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Hans-Christian Siebert

Publications and source records attributed to Hans-Christian Siebert.

9 recordsLinked to original sources
PubMed2004

Chemical biology of the sugar code.

A high-density coding system is essential to allow cells to communicate efficiently and swiftly through complex surface interactions. All the structural requirements for forming a wide array of signals with a system of minimal size are met by oligomers of carbohydrates. These molecules surpass amino acids and nucleotides by far in information-storing capacity and serve as ligands in biorecognition processes for the transfer of information. The results of work aiming to reveal the intricate ways in which oligosaccharide determinants of cellular glycoconjugates interact with tissue lectins and thereby trigger multifarious cellular responses (e.g. in adhesion or growth regulation) are teaching amazing lessons about the range of finely tuned activities involved. The ability of enzymes to generate an enormous diversity of biochemical signals is matched by receptor proteins (lectins), which are equally elaborate. The multiformity of lectins ensures accurate signal decoding and transmission. The exquisite refinement of both sides of the protein-carbohydrate recognition system turns the structural complexity of glycans--a demanding but essentially mastered problem for analytical chemistry--into a biochemical virtue. The emerging medical importance of protein-carbohydrate recognition, for example in combating infection and the spread of tumors or in targeting drugs, also explains why this interaction system is no longer below industrial radarscopes. Our review sketches the concept of the sugar code, with a solid description of the historical background. We also place emphasis on a distinctive feature of the code, that is, the potential of a carbohydrate ligand to adopt various defined shapes, each with its own particular ligand properties (differential conformer selection). Proper consideration of the structure and shape of the ligand enables us to envision the chemical design of potent binding partners for a target (in lectin-mediated drug delivery) or ways to block lectins of medical importance (in infection, tumor spread, or inflammation).

Binding Sites↗
PubMed2004

Tumor galectinology: insights into the complex network of a family of endogenous lectins.

Beta-Galactosides of cell surface glycoconjugates are docking sites for endogenous lectins of the galectin family. In cancer cells, primarily galectins-1 and -3 have been studied to date. With the emergence of insights into their role in growth control, resistance to or induction of apoptosis and invasive behavior the notion is supported that they can be considered as functional tumor markers. In principle, the same might hold true for the other members of the galectin family. But their expression in tumors has hitherto been a subject of attention only to a very limited extent. Pursuing our concept to define the complexity of the galectin network in cancer cells and the degree of functional overlap/divergence with diagnostic/therapeutic implications, we have introduced comprehensive RT-PCR monitoring to map their galectin gene expression. The data on so far less appreciated galectins in this context such as galectins-4 and -8 vindicate this approach. They, too, attach value to extend the immunohistochemical panel accordingly. Our initial histopathological and cell biological studies, for example on colon cancer progression, prove the merit of this procedure. Aside from the detection of gene expression profiles by RT-PCR, the detailed molecular biological monitoring yielded further important information. We describe different levels of regulation of galectin production in colon cancer cells in the cases of the tandem-repeat-type galectins-8 and -9. Isoforms for them are present with insertions into the peptide linker sequence attributed to alternative splicing. Furthermore, variants with distinct amino acid substitutions (galectin-8, Po66-CBP, PCTA-1, CocaI/II and galectin-9/ecalectin) and generation of multiple mRNA species, notably those coding for truncated galectin-8 and -9 versions with only one lectin site, justify to portray these two family members not as distinct individuals but as groups. In aggregate, the ongoing work to thoroughly chart the galectin network and to disentangle the individual functional contributions is expected to make its mark on our understanding of the malignant phenotype in certain tumor types.

Alternative Splicing↗
PubMed2003

Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins.

Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.

Acetylgalactosamine↗
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↗