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

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

Symmetric dithiodigalactoside: strategic combination of binding studies and detection of selectivity between a plant toxin and human lectins

Thioglycosides offer the advantage over O-glycosides to be resistant to hydrolysis. Based on initial evidence of this recognition ability for glycosyldisulfides by screening dynamic combinatorial libraries, we have now systematically studied dithiodigalactoside on a plant toxin (Viscum album agglutinin) and five human lectins (adhesion/growth-regulatory galectins with medical relevance e.g. in tumor progression and spread). Inhibition assays with surface-presented neoglycoprotein and in solution monitored by saturation transfer difference NMR spectroscopy, flanked by epitope mapping, as well as isothermal titration calorimetry revealed binding properties to VAA (Ka: 1560 ± 20 M-1). They were reflected by the structural model and the affinity on the level of toxin-exposed cells. In comparison, galectins were considerably less reactive, with intrafamily grading down to very minor reactivity for tandem-repeat-type galectins, as quantitated by radioassays for both domains of galectin-4. Model building indicated contact formation to be restricted to only one galactose moiety, in contrast to thiodigalactoside. The tested lycosyldisulfide exhibits selectivity between the plant toxin and the tested human lectins, and also between these proteins. Therefore, glycosyldisulfides have potential as chemical platform for inhibitor design

Prognostic significance of endogenous adhesion/growth-regulatory lectins in lung cancer

Objective: To determine the expression of endogenous adhesion/growth-regulatory lectins and their binding sites using labeled tissue lectins as well as the binding profile of hyaluronic acid as an approach to define new prognostic markers. Methods: Sections of paraffin-embedded histological material of 481 lungs from lung tumor patients following radical lung excision processed by a routine immunohistochemical method (avidin-biotin labeling, DAB chromogen). Specific antibodies against galectins-1 and - 3 and the heparin-binding lectin were tested. Staining by labeled galectins and hyaluronic acid was similarly visualized by a routine protocol. After semiquantitative assessment of staining, the results were compared with the pT and pN stages and the histological type. Survival was calculated by univariate and multivariate methods. Results: Binding of galectin-1 and its expression tended to increase, whereas the parameters for galectin-3 decreased in advanced pT and pN stages at a statistically significant level. The number of positive cases was considerably smaller among the cases with small cell lung cancer than in the group with non-small-cell lung cancer, among which adenocarcinomas figured prominently with the exception of galectin-1 expression. Kaplan-Meier computations revealed that the survival rate of patients with galectin-3-binding or galectin-1-expressing tumors was significantly poorer than that of the negative cases. In the multivariate calculations of survival lymph node metastases ( p < 0.0001), histological type ( p = 0.003), galectin-3-binding capacity ( p = 0.01), galectin-3 expression ( p = 0.03) and pT status ( p = 0.003) proved to be independent prognostic factors, not correlated with the pN stage. Conclusion: The expression and the capacity to bind the adhesion/growth regulatory galectin-3 is defined as an unfavorable prognostic factor not correlated with the pTN stage. Copyright (C) 2005 S. Karger AG, Basel

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

Differentiation-dependent glycosylation of cells in squamous cell epithelia detected by a mammalian lectin

The squamous stratified epithelia contain a proliferative (harboring mitotic activity) and a differentiating compartment. Due to the potential of protein-carbohyd rate interactions to regulate cellular activities we introduced a mammalian lectin to cyto- and histochemical analysis. We answer the questions of whether and to what extent this new probe can pinpoint differentiation-dependent glycosylation changes in sections and in culture of keratinocytes. Material and Methods: Purification and labeling enabled monitoring of galectin-3 reactivity in frozen sections of human and pig epidermis and basal cell carcinomas as well as in culture of keratinocytes. The staining pattern of the lectin was correlated with the staining profile of other cell markers including desmosomal proteins, beta(1) integrin, and the proliferation marker Ki-67. The Dolichos biflorus agglutinin (DBA) sharing binding reactivity of galectin-3 to the A type histoblood group epitope was used for comparison. Results: Both lectins exhibit suprabasal binding. However, their profiles were not identical, substantiated by lack of coinhibition. Strong DBA reactivity was also observed in a limited number of basal layer cells, namely in cells without the expression of the proliferation marker Ki-67. Cultured mitotic epidermal cells have no reactivity for DBA. Presence of ligands for this plant lectin was connected with decreased positivity of nuclei for Ki-67 and the occurrence of ring-shaped nucleoli, micronucleoli or absence of nucleoli. Considering colocalization the pattern of galectin-3-binding sites coincided with the presence of desmosomal proteins such as desmoplakin-1 and desmoglein but not beta(1) integrin, a potential ligand. Interestingly, studied basal cell carcinomas expressed no binding sites for galectin-3, while a limited number of cells were DBA-reactive. Conclusion: The expression of galectin-3-binding sites and also DBA-reactive glycoligands correlates with an increased level of differentiation and/or cessation of proliferation in the examined squamous stratified epithelia. Further application of tissue lectins for characterizing ligand expression and its modulation is an important step to reveal functional relevance