The 2.15 A crystal structure of CG-16, the developmentally regulated homodimeric chicken galectin

Differential developmental regulation of expression, fine-specificity differences in ligand recognition and disparate capacity for homodimerization are characteristics of the two currently known proto-type chicken galectins. The X-ray crystal structure of the first avian galectin, the homodimeric agglutinin from chicken liver (CG-16), has been solved in the absence of ligand in two crystal forms. Although the arrangement of lectin dimers in the two crystals is different, the structure of the monomers and their association into the extended beta-sandwich that characterises the dimer are virtually identical. The fold establishes a beta-sandwich motif composed of a five-stranded and a six-stranded beta-sheet evocative of proto-type mammalian galectins. The carbohydrate-binding site is occupied by six water molecules that take the place of the sugar in the complex. They help to stabilise in the absence of the ligand the spatial arrangement of the amino acid side-chains involved in sugar recognition. Docking of N-acetyllactosamine into the binding site reveals that three of these water molecules, which are in direct contact with the protein, occupy positions equivalent to the key sugar hydroxyl groups, namely the hydroxyls at positions 4 and 6 of the galactose unit and at position 3 of the N-acetylglucosamine unit. Crystallographic data are fully consistent with the binding features in solution previously derived from chemical mapping with deoxy, fluoro and O-methyl derivatives and laser photo-CIDNP (chemically induced dynamic nuclear polarisation) studies. The possible molecular basis for the monomeric character of the chicken intestinal galectin as well as potential mechanisms of oxidative inactivation by disulphide bridging are evaluated on the basis of the given structural information concerning the CG-16 dimer interface and the cysteine residues, respectively.     

Analysis of selected blood and immune cell responses to carbohydrate-dependent surface binding of proto- and chimera-type galectins

Cell surface glycans present docking sites to endogenous lectins. With growing insight into the diversity of lectin families it becomes important to answer the question on the activity profiles of individual family members. Focusing on galectins (-galactoside-binding proteins without Ca²⁺-requirement sharing the jelly-roll-like folding pattern), this study was performed to assess the potency of proto-type galectins (galectins-1 and -7 and CG-16) and the chimera-type galectin-3 to elicit selected cell responses by carbohydrate-dependent surface binding and compare the results. The galectins, except for galectin-1, were found to enhance detergent (SDS)-induced hemolysis of human erythrocytes to different degrees. Their ability to confer increased membrane osmofragility thus differs. Aggregation of neutrophils, thymocytes and platelets was induced by the proto-type galectin-1 but not -7, by CG-16 and also galectin-3. Cell-type-specific quantitative differences and the importance of the fine-specificity of the galectin were clearly apparent. In order to detect cellular responses based on galectin binding and bridging of cells the formation of haptenic-sugar-resistant (HSR) intercellular contacts (an indicator of post-binding signaling) was monitored. It was elicited by CG-16 and galectin-1 but not galectin-3, revealing another level at which activities of individual galectins can differ. Acting as potent elicitor of neutrophil aggregation, CG-16-dependent post-binding effects were further analyzed. Carbohydrate-dependent binding to the neutrophils' surface led to a sustained increase of cytoplasmic Ca²⁺ concentration in a dose-dependent manner. The ability of CG-16 to activate H₂O₂ generation by human peripheral blood neutrophils was primed by the Ca²⁺-ionophor ionomycin and by cytochalasin B. In a general context, these results emphasize that – besides plant lectins as laboratory tools – animal lectins can trigger cell reaction cascades, implying potential in vivo relevance for the measured activities. Within the family of galectins, the activity profiles depend on the target cell type and the individual galectin. Notably, proto-type galectins do not necessarily share a uniform capacity as elicitor.     

Characterization of ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also of the expression of calcyclin in thyroid lesions

The purpose of this study was to characterize ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also the expression of calcyclin in various benign and malignant thyroid lesions. The extent of the binding of eight glycochemical probes was quantitatively assessed using computer-assisted microscopy on 76 thyroid lesions including 10 not-otherwise-specified multinodular goiters (S_MNG), 11 multinodular goiters with adenomatous hyperplasia (AH_MNG), 8 normomacrovesicular (NM_ADE) and 12 microvesicular (MIC_ADE) adenomas, and 9 papillary (P_CAR), 10 follicular variants of papillary (FvarP_CAR), 7 follicular (F_CAR) and 9 anaplastic (A_CAR) carcinomas. The 8 histochemical probes included 5 animal lectins (including galectins and sarcolectin), 1 polyclonal antibody (raised against calcyclin) and 2 immunoglobulin G subfractions from human serum with selectivity to alpha- and beta-galactosyl residues. The results show that multinodular goiters with adenomatous hyperplasia exhibited histochemical characteristics intermediate to those of normal multinodular goiters and microvesicular adenomas. Normomacrovesicular adenomas behaved very distinctly from microvesicular ones. Microvesicular adenomas were more closely related to differentiated thyroid carcinomas than any other type of benign thyroid lesions of epithelial origin. Papillary and follicular carcinomas seemed to represent the two extremes of the same biological entity with the follicular variant of the papillary carcinoma serving as a biological link between these two extremes. Anaplastic carcinomas behaved in a significantly different manner when compared to the differentiated forms of thyroid carcinomas. The results suggest that the patterns of expression of the glycoconjugates investigated in the present study may constitute useful tools for characterizing lesions in the human thyroid.     

Thermodynamic parameters of the interaction of Urtica dioica agglutinin with N-acetylglucosamine and its oligomers

The interaction between Urtica dioica agglutinin (UDA) and N-acetylglucosamine (GlcNAc) and its (1-4)-linked oligomers was studied by fluorescence titration and isothermal titration microcalorimetry. UDA possesses one significant binding site that can be measured calorimetrically. This site is composed of three subsites, each subsite accommodating one GlcNAc residue. The interaction is enthalpically driven, and the binding area of UDA is characterized by a H of interaction for a given oligosaccharide considerably smaller than that of wheat germ agglutinin (WGA), despite the fact that they both belong to a family of proteins composed entirely of hevein domains. Relatively high Cp values of the UDA-carbohydrate interactions and more favorable entropy term compared to WGA suggest that binding of the carbohydrate ligands by UDA has a higher hydrophobic component than that of WGA.     

NMR investigations of protein-carbohydrate interactions: insights into the topology of the bound conformation of a lactose isomer and beta-galactosyl xyloses to mistletoe lectin and galectin-1.

A hallmark of oligosaccharides is their often limited spatial flexibility, allowing them to access a distinct set of conformers in solution. Viewing each individual or even the complete ensemble of conformations as potential binding partner(s) for lectins in protein-carbohydrate interactions, it is pertinent to address the question on the characteristics of bound state conformation(s) in solution. Also, it is possible that entering the lectin's binding site distorts the low-energy topology of a glycosidic linkage. As a step to delineate the strategy of ligand selection for galactosides, a common physiological docking point, we have performed a NMR study on two non-homologous lectins showing identical monosaccharide specificity. Thus, the conformation of lactose analogues bound to bovine heart galectin-1 and to mistletoe lectin in solution has been determined by transferred nuclear Overhauser effect measurements. It is demonstrated that the lectins select the syn conformation of lactose and various structural analogues (Galbeta(1-->4)Xyl, Galbeta(1-->3)Xyl, Galbeta(1-->2)Xyl, and Galbeta(1-->3)Glc) from the ensemble of presented conformations. No evidence for conformational distortion was obtained. Docking of the analogues to the modeled binding sites furnishes explanations, in structural terms, for exclusive recognition of the syn conformer despite the non-homologous design of the binding sites.     

Detection of binding sites for biotinylated neoglycoproteins and heparin (endogenous lectins) during cerebellar ontogenesis in the rat

Endogenous carbohydrate-binding sites were studied during rat cerebellar development on sections of fixed tissue using synthetic tools, biotinylated neoglycoproteins, in conjunction with subsequent avidinperoxidase staining. Neoglycoproteins were constructed by chemically coupling the histochemically pivotal carbohydrate moieties to an inert carrier protein. The sugar part of the neoglycoproteins included common constituents of the carbohydrate part of cellular glycoconjugates, namely mannose, galactose, fucose, N-acetyl-glucosamine, N-acetylgalactosamine and N-acetyl-neuraminic acid to probe for the presence of respective endogenous receptors. Heparin was biotinylated after mild cyanogen bromide activation and aminoalkylation. Specific positive reactions were obtained for all neoglycoproteins and heparin. The staining pattern with the individual probes disclosed variable developmental regulation. Consequently, these results suggest that recognition processes during cerebellar development may include several types of carbohydrate determinants. In two instances, the binding of neoglycoproteins could be compared to endogenous lectin-specific antibodies. Despite a significant extent of accordance the comparison revealed notable differences. These differences were attributed primarily to fixation and the presence of physiological ligands that can mask the active endogenous carbohydrate-binding proteins. In any case, histochemical application of labeled neoglycoproteins is valuable to discern the presence, localization and developmental pattern of binding sites for the carbohydrate part of glycoconjugates, on which further biochemical and cell biological studies can consequently be based.     

Anaerobic degradation of papermill sludge in a two-phase digester containing rumen microorganisms and colonized polyurethane foam

Summary The use of reticulated polyurethane foam as a support material for the immobilization of methanogenic associations and its application to the anaerobic treatment of fine particulate solid wastes was investigated. The colonization of polyurethane support particles in a continuous upflow reactor fed on a mixture of acetate, propionate and butyrate, was both rapid and dense. The combination of rumen microorganisms and colonized support particles in a two-phase digester resulted in an efficient anaerobic decomposition of papermill sludge.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

This review focuses on the mechanisms of stress response in the synovial tissue of rheumatoid arthritis. The major stress factors, such as heat stress, shear stress, proinflammatory cytokines and oxidative stress, are discussed and reviewed, focusing on their potential to induce a stress response in the synovial tissue. Several pathways of stress signalling molecules are found to be activated in the synovial membrane of rheumatoid arthritis; of these the most important examples are heat shock proteins, mitogen-activated protein kinases, stress-activated protein kinases and molecules involved in the oxidative stress pathways. The expression of these pathways in vitro and in vivo as well as the consequences of stress signalling in the rheumatoid synovium are discussed. Stress signalling is part of a cellular response to potentially harmful stimuli and thus is essentially involved in the process of synovitis. Stress signalling pathways are therefore new and promising targets of future anti-rheumatic therapies.