Wolfgang Voelter - Academia.edu (original) (raw)

Papers by Wolfgang Voelter

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2008

We have studied the stability and reassociation behaviour of native molecules of Rapana venosa he... more We have studied the stability and reassociation behaviour of native molecules of Rapana venosa hemocyanin and its two subunits, termed RvH1 and RvH2. In the presence of different concentrations of Ca 2+ and Mg 2+ ions and pH values, the subunits differ not only in their reassociation behaviour, but also in their formation of helical tubules and multidecamers. RvH1 revealed a greater stability at higher pH values compared to RvH2. Overall, the stability of reassociated RvH and its structural subunits was found to be pH-dependent. The increasing stability of native Hc and its subunits, shown by pH-induced CD transitions (acid and alkaline denaturation), can be explained with the formation of quaternary structure. The absence of a Cotton effect at temperatures 20-40°C in the pH-transition curves of RvH2 indicates that this subunit is stabilized by additional "factors", e.g.: non-ionic/hydrophobic stabilization and interactions of carbohydrate moieties. A similar behaviour was observed for the T-transition curves in a wide pH interval for RvH and its structural subunits. At higher temperatures, many of the secondary structural elements are preserved especially at neutral pH, even at extreme high temperatures above 90°C the protein structures resemble a "globule state".

Biochemistry, 2001

The oncoprotein MDM2 inhibits the tumor suppressor protein p53 by binding to the p53 transactivat... more The oncoprotein MDM2 inhibits the tumor suppressor protein p53 by binding to the p53 transactivation domain. The p53 gene is inactivated in many human tumors either by mutations or by binding to oncogenic proteins. In some tumors, such as soft tissue sarcomas, overexpression of MDM2 inactivates an otherwise intact p53, disabling the genome integrity checkpoint and allowing cell cycle progression of defective cells. Disruption of the MDM2/p53 interaction leads to increased p53 levels and restored p53 transcriptional activity, indicating restoration of the genome integrity check and therapeutic potential for MDM2/p53 binding antagonists. Here, we show by multidimensional NMR spectroscopy that chalcones (1,3-diphenyl-2-propen-1-ones) are MDM2 inhibitors that bind to a subsite of the p53 binding cleft of human MDM2. Biochemical experiments showed that these compounds can disrupt the MDM2/p53 protein complex, releasing p53 from both the p53/MDM2 and DNA-bound p53/MDM2 complexes. These results thus offer a starting basis for structure-based drug design of cancer therapeutics. 10.FIGURE 1: A representative collection of basic chalcone skeletons used in our study. Inhibition of MDM2 binding to p53 measured by ELISA (IC 50 values given on the left side of the slash) and by NMR titration experiments (K D values given on the right side of the slash). Compound D was studied as a negative control. For details refer to text.

In this study, twenty-nine 2-aminopyrimidine derivatives are synthesized in good to excellent yie... more In this study, twenty-nine 2-aminopyrimidine derivatives are synthesized in good to excellent yields by fusing 2-amino-4,6-dichloropyrimidine with different amines in the presence of triethylamine without using any solvent or catalyst. Nucleophilic substitution reactions of 2-amino-4,6-dichloropyrimidine with amines have also been performed in ethanol. Comparisons of the yields and reaction times for both solvent and solvent-free conditions have shown that the newly developed solvent-free protocol is high yielding, more efficient, and simpler compared to conventional methods.

Hemocyanins are giant biological macromolecules acting as oxygen-transporting glycoproteins. Most... more Hemocyanins are giant biological macromolecules acting as oxygen-transporting glycoproteins. Most of them are respiratory proteins of arthropods and mollusks, but besides they also exhibit protecting effects against bacterial, fungal and viral invasions. As discovered by 2-DGE proteomics analyses, several proteins including hemocyanins of hemocytes from virus-infected arthropods increased upon infection, confirming hemocyanin's role as part of the organism's defence system. Based on the structural analyses of molluscan Hcs it is suggested that the carbohydrate chains of the glycoproteins seem to interact with surface-exposed amino acid or carbohydrate residues of the viruses through van der Waals interactions.

Molluscan hemocyanins are glycoproteins with different quaternary and carbohydrate structures. It... more Molluscan hemocyanins are glycoproteins with different quaternary and carbohydrate structures. It was suggested that the carbohydrate chains of some Hcs are involved in their antiviral and antitumor effect, as well in the organization of the quaternary structure of the molecules. Using a well-known complex for saccharide sensing, positions and access to the carbohydrate chains in the native hemocyanins from Rapana venosa (RvH) and Helix luco-rum (HlH) and also their structural subunits (RvH1, RvH2 and β c HlH) and functional units (FUs) were analysed by fluorescence spectroscopy and circular dichroism. Almost no effect was observed in the fluorescence emission after titration of the complex with native RvH and HlH due to lack of free hydroxyl groups which are buried in the dideca-meric form of the molecules. Titration with the structural subunits β c HlH and RvH2, increasing of the emission indicates the presence of free hydroxyl groups compared to the native molecules. Complex titration with the structural sub-unit βc-HlH of H. lucorum Hcs leads to a 2.5 fold increase in fluorescence intensity. However, the highest emission was measured after titration of the complex with FU βcHlH-g. The result was explained by the structural model of β c HlH-g showing the putative position of the glycans on the surface of the molecule. The results of the fluorescent measurements are in good correlation with those of the circular dichroism data, applied to analyse the effect of titration on the secondary structure of the native molecules and functional units. The results also support our previously made suggestion that the N-linked oligosaccharide trees are involved in the quaternary organization of molluscan Hcs.

In this study, an improved, rapid, high yield synthesis of N,N'-4,4'-bis(benzyl-2-boronic acid)-b... more In this study, an improved, rapid, high yield synthesis of N,N'-4,4'-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV) is described. The obtained o-BVV is applied in a two-component saccharide sensing system (complex) where it serves as a fluorescence quencher and a saccharide receptor. This system was applied to different natural oligosaccharides isolated from molluscan Rapana venosa (RvH1-a) and arthropodan Carcinus aestuarii (CaeH) hemocyanins (Hcs) and cyclodextrins (CDs). The carbohydrate contents of both Hcs were calculated in our previous work to be 1,6 % and 7 % for CaeH and RvH1-a, respectively. We propose that the difference in fluorescence increase of the native CaeH and RvH1-a when titrating them with the complex is due to the fact that the carbohydrate content of CaeH is lower and the carbohydrate chains are buried in between the structural subunits of the native molecule , while the glycans of the functional unit RvH1-a are exposed on the surface of the molecule leading to a 4-fold fluorescence's intensity change.

o-Diphenol oxidase activities (o-diPO) of chemically modified functional unit RvH1-a of molluscan... more o-Diphenol oxidase activities (o-diPO) of chemically modified functional unit RvH1-a of molluscan hemocyanin Rapana venosa were studied using L-Dopa and dopamine as substrates. With L-Dopa as substrate the native FU RvH1-a did not show any o-diPO activity. Therefore the native FU RvH1-a was converted to enzymatic active form, after treatment with SDS, trypsin, urea and different values of pH when its o-diPO activity was studied. The highest artificial induction of o-diPO activity was observed after incubation of FU with 3.0 mM SDS, and RvH1-a shows both, dopamine (K M = 6.53 mM, k cat /K M = 1.29) and L-Dopa (K M = 2.0 mM, k cat /K M = 2.1) activity due to a more open active site of the enzyme and better access of the substrates. It was determined that the K M value of SDS-activated RvH1-a against dopamine is higher compared to those of hemocyanins from Helix vulgaris, Helix pomatia and native tyrosinase from Ipomoea batatas but much lower than that from Illex argentinus (ST94) tyrosinase and arthropodan hemocyanin from Carcinus aestuarii. The Km value of SDS-activated RvH1-a against L-Dopa is higher than those of hemocyanins from H. vulgaris and Cancer magister, but lower than that of the tyrosinase from Streptomyces albus.

The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin ... more The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin (HtH) were studied by mass spectral sequence analysis of the glycans.

Rapana venosa hemocyanin (RvH), a circulating glycoprotein of the marine snail, has a complex str... more Rapana venosa hemocyanin (RvH), a circulating glycoprotein of the marine snail, has a complex structure. To provide details on the stability of the protein, one functional unit, RvH2-e, was compared with the native molecule and the structural subunits, RvH1 and RvH2, via pH–T diagrams, typical phase portraits for stability and denaturation reversibility. By analyzing the T transition curves of RvH2-e at different pH values, several parameters of the thermodynamic functions were obtained. Increasing the temperature from 25 °C to 55 °C, the reversibility of the molecule of protein also increases, opening a reversibility window within the range of pH 4.0–8.0. On analyzing the pH transition curves, the start of the acid denaturation (below pH 6) and alkaline denaturation (above pH 9) was determined to be between 20 °C and 35 °C. For this range, the thermodynamic functions ΔH° and ΔG° for a standard temperature of 25 °C were calculated.

This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright a b s t r a c t The Zn-proteinase, isolated from Saccharomonospora canescens (NPS), shares many common features with thermolysin, but considerable differences are also evident, as far as the substrate recognition site is concerned. In substrates of general structure AcylAlaAlaPhe 4NA, this neutral proteinase cleaves only the arylamide bond (non-typical activity of Zn-proteinases), while thermolysin attacks the peptide bond Ala-Phe. Phosphoramidon is a powerful tight binding inhibitor for thermolysin and significantly less specific towards NPS. The K i-values (65 lM for NPS vs 0.034 lM for thermolysin) differ nearly 2000-folds. This implies significant differences in the specificity of the corresponding subsites. The carbohydrate moi-ety is supposed to accommodate in the S 1-subsite and the series of arabinopyranosides and glucopyrano-sides (12 compounds), which are assayed as inhibitors in a model system: NPS with SucAlaAlaPhe4NA as a substrate could be considered as mapping the S 1-subsite of NPS. Members of the series with an additional ring (3,4-epithio, 3,4-anhydro-derivatives) turned out to be reasonably good competitive inhibitors (K i % 0.1–0.2 mM are of the same order as the K i value for phosphoramidon). The structure of these compounds (8, 9, 11 and 12) seems to fit the size of the S 1-subsite and due to an appropriately oriented OH-group in addition, to protect the active site Zn 2+ .

Keywords: Glycoprotein Cu/Zn-superoxide dismutase MALDI–TOF–TOF Q-Trap Yeast Kluyveromyces marxia... more Keywords: Glycoprotein Cu/Zn-superoxide dismutase MALDI–TOF–TOF Q-Trap Yeast Kluyveromyces marxianus a b s t r a c t The primary structure of Cu/Zn-superoxide dismutase from Kluyveromyces marxianus NBIMCC 1984 was elucidated by N-terminal sequence analysis of the intact protein and by determination of the amino acid sequences of tryptic peptides by MALDI–TOF–TOF tandem mass spectrometry. The molecular mass of one subunit of the homodimer SOD, containing 152 amino acid residues, was calculated to be 15858.3 Da while a value of 17096.63 Da was obtained by MALDI–TOF MS. This difference is explained by the presence of N-glycosylation of one linkage site,-Asn-Ile/Leu-Thr-, and a glycan chain with the structure Hex 5 GlcNAc 2. Glycosylation of K. marxianus superoxide dismutase is a post-translational modification. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins. Therefore, in this paper, we introduce a covalent modification of Cu/Zn-SOD from K. marxi-anus NBIMCC 1984, by analysis of the enzymatic liberated N-glycan from the enzyme using MALDI– TOF and tandem mass spectrometry on a Q-Trap mass spectrometer. This is the first report of the structure of the oligosaccharide of a naturally-glycosylated superoxide dis-mutase, determined by mass spectrometry.

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthrop... more Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods with different quaternary structure. They are represented in the hemolymph of molluscs with one, two or three isoforms, as decameric, didecameric, multidecameric and tubules aggregates. We describe here the structure of the hemocyanin Helix lucorum (HlH), species in the series of molluscan hemocyanins. In contrast with other molluscan hemocyanins, three different hemocyanin isopolypeptides were isolated from the hemolymph of the garden snail H. lucorum, named as β-HlH, α D-HlH and α N-HlH. Their molecular masses were determined by size exclusion chromatography to be 1068 kDa (β-HlH) and 1079 kDa (α D-HlH, and α N-HlH). Native HlH exhibits a predominant didecameric structure as revealed by electron microscopy and additionally few tridecamers are shown in the electron micrographs of HlH resulting from the association of a further decamer with one didecamer. The three isoforms are represented mainly as homogeneous didecamers, but they have different behaviour after dissociation and reassociation in the pH-stabilizing buffer, containing 20 mM CaCl 2. All isoforms were reassociated into didecamers and tubules with different length, but in contrast to α D-HlH isoform, longer tubules were observed in β-HlH. Moreover the structure of β-HlH was analysed after limited proteolysis with trypsin followed by FPLC and HPLC separation of the cleavage products. Eight different functional units were identified by their N-terminal sequences and molecular masses. The protein characteristics, including UV absorption at 340 nm, fluorescence and CD spectra of the native molecule and its units confirmed the structure of multimer protein complexes.

In the present work the effect of application of RvH on ADCC as well as on mitogen responsibility... more In the present work the effect of application of RvH on ADCC as well as on mitogen responsibility of spleen lymphocytes in hamsters with progressing myeloid Graffi tumors are followed during 25 days after tumor transplantation. The in vitro ADCC tests were performed in presence of serum from Tumor-bearing Hamsters (TBH), rabbit anti-tumor Graffi serum or of normal hamster serum. RvH was injected s. c. twice in a dose of 0.5 mg per animal, determined previously as the optimal protective dose and Keyhole Limpet Hemocyanin (KLH) was used as a control. It was found that spleen lymphocyte ADCC decreased during tumor progression, while ADCC of spleen lymphocytes against own tumor cells increased about twofold in comparison to that of lymphocytes from untreated TBH after treatment of animals with KLH or RvH. The lymphocytes isolated from normal animals without treatment showed two times lower cytotoxic activity compared to those from RvH-or KLH-treated controls. RvH induced 3-5% higher ADCC compared to KLH in all combinations of sera and lymphocytes. Both, RvH and KLH showed stimulating effects on spleen lymphocytes from TBH to the B-cell mitogen LPS. Three mechanisms for the stimulation effect of RvH on lymphocytes in TBH can be hypothesized: i/hemocyanins stimulate the maturatiion of dendritic cells, the proliferation of Th1 and specific T-cytotoxic lymphocyte populations; ii/direct action of RvH on tumor cells by an apoptotic way; iii/indirect action of RvH by stimulation of cytokines.

Molluscan hemocyanins (Hcs) have recently received particular interest due to their significant i... more Molluscan hemocyanins (Hcs) have recently received particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. We have now analyzed the oligosaccharides and the carbohydrate linkage sites of the Rapana Venosa hemocyanin (RvH) using different approaches. We analyzed a number of glycopeptides by LC/ESI-MS/MS and identified the sugar chains and peptide sequences of 12 glycopeptides. Additionally, the potential carbohydrate linkage sites of 2 functional units, RvH-b and RvH-c, were determined by gene sequence analysis. Only RvH-c shows a potential N-glycosylation site. During this study, we discovered a highly conserved linker-intron, separating the coding exons of RVH-b and RvH-c. Following reports on antiviral properties from arthropod hemocyanin, we conducted a preliminary study of the antiviral activity of RvH and the functional units RvH-b and RvH-c. We show that the glycosylated FU RvH-c has antiviral properties against the respiratory syncytial virus (RSV), whereas native RvH and the nonglycosylated FU RvH-b have not. This is the first report of the fact that also molluscan hemocyanin functional units possess antiviral activity.

pH-T diagram is typical " phase portrait " for stability of functional unit RvH2-e. Using differe... more pH-T diagram is typical " phase portrait " for stability of functional unit RvH2-e. Using different techniques the T-transition curves at different pH for RvH2-e were analyzed and the parameters of the thermodynamic functions were obtained. Increasing temperature and within the T range 25-55˚C the reversibility increases and " opens a reversibility window " within the range of pH 5.5-9.0, for which were calculate at standard temperature the thermodynamic functions ΔH o and ΔG o exp. Molecular modeling of correct 3D structure of functional unit RvH2-e was done which allows us to fix most probably position of missing 9 residues now presented in existed x-ray model at very poor resolution of 3.30Å.

The present study aims to provide new information about the unusual location of Cu/Zn-superoxide ... more The present study aims to provide new information about the unusual location of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) in lower eukaryotes such as Wlamentous fungi. Humicola lutea, a high producer of SOD was used as a model system. Subcellular fractions [cytosol, mitochon-drial matrix, and intermembrane space (IMS)] were isolated and tested for purity using activity measurements of typical marker enzymes. Evidence, based on electrophoretic mobility, sensitivity to KCN and H 2 O 2 and immunoblot analysis supports the existence of Cu/Zn-SOD in mitochon-drial IMS, and the Mn-SOD in the matrix. Enzyme activity is almost equally partitioned between both the compartments , thus suggesting that the intermembrane space could be one of the major sites of exposure to superoxide anion radicals. The mitochondrial Cu/Zn-SOD was puriWed and compared with the previously published cytosolic enzyme. They have identical molecular mass, cyanide-and H 2 O 2-sensitivity, N-terminal amino acid sequence, glycosylation sites and carbohydrate composition. The H. lutea mito-chondrial Cu/Zn-SOD is the Wrst identiWed naturally gly-cosylated enzyme, isolated from IMS. These Wndings suggest that the same Cu/Zn-SOD exists in both the mito-chondrial IMS and cytosol.

In the present study the structures of two glycopeptides (G1 and G1 0), isolated from FU RvH 1-b ... more In the present study the structures of two glycopeptides (G1 and G1 0), isolated from FU RvH 1-b and two glycopeptides (G2 and G3), isolated from the structural subunit RvH 1 of Rapana venosa hemocyanin, were determined. To structurally characterize the site-specific carbohydrate heterogeneity and binding site of the N-linked glycopeptide(s), a combination of capillary reversed-phase chromatography and ion trap mass spectrometry was used. The amino acid sequences of glycopeptides G1 and G1 0 were determined by Edman degradation and MS/MS sequencing demonstrated that the oligosaccharides are linked to N-glycosylation sites. Two peptides (a glycosylated (G1) and non-glycosylated one) were identified in this fraction and no linkage sites were observed in the latter one. Based on the sequencing of the glycosylated fractions G1, G1 0 , G2 and G3, the carbohydrate structure Man(a1e6)Man(a1e3)Man(b1e4)GlcNAc(b1e4)[Fuc(a1e6)]GlcNAc-R could be identified for glycopeptides G1 and G3, and only the typical core structure Man(a1e6)Man(a1e3)Man(b1e4)GlcNAc(b1e4)GlcNAc-R was found for G1 0 and G2. The Fuc residue found in glycopeptides G1 and G3 is attached to N-acetyl-glucosamine of the carbohydrate core, as often found in other glycopro-teins.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2008

Biochemistry, 2001