membrane-bound proteins by plant and mammalian lectins

Liver plasma membrane contains four major (130-kDa, 120-kDa, 110-kDa and 100-kDa) sialic acid-containing glycopolypeptides that are able to undergo adenylylation, as well as phosphorylation (San José et al. (1990) J. Biol. Chem. 265; 20653-20661). To gain insight into the regulation of these processes, lectins are employed to probe the extent of influence of their interaction with membrane fractions for these reactions. We demonstrate that the beta-galactoside-specific lectins from bovine heart and mistletoe at low concentrations inhibit the adenylylation of this set of plasma membrane glycopolypeptides. The extent of phosphorylation of these polypeptides is also reduced although to a lesser degree. Concanavalin A, too, inhibits the adenylylation of the plasma membrane glycopolypeptides, although higher concentrations of this lectin were required, whereas wheat germ lectin has only a very small inhibitory effect. The adenylylable polypeptides were isolated by concanavalin A-agarose chromatography upon elution with mannose. In agreement with this result, control experiments with a panel of neoglycoproteins indicate that mannose residues appear to be required for the concanavalin A-induced inhibition of the adenylylation. Neoglycoproteins containing mannose 6-phosphate, lactose, fucose, or sialic acid instead of mannose lack the ability to protect the adenylylation from the inhibitory action of concanavalin A. In contrast, none of the above-mentioned neoglycoproteins, nor asialofetuin, nor galactose-containing saccharides protect the adenylylation against the inhibitory effect of both the mistletoe and bovine heart lectins, emphasizing the importance of either high affinity carbohydrate ligands in the overall process, or other ligand sites for the lectins beside carbohydrates to affect the regulation of the adenylylation system.This work was supported in part by grants (to A.V.) from the Dirección General de Investigación Científica y Técnica (PB 89-0079). and from the Consejería de Educación de la Comunidad de Madrid (C174-90 and 366/92) Spain, grants (to H.-J.G.) from the Dr. M. Scheel-Stiftung für Krebsforschung and the BMFT program Alternative Methoden der Krebsbekämpfung, Germany, and the Acciones Imegradas (42A) between Germany and Spain (to H.-J.G. andA.V.). E.S.J. is the recipient of a predoctoral fellowship from the Departamento de Educación, Universidades e Investigación del Gobierno Vasco.Peer Reviewe

Thio- and selenoglycosides as ligands for biomedically relevant lectins: Valency-activity correlations for benzene-based dithiogalactoside clusters and first assessment for (di)selenodigalactosides

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Lectins: getting familiar with translators of the sugar code

The view on the significance of the presence of glycans in glycoconjugates is undergoing a paradigmatic change. Initially mostly considered to be rather inert and passive, the concept of the sugar code identifies glycans as highly versatile platform to store information. Their chemical properties endow carbohydrates to form oligomers with unsurpassed structural variability. Owing to their capacity to engage in hydrogen (and coordination) bonding and C-H/π-interactions these "code words" can be "read" (in Latin, legere) by specific receptors. A distinct class of carbohydrate-binding proteins are the lectins. More than a dozen protein folds have developed carbohydrate-binding capacity in vertebrates. Taking galectins as an example, distinct expression patterns are traced. The availability of labeled endogenous lectins facilitates monitoring of tissue reactivity, extending the scope of lectin histochemistry beyond that which traditionally involved plant lectins. Presentation of glycan and its cognate lectin can be orchestrated, making a glycan-based effector pathway in growth control of tumor and activated T cells possible. In order to unravel the structural basis of lectin specificity for particular glycoconjugates mimetics of branched glycans and programmable models of cell surfaces are being developed by strategic combination of lectin research with synthetic and supramolecular chemistry