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Bone is a vascularized organic-inorganic composite tissue that shows a heavily-mineralized extracellular matrix (ECM) on the nanoscale. Herein, the nucleation of calcium phosphates during the biomineralization process was mimicked using negatively-charged cellulose nanocrystals (CNCs). These mineralized-CNCs were combined with platelet lysate to produce nanocomposite scaffolds through cryogelation to mimic bone ECM protein-mineral composite nature and take advantage of the bioactivity steaming from platelet-derived biomolecules. The nanocomposite scaffolds showed high microporosity (94-95%), high elasticity (recover from 75% strain cycles), injectability, and modulated platelet-derived growth factors sequestration and release. Furthermore, they increased alkaline phosphatase activity (up to 10-fold) and up-regulated the expression of bone-related markers (up to 2-fold), without osteogenic supplementation, demonstrating their osteoinductive properties. Also, the scaffolds promoted the chemotaxis of endothelial cells and enhanced the expression of endothelial markers, showing proangiogenic potential. These results suggest that the mineralized nanocomposite scaffolds can enhance bone regeneration by simultaneously promoting osteogenesis and angiogenesis.

Mise-en-jeu is the ontological equivalent of film’s mise-en-scène. As such, mise-en-jeu is a cinematographic language through which game designers communicate. It offers designers the ability to create and shape the aesthetics of videogames’ mediated space, the space of the cinematographic presentation.Our prior work on mise-en-jeu focused on the visual aspects of videogames. With that in mind, starting with an analysis of mise-en-scéne, this paper provides an understanding of how sound is relevant for meaning-making through mise-en-jeu. Since videogames make use of some of motion picture’s filming techniques, we first studied practitioners and academics in the history of film, approaching videogames afterwards.The results of this research show that sound in mise-en-jeu allows designers to provoke emotions in players and to assist those players in formulating meaning as intended by the designers. We also found that mise-en-jeu allows for the deconstruction and interpretation of the characteristics of various variables of videogames’ mediated space. Therefore it allows us to understand better the relationship between videogames as audiovisual artefacts and the potential meanings that emerge from playing them.

This research examines temporal continuities in videogames—the passing of time regardless of player action—and players’ understanding of it, taking into account videogame and media theory. It presents a chronology of schools of thought and an analysis of the application of the theory to two case studies, to determine the motives behind players’ actions over time.

A lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N-acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X-ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity. Proteins 2017; 85:969-975. © 2016 Wiley Periodicals, Inc.

Green roofs are systems that can help to solve some urban environmental problems. A multi-layer green roof has five layers: from waterproof membrane till substrate and vegetation. Substrate is arguably the most important component of green roofs but there is still much to opti-mize and exploit. Also, world needs to avoid uncontrolled dispersal of waste. This new approach can be applied to substrates production. The main objective of this study is to compare and eval-uate 8 substrates incorporating industrial wastes with a commercial substrate. Employed plants were Armeria welwitschii, Festuca glauca and Sedum sediforme. Alongside, the rhizosphere eu-karyotic biocenosis (protists communities) were observed in optical microscope. Preliminary re-sults revealed that some industrial wastes may replace commonly used materials on green roofs substrate increasing plant growth. With regards to eukaryotic biocenosis, the performed observa-tions, point out that, these organisms can be good bioindicators for green roof technology., This work has been co-financed by Compete 2020, Portugal 2020 and the European Union through the European Regional Development Fund – FEDER within the scope of the project EGR - EcoGreenRoof: Desenvolvimento de eco-materiais para coberturas verdes (POCI-01-0247-FEDER-033728).

CBMs (carbohydrate-binding modules) are a class of polypeptides usually associated with carbohydrate-active enzymatic sites. We have characterized a new member of the CBM40 family, coded from a section of the gene NanI from Clostridium perfringens . Glycan arrays revealed its preference towards α(2,3)-linked sialosides, which was confirmed and quantified by calorimetric studies. The CBM40 binds to α(2,3)-sialyl-lactose with a K d of ∼30 μM, the highest affinity value for this class of proteins. Inspired by lectins' structure and their arrangement as multimeric proteins, we have engineered a dimeric form of the CBM, and using SPR (surface plasmon resonance) we have observed 6–11-fold binding increases due to the avidity affect. The structures of the CBM, resolved by X-ray crystallography, in complex with α(2,3)- or α(2,6)-sialyl-lactose explain its binding specificity and unusually strong binding. * CBM, : carbohydrate-binding module; CFG, : Consortium for Functional Glycomics; GlcNAc, : N -acetylglucosamine; HBS-T, : Hepes-buffered saline with Tween 20; ITC, : isothermal titration calorimetry; LFA, : Limax flavus agglutinin; MAA, : Maackia amurensis agglutinin; MIC, : minimal inhibition concentration; MME, : monomethyl ether; Neu5Ac, : N -acetylneuraminic acid; PSL-I, : Polyporus squamosus lectin I; SiaOPr, : Neu5Ac derivative with a propargyl aglycon; 3′-SL, : 3′-sialyl-lactose; 6′-SL, : 6′-sialyl-lactose; 3′-SLN, : 3′-sialyl-lactosamine; 6′-SLN, : 6′-sialyl-lactosamine; SNA-I, : Sambucus nigra agglutinin I; SPR, : surface plasmon resonance; TSA, : thermal shift assay; WGA, : wheatgerm agglutinin

Creating a mobile urban tourism storytelling application presents several interactivity challenges on how to convey an engaging multimedia experience on-site. This article describes a methodology for fast prototyping of a multimedia mobile applications dedicated to urban tourism storytelling. The application can be a game that takes advantage of several location-based technologies, freely available geo-referenced media, and augmented reality for immersive gameplay. The goal is to create serious games for tourism that follow a main narrative but where the story can automatically adapt itself to the current location of the player, assimilate possible detours and allow posterior out-of-location playback. Adaptable stories can use dynamic information from map sources such as points of interest (POI), elevation or virtual buildings. The main focus is for these location-based storytelling games to create more engagement between the tourists and the urban environment. To explore this concept, an application was designed for the city of Porto: Unlocking Porto. This location-based game with a central, yet adaptable, story engages the player into the main sights following an augmented reality path while playing small games. The article discusses and presents solutions for media acquisition, interactive storytelling, game-design interface and multi-disciplinary coordination for mobile app development.

Glycosylation is a group of post-translational modifications that displays a large variety of structures and are implicated in a plethora of biological processes. Therefore, studying glycosylation requires different technical approaches and reliable tools, lectins being part of them. Here, we describe the use of the recombinant mushroom lectin PVL to discriminate O-GlcNAcylation, a modification consisting in the attachment of a single N-acetylglucosamine residue to proteins confined within the cytosolic, nuclear and mitochondrial compartments. Recombinant PVL (Psathyrella velutina lectin) (rPVL) displays significantly stronger affinity for GlcNAc over Neu5Ac residues as verified by thermal shift assays and surface plasmon resonance experiments, being therefore an excellent alternative to WGA (wheat germ agglutinin). Labeling of rPVL with biotin or HRP (horseradish peroxidase) allows its useful and efficient utilization by western blot. The staining of whole cell lysates with labeled-rPVL was dramatically decreased in response to O-GlcNAc transferase knockdown and seen to increase after pharmacological blockade of O-GlcNAcase. Also, HRP-rPVL seemed to be more sensitive than the anti-O-GlcNAc antibody RL2. Thus, rPVL is a potent new tool to selectively detect O-GlcNAcylated proteins. 27;2

A lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N-acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X-ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity. Proteins 2017; 85:969-975. © 2016 Wiley Periodicals, Inc.

The structurally related AB5-type heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are classified into two major types. The type I group includes cholera toxin (CT) and E. coli LT-I, whereas the type II subfamily comprises LT-IIa, LT-IIb and LT-IIc. The carbohydrate-binding specificities of LT-IIa, LT-IIb and LT-IIc are distinctive from those of cholera toxin and E. coli LT-I. Whereas CT and LT-I bind primarily to the GM1 ganglioside, LT-IIa binds to gangliosides GD1a, GD1b and GM1, LT-IIb binds to the GD1a and GT1b gangliosides, and LT-IIc binds to GM1, GM2, GM3 and GD1a. These previous studies of the binding properties of type II B-subunits have been focused on ganglio core chain gangliosides. To further define the carbohydrate binding specificity of LT-IIb B-subunits, we have investigated its binding to a collection of gangliosides and non-acid glycosphingolipids with different core chains. A high-affinity binding of LT-IIb B-subunits to gangliosides with a neolacto core chain, such as Neu5Gcα3- and Neu5Acα3-neolactohexaosylceramide, and Neu5Gcα3- and Neu5Acα3-neolactooctaosylceramide was detected. An LT-IIb-binding ganglioside was isolated from human small intestine and characterized as Neu5Acα3-neolactohexaosylceramide. The crystal structure of the B-subunit of LT-IIb with the pentasaccharide moiety of Neu5Acα3-neolactotetraosylceramide (Neu5Ac-nLT: Neu5Acα3Galβ4GlcNAcβ3Galβ4Glc) was determined providing the first information for a sialic-binding site in this subfamily, with clear differences from that of CT and LT-I.

11 páginas, 4 figurasd, 2 esquemas -- PAGS nros. 4354-4364, Our recent description of pulse sequences for the intramolecular relay of 1H polarization to the 19F reporter of a carbohydrate ligand after saturation transfer from a cognate lectin prompted us to test the applicability of this technique for inhibitor screening. By strategically combining synthetic organic chemistry and cell assays with 19F-NMR-based competition experiments, we document the validity of this approach. Two mannose-specific leguminous agglutinins as receptors, the α-methyl derivative of 2-deoxy-2-fluoro-D-mannopyranoside as sensor and synthetic mannosides selected to represent different inhibitory capacities were used to establish a test panel. Signal amplitudes were found to vary among the two related lectins, and their precipitation by glycodendrimers enabled inherent limits to be set. The obtained experimental basis was then broadened by assaying a galactopyranoside-binding plant toxin, using 6-deoxy-6-fluoro-D-galactopyranose as a sensor molecule. The easy identification of the two anomeric signals by 19F NMR spectroscopy enabled ready detection of the preference of this lectin for the α-anomer and allowed the two individual inhibition profiles to be deduced. These 19F NMR spectroscopic data were in accord with the activities of inhibitors used to protect cells from toxicity. Our results therefore provide an experimental basis for 19F-NMR-based inhibitor screening, This work was generously supported by the European Community (Marie Curie Research training grant MCRTN-CT-2005–19561), the Ministerio de Ciencia e Innovación (MICINN) (grant numbers CTQ2009-08536 and BFU2009-10052), and the Spanish Institute of Health Carlos III (ISCIII), under the initiative CIBER of Respiratory Diseases (CIBERES). The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 260600 (“GlycoHIT”)

A protocol based on saturation transfer difference (STD)-NMR spectroscopy competition experiments is presented as a fast compound-screening method (30 min acquisition time per compound) capable of evaluating ligand properties for the lectin site with the intention of selecting compounds for iterative refinement. Glycosides with variations in the aglycon and plant toxin viscumin (VAA) were studied systematically. The best hits were further analyzed via docking simulations to rationalize an increased affinity structurally. The strategy described can readily be extended to other medically relevant lectins.

This thesis deals with the design, formation and evaluation of dynamic systems constructed by means of sulfur-containing reversible reactions, in organic and aqueous media and under mild conditions. In a first part, the synthesis of thioglycoside derivatives, constituting the biologically relevant starting components of the dynamic systems, is described. In addition, the pD-profile of the mutarotation process in aqueous media for a series of 1-thioaldoses is reported and revealed an astonishing beta-anomeric preference for all the carbohydrate analogs under acidic or neutral conditions. In a second part, the phosphine-catalyzed or -mediated disulfide metathesis for dynamic system generation in organic or aqueous media is presented, respectively. The direct in situ 1H STD-NMR resolution of a dynamic carbohydrate system in the presence of a target protein (Concanavalin A) proved the suitability and compatibility of such disulfide metathesis protocols for the discovery of biologically relevant ligands. In a third part, hemithioacetal formation is demonstrated as a new and efficient reversible reaction for the spontaneous generation of a dynamic system, despite a virtual character of the component associations in basic aqueous media. The direct in situ 1H STD-NMR identification of the best dynamic beta-galactosidase inhibitors from the dynamic HTA system was performed and the results were confirmed by inhibition studies. Thus, the HTA product formed from the reaction between 1-thiogalactopyranose and a pyridine carboxaldehyde derivative provided the best dynamic inhibitor. In a fourth and final part, a dynamic drug design strategy, where the best inhibitors from the aforementioned dynamic HTA system were used as model for the design of non-dynamic (or “static”) beta-galactosidase inhibitors, is depicted. Inhibition studies disclosed potent leads among the set of ligands.

8 páginas, 8 figuras, 1 tabla -- PAGS nros. 5891-5898, A combined NMR, computational, and HPLC study of the inclusion of aromatic and fluoroaromatic compounds in cyclodextrins has been carried out with the aim of studying this process as a model for carbohydrate···aromatic interactions. NMR experiments showed that although β-CD formed an inclusion complex with benzene, no stable inclusion adduct was formed with hexafluorobenzene. MM3* calculations confirmed these data and, when extended to naphthalene, anthracene, phenanthrene, and their partially and fully fluorinated analogues, showed that β-CD formed inclusion adducts only with the hydrocarbons, whereas the partially fluorinated derivatives were shown to enter the cavity only with their non-fluorinated part, and the perfluoro compounds were not included. HPLC experiments, carried out by eluting these compounds through CD-modified HPLC columns, also confirmed these results, at least with the mono- and bicyclic arenes. Indeed, the elution of these derivatives showed that the efficiency of the inclusion, as determined by comparing HPLC retention times, decreased on passing from hydrocarbons to partially fluorinated compounds, and reached a minimum with perfluorinated derivatives. In contrast with this general trend, 1,2,3,4-tetrafluoroanthracene had longer retentions times on β- and γ-CD-modified HPLC columns than anthracene, possibly for solubility reasons. As a whole, these results were interpreted in terms of carbohydrate···aromatic interactions between the C5–H5 bonds on the hydrophobic faces of the sugars and the π electrons of the aromatic partner. The interactions seem to have an important dispersive component.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008), We thank the Spanish Ministerio de Educación y Ciencia (MEC) (grant CTQ-2006-10335) and the European Union (grant MRTN-CT2006-035546) for financial support

The characterization of the binding of reducing carbohydrates present as mixtures of anomers in solution to a sugar recepor (lectin) poses severe difficulties. In this situation, NMR spectroscopy enables the observation of signals for each anomer in the mixture by applying approaches based on ligand observation. Saturation transfer difference (STD) NMR allows fast and efficient screening of compound mixtures for reactivity to a receptor. Owing to the exceptionally favorable properties of 19F in NMR spectroscopy and the often complex 1H spectra of carbohydrates, 19F-containing sugars have the potential to be turned into versatile sensors for recognition. Extending the recently established 1H 1H STDre19F-NMR technique, we here demonstrate its applicability to measure anomeric selectivity of binding in a model system using the plant lectin concanavalin A (ConA) and 2-deoxy-2-fluoro-D-mannose. Indeed, it is also possible to account for the mutual inhibition between the anomers on binding to the lectin by means of a kinetic model. The monitoring of 19F-NMR signal perturbation disclosed the relative activities of the anomers in solution and thus enabled the calculation of their binding affinity towards ConA. The obtained data show a preference for the anomer that increases with temperature. This experimental approach can be extended to others systems of biomedical interest by testing human lectins with suitably tailored glycan derivatives., The authors acknowledge the financial support from MINECO, Spain (project CTQ2012-32025) and the EC-funded Marie Curie Initial Training Network GLYCOPHARM (PITN-GA-2012-317297) and thank the reviewers for their valuable input., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

3 páginas, 2 figuras -- PAGS nros. 5666-5668, As a novel approach for studying carbohydrate-lectin interactions spectroscopically, we combine the resolution and specificity of (19)F-detected NMR spectroscopy with the sensitivity of the saturation transfer difference (STD) technique. The resulting background-free (19)F-STD spectra open a promising perspective for broad application with medical relevance, for example, in drug discovery, Financial support by a bizkaia: Xede grant, an EC Marie Curie Research Training network (MRTN-CT2005-019561), the Ministry of Science and Innovation of Spain (CTQ2006- 10874-C02-01)

The glycome, that is, the cohort of carbohydrates within a cell or tissue, plays a key part in diverse biological interactions involved in health and disease. Glycans are structurally complex and notoriously difficult to analyze. Lectin microarrays, a sensitive and high-throughput method for glycomic profiling, provide a global view of the glycome. In recent work, this technology has been successfully applied to a wide range of studies, from identification of glycan-based stem cell markers to the detection of pathogens and early diagnosis of disease. This review focuses on advances in the field of lectin microarrays.

15 páginas, 7 figuras, By definition, adhesion/growth-regulatory galectins are known for their ability to bind beta-galactosides such as Gal beta(1 -> 4)Glc (lactose). Indications for affinity of human galectin-1 to alpha-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH-pi stacking. These issues are resolved by a combination of (15)N-(1)H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the alpha(1 -> 3/4)-linked digalactosides and also Gal alpha(1 -> 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the alpha(1 -> 4) disaccharide, however, the typical CH-pi interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of alpha-linkage in digalactosides has an impact on maintaining CH-pi interactions and the pattern of hydrogen bonding, explaining preference for the alpha(1 -> 3) linkage. Thus, this lectin is able to accommodate both alpha- and beta-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit., This work was sponsored by research grants from the National Cancer Institute (NIH grant # CA096090) to KHM, the Ministery of Science and Innovation of Spain (CTQ2009-08536) to JJ-B and the EC Seventh Framework Program (FP7/2007-2013) under grant agreement no. 260600 ("GlycoHIT") to JJ-B and H-JG.The authors wish to thank the Minnesota Supercomputing Institute (University of Minnesota) for providing computer resources. NMR instrumentation was provided with funds from the NSF (BIR-961477), the University of Minnesota Medical School and the Minnesota Medical Foundation., Minnesota Supercomputing Institute (University of Minnesota) for providing computer resources. NMR instrumentation was provided with funds from the NSF (BIR-961477), the University of Minnesota Medical School and the Minnesota Medical Foundation.

11 páginas, 8 figuras -- PAGS nrs. 5445-5455, 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 (K(a): 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 glycosyldisulfide 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., This work has been generously supported by grants CTQ2009-08536, SAF2008-00945, BFU2006-10288 and BFU2009-10052 from the Spanish Ministry, an EC Marie Curie Research Training Network grant (contract no. MRTN-CT-2005-019561), the EC research program GlycoHIT (contract ID 260600), the Verein zur Forderung des biologisch-technologischen Fortschritts in der Medizin e. V. and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII). We thank Drs B. Friday and S. Namirha for inspiring discussions as well as M. V. Lopez-Moyano for excellent technical assistance.

Detailed facts of importance to specialist readers are published as ”Supporting Information” at Wiley OnlineLibrary with the filename: cmdc_200900476_sm_miscellaneous_information.pdf, The growing awareness of the sugar code—i.e. the biological functionality of glycans—is leading to increased interest in lectins as drug targets. The aim of this study was to establish a strategic combination of screening procedures with increased biorelevance. As a model, we used a potent plant toxin (viscumin) and lactosides synthetically modified at the C6/C6′ positions and the reducing end aglycan. Changes in the saturation transfer difference (STD) in NMR spectroscopy, applied in inhibition assays, yielded evidence for ligand activity and affinity differences. Inhibitory potency was confirmed by the blocking of lectin binding to a glycoprotein-bearing matrix. In cell-based assays, iodo/azido-substituted lactose derivatives were comparatively active. Interestingly, cell-type dependence was observed, indicating the potential of synthetic carbohydrate derivative to interact with lectins in a cell-type (glycan profile)-specific manner. These results are relevant to research into human lectins, glycosciences, and beyond., Financial support by an EC Marie Curie Research Training network (MRTN-CT2005–019561), the Ministry of Science and Innovation of Spain (CTQ2006–10874-C02–01), and the research initiative LMUexcellent is gratefully acknowledged.

Supplementary data can be obtained at: http://www.sciencedirect.com/science/MiamiMultiMediaURL/1-s2.0-S0003269709006071/1-s2.0-S0003269709006071-mmc1.doc/272363/html/S0003269709006071/4a58356491e3b2153684edba66691f9a/mmc1.doc, Small molecules are difficult to detect in protein solutions, whether they originate from elution during affinity chromatography (e.g., imidazole, lactose), buffer exchange (Tris), stabilizers (e.g., beta-mercaptoethanol, glycerol), or excess labeling reagents (fluorescent reagents). Mass spectrometry and high-pressure liquid chromatography (HPLC) often require substantial efforts in optimization and sample manipulation to provide sufficient sensitivity and reliability for their detection. One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) could, in principle, detect residual amounts of small molecules in protein solutions down to equimolecular concentrations with the protein. However, at lower concentrations, the NMR signals of the contaminants can be hidden in the background spectrum of the protein. As an alternative, the 1D diffusion difference protocol used here is feasible. It even improves the detection level, picking up NMR signals from small-molecule contaminants at lower concentrations than the protein itself. We successfully observed 30 microM imidazole in the presence of four different proteins (1-1.5 mg/ml, 6-66 kDa, 25-250 microM) by 1D diffusion-ordered spectroscopy (DOSY) difference and 1-h total acquisition time. Of note, imidazole was not detected in the corresponding 1D (1)H NMR spectra. This protocol can be adapted to different sample preparation procedures and NMR acquisition methods with minimal manipulation in either deuterated or nondeuterated buffers., D.-J.S. and P.G. acknowledge the financial support of their groups from the Instituto de Tecnologia Química e Biológica (ITQB). J.P.R., S.A., H.-J.G., and J.J.-B. acknowledge generous funding from an EC Marie Curie Research Training Network grant (MRTN-CT-2005-019561). The Madrid group also thanks the Ministry of Science of Innovation of Spain (CTQ2006-10874-C02-01 and CTQ2009-8536). The 500-MHz NMR spectrometer used in this study is part of the Portuguese National NMR Network acquired with funds from Fundação para a Ciência e a Tecnologia (Portugal) and Fundo Europeo de Desenvolvimento Regional (FEDER).

Human tandem-repeat-type galectin-9 is a potent adhesion/growth-regulatory effector via lectin capacity of its N- and C-terminal domains. This bioactivity prompted further crystallographic study of the N-domain, combined with analysis in solution. Binding of lactose markedly increased the N-domain's resistance to thermal denaturation. Crystallography revealed its intimate contact profile, besides detecting an extension of the beta-sandwich fold by an antiparallel beta-strand F0 aligned to the C-terminal F1 strand. Ligand accommodation in its low-energy conformation leads to a movement of Arg87's side chain. As consequence, the ligand's glucose moiety and Arg87 become hydrogen bonded. The resulting predictions for spatial parameters in solution were verified by determining (a) the pattern of magnetization transfer from the protein to protons of lactose and Forssman disaccharide by NMR spectroscopy and (b) the ellipticity changes at wavelengths characteristic for Trp/Tyr residues in near-UV CD spectroscopy. Whereas solid-phase assays confirmed a previously noted tendency for homo- and heterotypic aggregation, gel filtration and ultracentrifugation disclosed monomeric status in solution, in line with crystallographic data. Using cell mutants with defects in glycosylation, this lectin domain was shown to preferentially bind N-glycans without alpha2,3-sialylation. Since proximal promoter sequences were delineated to diverge markedly among galectin genes and resulting differences in expression profiles were exemplarily documented immunohistochemically, the intrafamily diversification appears to have assigned this protein to a characteristic expression and activity profile among galectins. Our data thus take the crystallographic information to the level of the lectin in solution and in tissues by a strategic combination of spectroscopic and cell/histochemical assays.