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Hans-Christian Siebert

Publications and source records attributed to Hans-Christian Siebert.

5 recordsLinked to original sources
PubMed2003

Detection of ligand- and solvent-induced shape alterations of cell-growth-regulatory human lectin galectin-1 in solution by small angle neutron and x-ray scattering.

The bioactivity of galectin-1 in cell growth regulation and adhesion prompted us to answer the questions whether ligand presence and a shift to an aprotic solvent typical for bioaffinity chromatography might alter the shape of the homodimeric human lectin in solution. We used small angle neutron and synchrotron x-ray scattering studies for this purpose. Upon ligand accommodation, the radius of gyration of human galectin-1 decreased from 19.1 +/- 0.1 A in the absence of ligand to 18.2 +/- 0.1 A. In the aprotic solvent dimethyl sulfoxide, which did not impair binding capacity, galectin-1 formed dimers of a dimer, yielding tetramers with a cylindrical shape. Intriguingly, no dissociation into subunits occurred. In parallel, NMR monitoring was performed. The spectral resolution was in accord with these data. In contrast to the properties of the human protein, a nonhomologous agglutinin from mistletoe sharing galactose specificity was subject to a reduction in the radius of gyration from approximately 62 A in water to 48.7 A in dimethyl sulfoxide. Evidently, the solvent caused opposite responses in the two tested galactoside-binding lectins with different folding patterns. We have hereby proven that ligand presence and an aprotic solvent significantly affect the shape of galectin-1 in solution.

Computer Simulation↗
PubMed2003

Suitability of binary mixtures of water with aprotic solvents to turn hydroxyl protons of carbohydrate ligands into conformational sensors in NOE and transferred NOE experiments.

The structural analysis of protein-carbohydrate interactions is essential for the long-range aim to sort out entropic/ enthalpic factors in the binding process. Of conspicuous clinical interest, this work can also offer the perspective to devise new classes of therapeuticals which interfere with disease-related glycan recognition. We have shown that it is possible to use exchangeable hydroxyl protons of carbohydrate ligands as conformational sensors for defining their bound-state topology by measurements in dimethyl sulfoxide(d6) (Siebert et al. (2000) ChemBioChem, 1, 181-195). However, the proteins are required to maintain binding capacity in the aprotic solvent. To define conditions to limit its harmful effect on sensitive protein structures while still being able to pick up solvent-exchangeable hydroxyl signals we systematically tested binary solvent mixtures of dimethyl sulfoxide and acetone with water. These solvent mixtures did not preclude to monitor hydroxyl protons of carbohydrate ligands even at temperatures well above 0 degrees C. Notably, hydrogen bonding of the two tested disaccharides (Galbeta1-4Glcalpha/beta and Galalpha1-3Galalpha/beta or Galalpha1-3Galbeta1-OCH(3)), which are common lectin ligands, resembled the situation under physiological conditions. Also, a refined topological description for hydroxyl positioning could be achieved for Galalpha1-3Gal. At least equally important, this approach worked for elucidation of the mistletoe-lectin-bound topology of lactose in its syn-conformation with indication for formation of a characteristic interresidual hydrogen bond. These measurements were performed in a binary dimethyl sulfoxide(d6):water mixture (6:4 ratio, v/v) at -12 degrees C and encourage to pursue this line of investigation by monitoring in the course of stepwise temperature increases. Our experiments reveal that binary mixtures have favorable properties for the conformational analysis of the free- and bound-state topologies of bioactive ligands.

Acetone↗
PubMed2002

The sugar code: functional lectinomics.

Analysis of the genome and proteome assumes the focus of attention in efforts to relate biochemical coding with cell functionality. Among other chores in energy metabolism, the talents of carbohydrates to establish a high-density coding system give reason for a paradigmatic shift. The sequence complexity of glycans and glycan-processing enzymes (glycosyltransferases, glycosidases and enzymes introducing substituents such as sulfotransferases), the growing evidence for the importance of glycans from transgenic and knock-out animal models and the correlation of defects in glycosylation with diseases are substantial assets to portray oligosaccharides as code words in their own right. Matching the pace of progress in the work on glycoconjugates, the increasing level of refinement of our knowledge about lectins (definition of this term: carbohydrate-binding proteins, excluding sugar-specific antibodies, receptors of free mono- or disaccharides for transport or chemotaxis and enzymes modifying the bound carbohydrate) epitomizes the sphere of action of the sugar code (functional lectinomics). It encompasses, among other activities, intra- and intercellular transport processes, sensor branches of innate immunity, regulation of cell-cell (matrix) adhesion or migration and positive/negative growth control with implications for differentiation and malignancy. The Q & A approach taken in this review lists a series of arguments in a stepwise manner to make the reader wonder why it is only a rather recent process that the concept of the sugar code has taken root in deciphering the mechanistic versatility of biological information storage and transfer.

Animals↗
PubMed2002

Analysis of protein-carbohydrate interaction at the lower size limit of the protein part (15-mer peptide) by NMR spectroscopy, electrospray ionization mass spectrometry, and molecular modeling.

Structural analysis of minimally sized lectins will offer insights into fundamentals of intermolecular recognition and potential for biomedical applications. We thus moved significantly beyond the natural limit of lectin size to determine the structure of synthetic mini-lectins in solution, their carbohydrate selectivity and the impact of ligand binding on their conformational behavior. Using three disaccharide (Thomsen-Friedenreich antigen; Gal beta 1,3GalNAc alpha 1,R)-binding pentadecapeptides without internal disulfide bridges as role models, we successfully tested a combined strategy with different techniques of NMR spectroscopy, electrospray ionization mass spectrometry, and molecular modeling. In solution, the peptides invariably displayed flexibility with rather limited restrictions, shown by NMR experiments including nearly complete resonance assignments and molecular dynamics simulations. The occurrence of aromatic/nonpolar amino acids in the sequence did not lead to formation of a hydrophobic core known from microbial chitinase modules. Selectivity of disaccharide binding was independently observed by mass spectrometry and NMR analysis. Specific ligand interaction yielded characteristic NMR signal alterations but failed to reduce conformational flexibility significantly. We have thereby proven effectiveness of our approach to analyze even low-affinity interactions (not restricted to carbohydrates as ligands). It will be useful to evaluate the impact of rational manipulation of lead peptide sequences.

Amino Acid Sequence↗