Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells.

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-ß1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-ß1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-ß1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-ß1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-ß1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.

Murine homodimeric adhesion/growth-regulatory galectins-1, -2 and -7: comparative profiling of gene/ promoter sequences by database mining, of expression by RT-PCR/immunohistochemistry and of contact sites for carbohydrate ligands by computational chemistry.

Following the detection of individual members of the family of galectins it is an obvious challenge to define the extent of functional overlap/divergence among these proteins. As a step to address this issue a comparative profiling has been started in the mouse as a model organism, combining sequence analysis, expression patterns and structural features in the cases of the homodimeric galectins-1, -2 and -7. Close relationship was apparent at the level of global gene organization. Scrutiny of the proximal promoter regions for putative transcription-factor-binding sites by two search algorithms uncovered qualitative and quantitative differences with potential to influence the combinatorial functionality of regulatory sequences. RT-PCR mapping with samples from an array of 17 organs revealed significant differences, separating rather ubiquitous gene expression of galectin-1 from the more restricted individual patterns of galectins-2 and -7. Using specific antisera obtained by affinity depletion including stringent controls to ascertain lack of cross-reactivity these results were corroborated at the level of galectin localization in fixed tissue sections. Nuclear presence was seen in the case of galectin-1. In addition to nonidentical expression profiles the mapping of the carbohydrate recognition domains of galectins-1 and -7 by homology modelling and docking of naturally occurring complex tetra- and pentasaccharides disclosed a series of sequence deviations which may underlie disparate affinities for cell surface glycans/glycomimetic peptides. In view of applicability the presented data can serve as useful reference to delineate changes with respect to disease and in genetically engineered models. To enable more general conclusions on the galectin network it is warranted to further pursue this combined approach within this lectin family.

Distinct differences in binding capacity to saccharide epitopes in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas, and glioblastomas.

We monitored the expression of glycan-binding sites on a panel of 10 biotinylated neoglycoconjugates by means of quantitative computer-assisted microscopy to further study the molecular mechanisms in the extensive infiltration of the surrounding brain parenchyma by most astrocytic tumors. Three distinct histological compartments were analyzed for each of the 108 astrocytic tumors (15 pilocytic astrocytomas (WHO grade I), 25 astrocytomas (WHO grade II), 30 anaplastic astrocytomas (WHO grade III), and 38 glioblastomas (WHO grade IV) included in our series. These compartments were tumors (nonperivascular tumor astrocytes), perivascular tumor astrocytes, and blood vessel walls. Clear differences were observed between the pilocytic and the diffuse astrocytic tumors. Furthermore, malignant progression in the latter category was paralleled by a decrease in cells' ability to bind distinct sugar epitopes, especially the D-GalNAc(alpha1-3)-D-GalNAc-beta1-R determinant of the Forssman pentasaccharide in tumors, the alpha-L-fucose in perivascular tumor areas, and the beta-D-glucose in tumor vessel walls. Markedly, the level of binding site expression for alpha-D-mannose decreased in the tumors, the perivascular tumor areas, and the vessel walls. These glycohistochemical results imply the functional relevance of protein-carbohydrate interactions in this tumor system.

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.

Towards defining the role of glycans as hardware in information storage and transfer: basic principles, experimental approaches and recent progress.

The term 'code' in biological information transfer appears to be tightly and hitherto exclusively connected with the genetic code based on nucleotides and translated into functional activities via proteins. However, the recent appreciation of the enormous coding capacity of oligosaccharide chains of natural glycoconjugates has spurred to give heed to a new concept: versatile glycan assembly by the genetically encoded glycosyltransferases endows cells with a probably not yet fully catalogued array of meaningful messages. Enciphered by sugar receptors such as endogenous lectins the information of code words established by a series of covalently linked monosaccharides as letters for example guides correct intra- and intercellular routing of glycoproteins, modulates cell proliferation or migration and mediates cell adhesion. Evidently, the elucidation of the structural frameworks and the recognition strategies within the operation of the sugar code poses a fascinating conundrum. The far-reaching impact of this recognition mode on the level of cells, tissues and organs has fueled vigorous investigations to probe the subtleties of protein-carbohydrate interactions. This review presents information on the necessarily concerted approach using X-ray crystallography, molecular modeling, nuclear magnetic resonance spectroscopy, thermodynamic analysis and engineered ligands and receptors. This part of the treatise is flanked by exemplarily chosen insights made possible by these techniques.

Structural basis for chitin recognition by defense proteins: GlcNAc residues are bound in a multivalent fashion by extended binding sites in hevein domains.

BACKGROUND: Many plants respond to pathogenic attack by producing defense proteins that are capable of reversible binding to chitin, a polysaccharide present in the cell wall of fungi and the exoskeleton of insects. Most of these chitin-binding proteins include a common structural motif of 30 to 43 residues organized around a conserved four-disulfide core, known as the 'hevein domain' or 'chitin-binding' motif. Although a number of structural and thermodynamic studies on hevein-type domains have been reported, these studies do not clarify how chitin recognition is achieved. RESULTS: The specific interaction of hevein with several (GlcNAc)(n) oligomers has been studied using nuclear magnetic resonance (NMR), analytical ultracentrifugation and isothermal titration microcalorimetry (ITC). The data demonstrate that hevein binds (GlcNAc)(2-4) in 1:1 stoichiometry with millimolar affinity. In contrast, for (GlcNAc)(5), a significant increase in binding affinity is observed. Analytical ultracentrifugation studies on the hevein-(GlcNAc)(5,8) interaction allowed detection of protein-carbohydrate complexes with a ratio of 2:1 in solution. NMR structural studies on the hevein-(GlcNAc)(5) complex showed the existence of an extended binding site with at least five GlcNAc units directly involved in protein-sugar contacts. CONCLUSIONS: The first detailed structural model for the hevein-chitin complex is presented on the basis of the analysis of NMR data. The resulting model, in combination with ITC and analytical ultracentrifugation data, conclusively shows that recognition of chitin by hevein domains is a dynamic process, which is not exclusively restricted to the binding of the nonreducing end of the polymer as previously thought. This allows chitin to bind with high affinity to a variable number of protein molecules, depending on the polysaccharide chain length. The biological process is multivalent.

NMR investigations of protein-carbohydrate interactions: studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N',N"-triacetylchitotriose.

Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N', N"-triacetylchitotriose has been analyzed by (1)H-NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target to solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was extended to assessment of the topology of the complex between pseudohevein and N, N',N"-triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton-proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand-induced conformational change. The average backbone (residues 3-41) RMSD of the 20 refined structures was 1.14 A, whereas the heavy atom RMSD was 2.18 A. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectin's capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyr-substitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin-1 and a mammalian C-type lectin. A comparison of the three-dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in solution has been performed, providing a data source about structural variability of the hevein domains. The experimentally derived structures and the values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems.

NMR investigations of protein-carbohydrate interactions binding studies and refined three-dimensional solution structure of the complex between the B domain of wheat germ agglutinin and N,N', N"-triacetylchitotriose.

The specific interaction of the isolated B domain of wheat germ agglutinin (WGA-B) with N,N',N"-triacetylchitotriose has been analyzed by 1H-NMR spectroscopy. The association constants for the binding of WGA-B to this trisaccharide have been determined from both 1H-NMR titration experiments and microcalorimetry methods. Entropy and enthalpy of binding have been obtained. The driving force for the binding process is provided by a negative DeltaH which is partially compensated by negative DeltaS. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 327 protein proton-proton distance constraints. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the refined solution conformation of this protein/carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein NOEs were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 35 refined structures was 1.05 A, while the heavy atom rmsd was 2.10 A. Focusing on the bound ligand, two different orientations of the trisaccharide within WGA-B binding site are possible. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to both complexes. A comparison of the three-dimensional structure of WGA-B in solution to that reported in the solid state and to those deduced for hevein and pseudohevein in solution has also been performed.

Medicinal chemistry based on the sugar code: fundamentals of lectinology and experimental strategies with lectins as targets.

Theoretical calculations reveal that oligosaccharides are second to no other class of biochemical oligomery in terms of coding capacity. As integral part of cellular glycoconjugates they can serve as recognitive units for receptors (lectins). Having first been detected in plants, lectins are present ubiquitously. Remarkably for this field, they serve as bacterial and viral adhesins. Following a description of these branches of lectinology to illustrate history, current status and potential for medicinal chemistry, we document that lectins are involved in a wide variety of biochemical processes including intra- and intercellular glycoconjugate trafficking, initiation of signal transduction affecting e. g. growth regulation and cell adhesion in animals. It is thus justified to compare crucial carbohydrate epitopes with the postal code ensuring correct mail routing and delivery. In view of the functional relevance of lectins the design of high-affinity reagents to occupy their carbohydrate recognition domains offers the perspective for an attractive source of new drugs. Their applications can be supposed to encompass the use as cell-type-selective determinant for targeted drug delivery and as blocking devices in anti-adhesion therapy during infections and inflammatory disease. To master the task of devising custom-made glycans/glycomimetics for this purpose, the individual enthalpic and entropic contributions in the molecular rendezvous between the sugar receptor under scrutiny and its ligand in the presence of solvent molecules undergoing positional rearrangements need to be understood and rationally exploited. As remunerative means to this end, cleverly orchestrated deployment of a panel of methods is essential. Concerning the carbohydrate ligand, its topological parameters and flexibility are assessed by the combination of computer-assisted molecular-mechanics and molecular-dynamics calculations and NMR-spectroscopic measurements. In the presence of the receptor, the latter technique will provide insights into conformational aspects of the bound ligand and into spatial vicinity of the ligand to distinct side chains of amino acids establishing the binding site in solution. Also in solution, the hydrogen-bonding pattern in the complex can be mapped with monodeoxy and monofluoro derivatives of the oligosaccharide. Together with X-ray crystallographic and microcalorimetric studies the limits of a feasible affinity enhancement can be systematically probed. With galactoside-binding lectins as instructive mo del, recent progress in this area of drug design will be documented, emphasizing the general applicability of the outlined interdisciplinary approach.

A new combined computational and NMR-spectroscopical strategy for the identification of additional conformational constraints of the bound ligand in an aprotic solvent.

This study documents the feasibility of switching to an aprotic medium in sugar receptor research. The solvent change offers additional insights into mechanistic details of receptor--carbohydrate ligand interactions. If a receptor retained binding capacity in an aprotic medium, solvent-exchangeable protons of the ligand would not undergo transfer and could act as additional sensors, thus improving the level of reliability in conformational analysis. To probe this possibility, we first focused on hevein, the smallest lectin found in nature. The NMR-spectroscopic measurements verified complexation, albeit with progressively reduced affinity by more than 1.5 orders of magnitude, in mixtures of up to 50% dimethyl sulfoxide (DMSO). Since hevein lacks the compact beta-strand arrangement of other sugar receptors, such a structural motif may confer enhanced resistance to solvent exchange. Two settings of solid-phase activity assays proved this assumption for three types of alpha- and/or beta-galactoside-binding proteins, that is, a human immunoglobulin G (IgG) subfraction, the mistletoe lectin, and a member of the galectin family of animal lectins. Computer-assisted calculations and NMR experiments also revealed no conspicuous impact of the solvent on the conformational properties of the tested ligands. To define all possible nuclear Overhauser effect (NOE) contacts in a certain conformation and to predict involvement of exchangeable protons, we established a new screening protocol applicable during a given molecular dynamics (MD) trajectory and calculated population densities of distinct contacts. Experimentally, transferred NOE (tr-NOE) experiments with IgG molecules and the disaccharide Gal'alpha1-3Galbeta1-R in DMSO as solvent disclosed that such an additional crosspeak, that is, Gal'OH2--GalOH4, was even detectable for the bound ligand under conditions in which spin diffusion effects are suppressed. Further measurements with the plant lectin and galectins confirmed line broadening of ligand signals and gave access to characteristic crosspeaks in the aprotic solvent and its mixtures with water. Our combined biochemical, computational, and NMR-spectroscopical strategy is expected to contribute notably to the precise elucidation of the geometry of ligands bound to compactly folded sugar receptors and of the role of water molecules in protein--ligand (carbohydrate) recognition, with relevance to areas beyond the glycosciences.

Conformer selection and differential restriction of ligand mobility by a plant lectin--conformational behaviour of Galbeta1-3GlcNAcbeta1-R, Galbeta1-3GalNAcbeta1-R and Galbeta1-2Galbeta1-R' in the free state and complexed with galactoside-specific mistletoe lectin as revealed by random-walk and conformational-clustering molecular-mechanics.

To study conformational parameters of ligands before and after complex formation with the galactoside-binding agglutinin of Viscum album L. (VAA) in solution, combined computer-assisted random walk molecular mechanics (RAMM) calculations extended by conformational clustering analysis (CCA), molecular dynamics (MD) simulations as well as two-dimensional rotating-frame nuclear Overhauser effect (ROE) and two-dimensional nuclear Overhauser effect (NOE) spectroscopy NMR experiments were employed. Derivatives of the naturally occurring disaccharides Galbeta1-3GlcNAcbeta1-R and Galbeta1-3GalNAcbeta1-R as well as of a synthetic high-affinity binding partner, i.e. the disaccharide Galbeta1-2Galbeta1-R', were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between phi/psi combinations of (40 degrees/60 degrees) and (40 degrees/-60 degrees). Calculations of the de-N-acetylated sugars revealed that presence of this group did not markedly influence the distribution of low-energy conformers in the phi, psi, epsilon plot. Occupation of side minima at phi/psi (180 degrees/0 degrees) or (0 degrees/180 degrees) is either unlikely or low according to the results of MD simulations and RAMM calculations extended by CCA. Notably, these side minima define conformations which are not stable during a MD simulation. Transitions to other minima occur already a few picoseconds after the start of the simulation. NMR experiments of the free-state ligand confirmed the validity of the data sets obtained by the calculations. Following the description of the conformational space in the free-state NMR experiments were performed for these disaccharides complexed with VAA. They yielded two interresidual contacts for Galbeta1-3GlcNAcbeta1-R and Galbeta1-2Galbeta1-R'. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the free state. One- and two-dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments at different mixing times excluded the influence of spin-diffusion effects. When the NOE build-up curves in the three studied cases were compared, the residual mobility of the penultimate carbohydrate unit of Galbeta1-3GalNAcbeta1-R was observed to be higher than that of the respective hexopyranose unit of the other two bound ligands. Due to the availability of the conformational parameters of Galbeta1-2Galbeta1-R' in association with a galectin, namely the beta-galactoside-binding protein from chicken liver, it is remarkable to note that this ligand displays different conformations in the binding sites of either the plant or the animal lectin. They correspond to local energy-minimum conformations in the phi,psi, epsilon plot and substantiate differential conformer selection by these two lectins with identical nominal monosaccharide specificity.

Involvement of laser photo-CIDNP (chemically induced dynamic nuclear polarization)-reactive amino acid side chains in ligand binding by galactoside-specific lectins in solution.

For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-1 and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-1/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of bovine galectin-1 is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82/Tyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When single-site mutants of EcorL ([Ala106]EcorL, [Ala108]EcorL, [Ala229]EcorL) were subjected to molecular-dynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.

Role of aromatic amino acids in carbohydrate binding of plant lectins: laser photo chemically induced dynamic nuclear polarization study of hevein domain-containing lectins.

Carbohydrate recognition by lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies accessibility of the respective groups to the light-absorbing dye. In principle, this technique is suitable to monitor surface properties of a receptor and the effect of ligand binding if CIDNP-reactive amino acids are affected. The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial study. It focuses on a series of N-acetylglucosamine-binding plant lectins of increasing structural complexity (hevein, pseudohevein, Urtica dioica agglutinin and wheat germ agglutinin and its domain B), for which structural NMR- or X-ray crystallographic data permit a decision of the validity of the CIDNP method-derived conclusions. On the other hand, the CIDNP data presented in this study can be used for a rating of our molecular models of hevein, pseudohevein, and domain B obtained by various modeling techniques. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate ligand is bound, CIDNP signals of side chain protons of tyrosine, tryptophan, or histidine residues are altered, for example, they are broadened and of reduced intensity or disappear completely. In the case of UDA, the appearance of a new tryptophan signal upon ligand binding was interpreted as an indication for a conformational change of the corresponding indole ring. Therefore, CIDNP represents a suitable tool to study protein-carbohydrate interactions in solution, complementing methods such as X-ray crystallography, high-resolution multidimensional nuclear magnetic resonance, transferred nuclear Overhauser effect experiments, and molecular modeling.

Carbohydrate-protein interaction studies by laser photo CIDNP NMR methods.

The side chains of tyrosine, tryptophan and histidine are able to produce CIDNP (Chemically Induced Dynamic Nuclear Polarization) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies surface accessibility of the respective groups to the light-absorbing dye. In principle, this technique allows the monitoring of the effect of ligand binding to a receptor and of site-directed mutagenesis on conformational aspects of any protein if CIDNP-reactive amino acids are involved. The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial model study for several N-acetyl-glucosamine-binding lectins of increasing structural complexity as well as for a wild type bacterial sialidase and its mutants. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate is bound, CIDNP signals of side chain protons of tyrosine, tryptophan or histidine residues can be broadened and of reduced intensity. This is the case for hevein, pseudo-hevein, the four hevein domains-containing lectin wheat germ agglutinin (WGA) and the cloned B-domain of WGA 1 (domB) representing one hevein domain. This response indicates either a spatial protection by the ligand or a ligand-induced positioning of formerly surface-exposed side chains into the protein's interior part, thereby precluding interaction with the photo-activated dye. Some signals of protons from the reactive side chains can even disappear when the lectin-ligand complexes are monitored. The ligand binding, however, can apparently also induce a conformational change in a related lectin that causes the appearance of a new signal, as seen for Urtica dioica agglutinin (UDA) which consists of two hevein domains. Additionally, the three CIDNP-reactive amino acids are used as sensors for the detection of conformational changes caused by pH variations or by deliberate amino acid exchanges, as determined for the isolectins hevein and pseudo-hevein as well as for the cloned small sialidase of Clostridium perfringens and two of its mutants. Therefore, CIDNP has proven to be an excellent tool for protein-carbohydrate binding studies and can be established in glycosciences as a third biophysical method beside X-ray-crystallography and high-resolution multidimensional NMR studies which provides reliable information of certain structural aspects of carbohydrate-binding proteins in solution.

Comparison between intact and desialylated human serum amyloid P component by laser photo CIDNP (chemically induced dynamic nuclear polarization) technique: an indication for a conformational impact of sialic acid.

The human pentraxin serum amyloid P component (SAP) exhibits no microheterogeneity in its complex di-antennary glycan. To elucidate whether the removal of sialic acids from this glycoprotein might affect the accessibility of certain amino acid residues of the protein we employed the laser photo CIDNP approach as a sensitive tool. The CIDNP effect is generated by the interaction of a photoexcited dye with reactive amino acids and results in enhanced absorption- or emission-signals which can be observed for the three aromatic amino acids histidine, tryptophan, and tyrosine if they are accessible to the dye. Therefore, this technique can be applied to explore surface exposure of these amino acid residues. The respective spectra of SAP and enzymatically desialylated SAP were determined. Six tryptophan/histidine signals and one tyrosine signal are present in the aromatic part of the CIDNP difference spectrum of SAP. The corresponding spectrum of desialylated SAP shows remarkable alterations. The chemical shift of one Trp/His-characteristic signal is decreased by 0.1 ppm. One Trp/His-signal disappeared and a new one was formed in the CIDNP difference spectrum of desialylated SAP, while the other signals were unaffected. The Tyr signal has a clearly enhanced intensity in desialylated SAP. Therefore, the removal of sialic acid moieties from the single N-glycan of each monomer apparently affects surface presentation of distinct CIDNP-reactive amino acids of SAP [1]. A conformational change of the protein part of SAP in relation with a different orientation of the desialylated oligosaccharide chain in comparison to the complete one is a possible explanation of our CIDNP results.

Molecular dynamics-derived conformation and intramolecular interaction analysis of the N-acetyl-9-O-acetylneuraminic acid-containing ganglioside GD1a and NMR-based analysis of its binding to a human polyclonal immunoglobulin G fraction with selectivity for O-acetylated sialic acids.

The influence of 9-O-acetylation of GD1a, yielding GD1a (eNeu5,9Ac2) with a 9-O-acetylated sialic acid moiety linked to the outer galactose residue, on the spatial extension and mobility of the carbohydrate chain and on recognition by a natural human antibody is analysed. To study a potential impact of the O-acetyl group on the overall conformation of the carbohydrate chain, molecular dynamics (MD) simulations of oligosaccharide chain fragments of increasing length starting from the non-reducing end have been carried out for the first time in this study. They revealed a considerable loss in chain flexibility after addition of the internal N-acetylneuraminic acid onto the chain. Besides MD calculations with different dielectric constants, the conformational behaviour of the complete oligosaccharide chain of the 9-O-acetylated GD1a ganglioside was simulated in the solvents water and dimethyl sulfoxide. These solvents were also used in NMR measurements. The results of this study indicate that 9-O-acetylation at the terminal sialic acid does not influence the overall conformation of the ganglioside. An extended interaction analysis of energetically minimized conformations of GD1a (eNeu5,9Ac2) and GD1a, obtained during molecular dynamics simulations, allowed assessment of the influence of the different parts of the saccharide chains on spatial flexibility. Noteworthy energetic interactions, most interestingly between the 9-O-acetyl group and the pyranose ring of N-acetylgalactosamine, were ascertained by the calculations. However, the strength of this interaction does not force the ganglioside into a conformation, where the 9-O-acetyl group is no longer accessible. Binding of GD1a (eNeu5,9Ac2) to proteins, which are specific for 9-O-acetylated sialic acids, should thus at least partially be mediated by the presence of this group. To experimentally prove this assumption, a NMR study of 9-O-acetylated GD1a in the presence of an affinity-purified polyclonal IgG fraction from human serum with preferential binding to 9-O-acetylated sialic acid was performed. The almost complete disappearance of the intensity of the 9-O-acetyl methyl signal of the GD1a (eNeu5,9Ac2) clearly indicates that the assumed interaction of the 9-O-acetyl group with the human protein takes place.

[The mistletoe myth--claims, reality and provable perspectives]. / Mythos Mistel--Anspruch, Wirklichkeit und prüfbare Perspektive.

Intuition guided R. Steiner to refer to mistletoe as the future remedy for cancer. He proposed that its spiritual qualities support re-establishment of the harmonious integration of the alleged four different entities of human existence in a patient. The assumption of potency without chemical basis is derived from the dogmatic system of anthroposophic reasoning. It explains the evidently similar claim of clinical efficiency for the proprietary mistletoe extracts despite the lack of information on the actual contents of the complex mixtures and despite the conspicuous diversity of methods of manufacture for these products. Thorough scientific analysis of the published clinical experience does not justify this claim. Due to the increasing reference to defined substances in advertisements for commercial extracts, they should no longer avoid rigorous testing according to common quality standards. Interdisciplinary research efforts on the immunomodulatory galactoside-binding lectin illustrate how to yield a clinically testable substance from an ill-defined extract, thereby providing a notable example for rational investigation of unconventional treatment modalities.

NMR-based, molecular dynamics- and random walk molecular mechanics-supported study of conformational aspects of a carbohydrate ligand (Gal beta 1-2Gal beta 1-R) for an animal galectin in the free and in the bound state.

The binding of a carbohydrate to a lectin may affect the conformation of the ligand. To address this question for the galectin from chicken liver, the conformation of Gal beta 1-2Gal beta 1-R was analyzed in the free and in the galectin-bound state with 2D-ROESY- and 1D- as well as 2D-transferred NOE-experiments. A computer-assisted analysis of spatial parameters of the ligand by molecular dynamics (MD) and random walk molecular mechanics (RAMM) calculations, taking different dielectric constraints from epsilon = 1 to epsilon = 80 and various force fields into account, were instrumental to define the energetic minima of the free state. NMR-derived interresidual distance constraints enabled a conformational mapping. The two overlapping interresidual distance constraints obtained from transferred-NOE experiments of the galectin-ligand complex clearly support the notion that the conformation of the disaccharide in the bound state is at least very close to its global energy minimum state in solution.

Effect of enzymatic desialylation of human serum amyloid P component on surface exposure of laser photo CIDNP (chemically induced dynamic nuclear polarization)--reactive histidine, tryptophan and tyrosine residues.

The human pentraxin serum amyloid P component (SAP) exhibits no microheterogeneity in its complex di-antennary glycan. To elucidate whether the removal of sialic acids from this glycoprotein might affect the accessibility of certain amino acid residues of the protein we employed the laser photo CIDNP approach as a sensitive tool. The CIDNP effect is created by the interaction of a photoexcited dye with reactive amino acids and results in enhanced absorption- or emission-signals which can be observed for the three aromatic amino acids histidine, tryptophan, and tyrosine if they are accessible to the dye. Therefore, this technique can be applied to explore surface exposure of these residues. The respective spectra of SAP and enzymatically desialylated SAP were determined. Six tryptophan/histidine signals and one tyrosine signal are present in the aromatic part of the CIDNP difference spectrum of SAP. The corresponding spectrum of desialylated SAP shows remarkable alterations. The chemical shift of one tryptophan/histidine-characteristic signal is decreased by 0.1 ppm. One tryptophan/histidine signal disappeared and a new one was formed in the CIDNP difference spectrum of desialylated SAP, while the other signals were unaffected. The tyrosine signal has a clearly enhanced intensity in desialylated SAP. Therefore, the removal of sialic acid moieties from the single N-glycan of each monomer apparently affects surface presentation of distinct CIDNP-reactive amino acids of SAP.