Biomedical subjects

C W von der Lieth

Publications and source records attributed to C W von der Lieth.

At least 19 recordsLinked to original sources

PubMed2002

Glycosylation of proteins: a computer based method for the rapid exploration of conformational space of N-glycans.

Inspection of protein databases suggests that as many as 70% of proteins have potential N-glycosylation sites. Unfortunately glycoproteins often refuse to crystallize and NMR techniques do not allow an unambiguous determination of the complete conformation of the sugar part. Therefore, time-consuming complex simulation methods are often used to explore the conformational space of N-glycans. The generation of a comprehensive data base describing the conformational space of larger fragments of N-glycans taking into account the effects of branching is presented. High-temperature molecular dynamics simulations of essential N-glycan fragments are performed until conformational equilibrium has been reached. Free energy landscapes are calculated for each glycosidic linkage. All possible conformations for each N-glycan fragment are automatically assigned, ranked according to their relative population and stored in a database. These values are recalled for the generation of a complete set of all possible conformations for a given N-glycan topology. The constructed conformations are ranked according to their energy content. Since this approach allows to explore the complete conformational space of a given N-glycan within a few minutes of CPU-time on a standard PC, it is well suited to be used as a Web-Based application.

Carbohydrate Conformation↗

PubMed2001

Monosaccharide-linked inhibitors of O(6)-methylguanine-DNA methyltransferase (MGMT): synthesis, molecular modeling, and structure-activity relationships.

A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.

Cell-Free System↗

PubMed2001

LINUCS: linear notation for unique description of carbohydrate sequences.

The use of proteomics databases has become indispensable for daily work of molecular biologists, but this situation has not yet been achieved for carbohydrate applications. One obvious reason is that existing data collections are only rarely annotated and no cross-linking to other resources exists. The existence of a generally accepted linear, canonical description for carbohydrates which can be readily processed by computers will enable efficient automatic cross-linking of distributed carbohydrate data collections by serving as a unique and unambiguous database access key. Various possibilities to derive a canonical notation are discussed. They can be divided into attempts that require structure description alone and alternatives that profit from the fact that a preferred graph direction (non-reducing to reducing end) exists within the structure. To open a fruitful discussion among glycoscientists a possible solution is presented where the reducing monosaccharide unit is selected as graph root and linkage information is used to define the priority of the various branches. A Web interface (http://www.dkfz.de/spec/linucs/) has been created that directly converts the commonly used extended representation of complex carbohydrates into the preferred canonical description or into its inverted form.

Algorithms↗

PubMed2001

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.

Cell Communication↗

PubMed2000

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.

Animals↗

PubMed1999

Molecular dynamics study of the proposed beta-hairpin form of the switch domain from HIV1 gp120 alone and complexed with an inhibitor of CD4 binding.

The strong tendency of beta-hairpin peptides to aggregate can prevent their structural resolution. The polar form of the switch peptide (LAV 15mer) at the CD4-binding domain of HIV1 gp120 is such a peptide, and NMR investigations of its interaction with a class of CD4-binding inhibitors developed in this laboratory have been hindered. Detailed knowledge of the interaction is required for the development of more potent switch inhibitors, that act by disrupting the cooperative folding transition necessary for binding to the CD4 receptor. In carrying out molecular dynamics simulation of the free peptide under polar conditions, we found that the properties of the resulting structure agree closely with those observed by circular dichroism. The same conditions, used to model the peptide/ inhibitor complex, produced a stable bimolecular structure with specific interactions between the inhibitor and side chains on the peptide, (e.g., Trp12 and the LPCR tetrad), known to control the folding transition. These help explain existing data on the relative potency of inhibitor derivatives and provide a basis for improved inhibitor design.

Algorithms↗

PubMed1999

SWEET - WWW-based rapid 3D construction of oligo- and polysaccharides.

UNLABELLED: SWEET is a WWW-based tool which rapidly converts the commonly used carbohydrate sequence information directly into a preliminary but reliable 3D model which can be visualised and written to files in several ways. AVAILABILITY: SWEET is accessible via the Internet at http://www.dkfz-heidelberg.de/spec/. CONTACT: a. bohne@dkfz-heidelberg.de or w.vonderlieth@ dkfz-heidelberg.de SUPPLEMENTARY INFORMATION: The current version of SWEET generates only one conformation out of a manifold. Several authors have analysed possible conformations of high-mannose N-linked glycans using a combination of NMR methods and computational approaches showing that such molecules are rather flexible populating normally several conformations for each glycosidic linkage. The displayed model exhibits for all glycosidic linkages a conformation which is in accordance with the reported variations of Phi, psi and omega values for specific linkage (see http://www.dkfz-heidelberg. de/spec/sweet2/doc/input/sba_example.html).

Carbohydrate Conformation↗

PubMed1998

Elongation of the N-acyl side chain of sialic acids in MDCK II cells inhibits influenza A virus infection.

The interaction of influenza A virus with sialyated receptor components is one of the best characterized ligand-receptor interactions. We pretreated MDCK II host cells with three different N-acyl-modified sialic acid precursor analogues, N-propanoyl, N-butanoyl or N-pentanoyl D-mannnosamine. Cellular sialic acid biosynthesis yielded 18-35% of new, modified sialic acids on cell surface glycoconjugates, N-propanoyl, N-butanoyl or N-pentanoyl neuraminic acid, respectively. The elongation of the N-acyl group of sialic acids resulted in an inhibition of influenza A virus (strain X31) binding and subsequent infection of up to 80%. In contrast, the sialic acid-independent infection of vesicular stomatitis virus was unaffected in these cells. Molecular modeling studies based on the crystal structure of the influenza A virus hemagglutinin complexed with sialyllactose suggest a steric hindrance of hemagglutinin binding to aliphatically elongated N-acyl groups. We propose that biosynthetic sialic acid modification in conjunction with molecular modeling is a potent tool to further analyze the influenza A virus-receptor interaction.

Animals↗

PubMed1998

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.

Binding Sites↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed1997

Efficient modelling protocols for oligosaccharides: from vacuum to solvent.

The determination of conformational preferences of oligosaccharides is best approached by describing their preferred conformations on potential energy surfaces as a function of the glycosidic linkage phi, psi torsional angles. For proper molecular mechanics modelling the flexibility of the rotatable pendant groups must also be considered. The so called adiabatic maps partially mimic the flexibility within the 10 dimensional conformational space of the pendant groups of the given disaccharide. These molecular mechanics maps are considered to be the state-of-the art of the phi, psi potential energy surface of disaccharides recently calculated. The RAMM (RAndom Molecular Mechanics) method was shown to be able to calculate such profiles automatically. Additionally, based on the continuum solvent approach, RAMM allows the calculation of the effects of solvent on conformational energy profiles. Molecular dynamics simulations are also useful tools to study the influence of solvent on conformational behaviour of oligosaccharides. The capability of the RAMM calculational protocol to locate low-energy conformers on the multidimensional potential energy hypersurfaces of disaccharides is illustrated and compared with molecular dynamics simulations with and without inclusion of the solvent.

Carbohydrate Conformation↗

PubMed1997

Consequences of a subtle sialic acid modification on the murine polyomavirus receptor.

Polyomaviruses are small, nonenveloped DNA tumor viruses with restricted host ranges. Virus binding to cell surface receptors is one determinant of viral tropism. Although murine polyomavirus is among the best characterized viruses, little is known about the sialic acid-containing receptor and its interaction with viral particles. By using nonradioactive virus binding assays as recently described for the B-lymphotropic papovavirus, murine polyomavirus particles were found to bind in a saturable and noncooperative manner to 25,000 receptors per 3T6 mouse fibroblast. The virus-receptor interaction at 4 degrees C was of high affinity (Kd = 1.8 x 10(-11) M), very fast (k1 = 1.7 x 10(7) M(-1) s(-1)), and stable (half-life = 38 min). Elongation of the N-acyl side chain of sialic acid by biosynthetic modulation with synthetic precursor analogs has been shown for other polyomaviruses to influence both sialic acid-dependent binding and infection (O. T. Keppler, P. Stehling, M. Herrmann, H. Kayser, D. Grunow, W. Reutter, and M. Pawlita, J. Biol. Chem. 270:1308-1314, 1995). In 3T6 cells in which about one-third of the sialic acids were modified, infection and binding of polyomavirus particles were significantly reduced. The number of receptors per cell was decreased to 18,000, with the remaining receptors displaying the same affinity as in untreated cells. Molecular modeling studies based on the three-dimensional structure of a mouse polyomavirus-sialyllactose complex recently solved by T. Stehle and coworkers (T. Stehle, Y. W. Yan, T. L. Benjamin, and S. C. Harrison, Nature 369:160-163, 1994) were performed. They suggest that the elongation of the N-acyl side chain by a single methylene group leads to steric hinderence, with the peptide backbone of a loop walling the tip of the shallow sialic acid binding groove. This collision appears to be incompatible with functional binding. The data are taken as a basis to discuss possible features of the organization and topology of the cellular receptor for mouse polyomavirus.

Animals↗

PubMed1997

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.

Amino Acids↗

PubMed1996

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.

Animals↗

PubMed1996

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.

Acetylation↗

PubMed1996

[The mistletoe myth--claims, reality and provable perspectives].

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.

Adjuvants, Immunologic↗

PubMed1994

Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid.

Synthetic oligonucleotides (18-mers) containing either a single deoxyadenosine residue or a single deoxyguanosine residue were treated with aristolochic acid I (AAI) or aristolochic acid II (AAII), the main components of the plant carcinogen aristolochic acid (AA). These reactions resulted in the formation of site-specifically adducted oligonucleotides containing the two known AAI-DNA adducts (dA-AAI, dG-AAI) or the two known AAII-DNA adducts (dA-AAII, dG-AAII) at position 15 from the 3' end. Using HPLC chromatography, the oligonucleotides were purified and subsequently shown to contain the adducts of interest by 32P-postlabelling. The adducted oligonucleotides were used as templates in primer (11-mer) extension reactions catalysed by modified bacteriophage T7 DNA polymerase (Sequenase). Regardless of the type of DNA adduct examined, DNA synthesis was blocked predominantly (80-90%) at the nucleotide 3' to each adduct, although primer extension to the full length of the template was noted with unmodified control templates. However, 15 nucleotide products, indicating blocking of DNA synthesis after incorporation of a nucleotide opposite the adduct and translesional synthesis products were formed in all cases in different amounts, depending on the adduct structure. When a 14-mer primer together with high dNTP concentrations was used to examine nucleotide incorporation directly across from the four different purine adducts we found that the deoxyadenosine adducts (dA-AAI and dA-AAII) allowed incorporation of dAMP and dTMP equally well, whereas the deoxyguanosine adducts (dG-AAI and dG-AAII) allowed preferential incorporation of dCMP. Molecular dynamic simulations showed that the aristolactam moiety of all adducts exhibit a strong stacking, with the adenine residue at the 3' end of the 14-mer primer. These studies demonstrate that all AA purine adducts provide severe blocks to DNA replication and that the guanine adducts may not be very efficient mutagenic lesions. In contrast, the translesional bypass past adenine adducts of the aristolochic acids suggests a mutagenic potential resulting from dAMP incorporation by polymerase. AT-->TA transversion mutations would be the mutagenic consequences of AA adenine adducts, which are consistent with the activating mutations of c-ras genes found in AA-induced tumours of rodents.

Aristolochic Acids↗

PubMed1993

Solution conformation of the tetrasaccharide glycoside Xyl beta 1-(Man alpha 1-3)2Man beta 1-4Glc beta 1R from the mollu-series glycosphingolipids.

The conformation of Xyl beta 1-(Man alpha 1-3)2Man beta 1-4Glc beta 1-OCH2CH(N3)CH(OH) CH = CHC13H27 tetrasaccharide glycoside in Me2SO-d6 was investigated with use of a distance mapping procedure based on rotating-frame NOE and hydrogen bond constraints, and Molecular Mechanics calculations. The Man alpha 1-3Man beta linkage was found to be well defined in a single conformation, whereas some flexibility could be assumed for the remaining two glycosidic linkages. Similar conclusions, though based on a smaller number of constraints, were drawn for the title compound anchored in mixed D2O/dodecylphosphocholine-d38 micelles.

Carbohydrate Conformation↗