Biomedical subjects
Publications and source records attributed to H Decker.
Hemocyanin sequences allineament shows the presence of highly invariant regions especially in the active site and in the tight intersubunits interaction sites. Comparing the aminoacids in contact regions between monomers is possible to interpret the stability of hexamers.
Hemocyanin from the tarantula Eurypelma californicum is a large respiratory protein with an exceptional high cooperativity. In contrast to hemocyanins from other species, no physiological allosteric effectors other than protons have been identified so far for this 24-meric oligomer. Here we report for the first time the mediating effects of water activity on the oxygen binding properties of a hemocyanin. Oxygen binding curves were measured in presence of several concentrations of glycine and sucrose since both substances reduce water activity. A pronounced shift of the p(50) was observed in both cases but in different directions: adding sucrose shifts the p(50) towards lower values whereas presence of glycine shows the same tendency as for human hemoglobin. Furthermore, prolonged incubation in sucrose slightly distorts the oxygen binding characteristics of spider hemocyanin. Therefore, only the influence of glycine was further analysed. An analysis based on the nested MWC model indicates, that presence of glycine leads to a preferential population of the two states with lower oxygen affinity (tR and tT) compared to the high affinity states rT and rR. The results corroborate the presence of hierarchically organized interactions in this hemocyanin.
We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.
The echinocandin-type antimycotic mulundocandin and its derivatives are produced by the filamentous fungus Aspergillus sydowii (strain FH2551). These agents have been considered as a potential drug to treat immunocompromised patients who suffer from severe opportunistic fungal infections. In order to generate strains with a modified mulundocandin biosynthesis, we developed molecular tools for genetic engineering of A. sydowii as an alternative to conventional strain improvement procedures. For our experiments, we used strain FH2551, which was discriminated from other Aspergillus strains by determining the sequence of the two internal transcribed spacers (ITS1 and ITS2) of the rDNA locus. In addition, the electrophoretic karyotype of A. sydowii was established using pulsed-field gel electrophoresis (PFGE), leading to a calculated genomic size of about 40 Mb. For gene mapping, chromosomes were subjected to PFGE either unrestricted or after incubation with rare cutting enzymes and probed with heterologous genes. Using the bacterial hygromycin B phosphotransferase gene as a selectable marker for transformation of A. sydowii, we generated transformants with single and multiple copies of plasmid DNA. Subsequently, the heterologous lacZ and gfp genes were efficiently transferred and expressed in A. sydowii. The majority of lacZ-transformants showed more than 6 pkat beta-galactosidase activity/mg protein, while the control strains had no significant background activity. Fluorescence microscopy of gfp-transformants demonstrated that the green-fluorescent protein is present in a stable and active form in the cytoplasm of vegetative hyphae and conidiospores.
The oxygen binding behaviour of hemocyanins from Crustacea is regulated by small organic compounds such as urate and L-lactate. We investigated the binding characteristics of urate and the related compound caffeine to the 2 x 6-meric hemocyanin of A. leptodactylus under fully oxygenated conditions employing isothermal titration calorimetry (ITC). An analysis of urate and caffeine binding based on a model of n identical binding sites resulted in approximately four binding sites for caffeine and eight for urate. This result suggests that the binding process for these effectors is more complex than this most simple model. Therefore, we introduced a number of alternative models. Displacement experiments helped to select the appropriate model. Based on these experiments, at least two different types of binding sites for urate and caffeine exist on the 2 x 6-meric hemocyanin of A. leptodactylus. The two binding sites differ strongly in their specificity towards the two analogues. It can be hypothesized that two different subunit types (beta and gamma) are responsible for the two types of binding sites.
Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.
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Phenoloxidase, widely distributed among animals, plants, and fungi, is involved in many biologically essential functions including sclerotization and host defense. In chelicerates, the oxygen carrier hemocyanin seems to function as the phenoloxidase. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab Limulus polyphemus and the tarantula Eurypelma californicum, exhibit O-diphenoloxidase activity induced by submicellar concentrations of SDS, a reagent frequently used to identify phenoloxidase activity. The enzymatic activity seems to be restricted to only a few of the heterogeneous subunits. These active subunit types share similar topological positions in the quaternary structures as linkers of the two tightly connected 2 x 6-mers. Because no other phenoloxidase activity was found in the hemolymph of these animals, their hemocyanins may act as a phenoloxidase and thus be involved in the primary immune response and sclerotization of the cuticle. In contrast, hemolymph of a more recent arthropod, the crab Cancer magister, contains both hemocyanin with weak phenoloxidase activity and another hemolymph protein with relatively strong phenoloxidase activity. The chelicerate hemocyanin subunits showing phenoloxidase activity may have evolved into a separate phenoloxidase in crustaceans.
The respiratory protein of the keyhole limpet, Megathura crenulata, the hemocyanin (KLH), commonly used as an immunogen, binds oxygen cooperatively, which implies the existence of different conformations. For the first time, two different conformations of KLH1 were detected upon oxygenation, a deoxy and an oxy state, using small-angle neutron scattering. Rearrangements in the quaternary structure of KLH1 were predicted from the different radii of gyration and the shifts of the minima and maxima in the scattering curves. Upon oxygenation, KLH1 becomes smaller and more compact. Model reconstruction of KLH1 indicates a hollow cylinder with two rings located close to both ends, which move slightly together upon oxygenation.
Elloramycin A (1) belongs to a small family of naphthacenequinones characterized by a unique highly hydroxylated cyclohexenone moiety. A cosmid clone 16F4, harboring genes for the production of 1 from Streptomyces olivaceus Tü2353, has been previously isolated. DNA sequence analysis of a 3.2-kb fragment from 16F4 revealed four open reading frames--the elmGHIJ genes. Heterologous expressions of the elmGHI genes in either Escherichia coli or Streptomyces lividans, followed by biochemical characterizations of the ElmGHI proteins, established ElmG as tetracenomycin B2 oxygenase, ElmH as tetracenomycin F1 monooxygenase, and ElmI as tetracenomycin F2 cyclase. These results provide direct biochemical evidence for the hypothesis that the biosynthesis of 1 in S. olivaceus parallels that of tetracenomycin C (2) in Streptomyces glaucescens and support the notion that the biosynthesis of the highly hydroxylated cyclohexenone moiety in other polyketides most likely follows the same paradigm as the tetracenomycin B2 or A2 oxygenase.
Haemocyanin serves as an oxygen carrier in the haemolymph of decapod crustaceans. The oxygen-binding behaviour of the pigment is modulated by the two major anaerobic metabolites, l-lactate and urate. The binding of these two metabolites to haemocyanin has been investigated mainly indirectly by following the effector-induced changes in the oxygen-binding properties of the respiratory pigment. Only a few direct investigations of effector binding, employing ultracentrifugation techniques and equilibrium dialysis, have been carried out. No evidence for cooperative binding for either effector was detected using these methods. However, isothermal titration calorimetry (ITC) offers a useful tool to gain additional insight into the binding of effectors to these highly allosterically regulated macromolecules. By applying the ITC method to the fully oxygenated dodecameric haemocyanin of the lobster Homarus vulgaris, cooperativity in binding has been found for the urate analogue caffeine but not for urate itself: using urate and the urate analogue caffeine as ligands, two conformations of the oxygenated pigment were detected.
The new pluramycin-type antibiotics pluraflavin A, C43H54N2O14, pluraflavin B, C43H56N2O15, and pluraflavin E, C36H41NO14 were isolated from cultures of the Saccharothrix species DSM 12931. The structures of the novel compounds were elucidated with the aid of 2D NMR and mass spectrometric investigations. The characteristic structural element of pluraflavins A and B is an additional 4-epi-vancosamine unit at position 13 of the anthraquinone-gamma-pyrone ring system. Pluraflavin E has a carboxyl group in this position. Pluraflavin A has a reactive dimethyl epoxide side chain at position 2 of the anthraquinone-gamma-pyrone aglycon, which may explain the high activity of the antibiotic. The outstanding biological characteristic of pluraflavin A is its powerful, organ-dependent cytostatic action: the IC50 in the colon carcinoma proliferation assay is in the subnanomolar range.
Hemocyanins are large oligomeric respiratory proteins found in many arthropods and molluscs. The hemocyanin of the tarantula Eurypelma californicum is a 24-mer protein complex with molecular mass of 1, 726,459 Da that consists of seven different polypeptides (a-g), each occupying a distinct position within the native molecule. Here we report the complete molecular structure of the E. californicum hemocyanin as deduced from the corresponding cDNAs. This represents the first complex arthropod hemocyanin to be completely sequenced. The different subunits display 52-66% amino acid sequence identity. Within the subunits, the central domain, which bears the active center with the copper-binding sites A and B, displays the highest degree of identity. Using a homology modeling approach, the putative three-dimensional structure of individual subunits was deduced and compared. Phylogenetic analyses suggest that differentiation of the individual subunits occurred 400-550 million years ago. The hemocyanin of the stemline Chelicerata was probably a hexamer built up of six distinct subunit types a, b/c, d, e, f, and g, whereas that of the early Arachnida was originally a 24-mer that emerged after the differentiation of subunits b and c.
Hemocyanin serves as an oxygen carrier in the hemolymph of the European lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigment under deoxygenated conditions. Urate binds to two identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M(-1) and an enthalpy change DeltaH degrees of -32.3 kcal mol(-1). Caffeine binds cooperatively to hemocyanin with two microscopic binding constants: K(1) = 14 100 M(-1) and K(2) = 40 400 M(-1). The corresponding enthalpy changes in binding are as follows: DeltaH degrees (1) = -23.3 kcal mol(-1) and DeltaH degrees (2) = -27.1 kcal mol(-1). The comparison of urate and caffeine binding to the oxygenated pigment indicates the existence of two protein conformations for oxygen-saturated hemocyanin. Since effector binding is not influenced by protons, four different conformations are required to create a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lobster hemocyanin, employing the nesting model.
Fibroblast growth factor receptor 3 (FGFR3) is a glycoprotein that belongs to the family of tyrosine kinase receptors. Specific mutations in the FGFR3 gene are associated with autosomal dominant human skeletal disorders such as hypochondroplasia, achondroplasia, and thanatophoric dysplasia. Hypochondroplasia (HCH), the mildest form of this group of short-limbed dwarfism disorders, results in approximately 60% of cases from a mutation in the intracellular FGFR3-tyrosine kinase domain. The remaining cases may either be caused by defects in other FGFR gene regions or other yet unidentified genes. We describe a novel HCH mutation, the first found outside the common mutation hot spot of this condition. This point mutation, an N328I exchange in the extracellular Ig domain III of the receptor, seems to be unique as it affects a putative N-glycosylation site that is conserved between different FGFRs and species. The amino acid exchange itself most probably has no impact on the three-dimensional structure of the receptor domain, suggesting that the phenotype is the result of altered receptor glycosylation and its pathophysiological consequences.
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The enzymes tyrosinase, catecholoxidase and hemocyanin all share similar active sites, although their physiological functions differ. Hemocyanins serve as oxygen carrier proteins, and tyrosinases and catecholoxidases (commonly referred to as phenoloxidases in arthropods) catalyze the hydroxylation of monophenols or the oxidation of o-diphenols to o-quinones, or both. Tyrosinases are activated in vivo by limited proteolytic cleavage, which might open up substrate access to the catalytic site. It has recently been demonstrated that if hemocyanins are subjected to similar proteolytic treatments (in vitro) they also exhibit at least catecholoxidase reactivity. On the basis of their molecular structures, hemocyanins are used as model systems to understand the substrate-active-site interaction between catecholoxidases and tyrosinases.
When Astacus leptodactylus were kept at various temperatures for several weeks, different ratios between di-hexameric and hexameric hemocyanins were observed in their hemolymph. The higher the temperature the more hexamers were present. This long-term adaptation to different temperatures or/and to temperature-induced pH-shifts as observed in the hemolymph has different effects on the expression of subunit types building up hexamers and those which covalently link two hexamers within the di-hexamers. The oxygen binding behaviour of di-hexameric hemocyanins from cold and warm adapted animals do not show differences with respect to affinity, Bohr effect and cooperativity.