Therapeutically targeting protein-glycan interactions.

Glycosylation is the most common form of post-translational modifications by which oligosaccharide side chains are covalently attached to specific residues of the core protein. Especially O-linked glycan structures like the glycosaminoglycans were found to contribute significantly to many (patho-)biological processes like inflammation, coagulation, cancer and viral infections. Glycans exert their function by interacting with proteins thereby changing the structure of the interacting proteins and consequently modulating their function. Given the complex nature of cell-surface and extracellular matrix glycan structures, this therapeutic site has been neglected for a long time, the only exception being the antithrombin III-glycan interaction which has been successfully targeted by unfractionated and low-molecular weight heparins for many decades. Due to the recent breakthrough in the '-ome' sciences, among them proteomics and glycomics, protein-glycan interactions became more amenable for therapeutic approaches so that novel inhibitors of this interaction are currently in preclinical and clinical studies. An overview of current approaches, their advantages and disadvantages, is given and the promising potential of pharmacologically interfering with protein-glycan interactions is highlighted here.

Developing chemokine mutants with improved proteoglycan affinity and knocked-out GPCR activity as anti-inflammatory recombinant drugs.

The interaction of chemokines and GAGs (glycosaminoglycans) on endothelial surfaces is a crucial step for establishing a chemotactic gradient which leads to the functional presentation of chemokines to their GPCRs (G-protein-coupled receptors) and thus to activation of approaching leucocytes. Based on molecular modelling, biophysical investigations, cell-based and in vivo experiments, we have developed a novel concept for therapeutically interfering with chemokine-GAG interactions, namely dominant-negative chemokine mutants with improved GAG binding affinity and knocked-out GPCR activity. These recombinant proteins displace their wild-type chemokine counterparts from the natural proteoglycan co-receptors without being able to activate leucocytes via GPCRs. Our mutant chemokines therefore represent the first protein-based GAG antagonists with high therapeutic potential in inflammatory diseases.

Ligand affinity, homodimerization, and ligand-induced secondary structural change of the human vitamin d receptor.

The intrinsic tryptophan fluorescence signal of the full-length nuclear receptor hVDR was used to directly determine the dissociation constants, K(d), of two ligands yielding K(d) = 32 nM for 1alpha,25(OH)(2)D(3) and K(d) = 322 nM for 25(OH)D(3). Ligand binding was accompanied by a conformational change in the alpha-helical part of hVDR as revealed by CD spectroscopy. In addition, the presence of calcitriol was found to be a necessary prerequisite for the homodimerisation of hVDR which was monitored using fluorescence anisotropy. We conclude that the observed ligand-induced structural change of hVDR is conditional for dimerisation of the protein.

Electrochemistry of pterin cofactors and inhibitors of nitric oxide synthase.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS), but its function is not fully understood. Specifically, it is unclear whether BH4 participates directly in electron transfer. We investigated the redox properties of BH4 and several other pteridines with cyclic voltammetry and Osteryoung square wave voltammetry. BH4 was oxidized at a potential of +0.27 V vs normal hydrogen electrode (NHE); the corresponding reductive signal after the reversal of the scan direction was very small. Instead, reduction occurred at a potential of -0.16 V vs NHE; there was no corresponding oxidative signal. These two transitions were interdependent, indicating that the reductive wave at -0.16 V represented the regeneration of BH4 from its product of oxidation at +0.27 V. Similar voltammograms were obtained with tetrahydroneopterin and 6,7-dimethyltetrahydropterin, both of which can substitute for BH4 in NOS catalysis. Completely different voltammograms were obtained with 7,8-dihydrobiopterin, sepiapterin, 2'-deoxysepiapterin, and autoxidized BH4. These 7,8-dihydropterins, which do not sustain NOS catalysis, were oxidized at much higher potentials (+0.82-1.04 V vs NHE), and appreciable reduction did not occur between +1.2 and -0.8 V, in line with the concept of a redox role for BH4 in NOS catalysis. However, the electrochemical properties of the potent pterin-site NOS inhibitor 4-amino-BH4 resembled those of BH4, whereas the active pterin cofactor 5-methyl-BH4 was not re-reduced after oxidation. We conclude that the 2-electron redox cycling of the pterin cofactor between BH4 and quinonoid dihydrobiopterin is not essential for NO synthesis. The data are consistent with 1-electron redox cycling between BH4 and the trihydrobiopterin radical BH3(*).

The influence of ATP on the association and unfolding of the tyrosine repressor ligand response domain of Haemophilus influenzae.

The secondary structure of the ligand response domain of the Haemophilus influenzae tyrosine repressor, TyrR(lrd), was investigated using CD spectroscopy which revealed 42.5% alpha-helix, 17.6% beta-sheet, and 39.9% loops. Quaternary structure analysis by fluorescence anisotropy showed that TyrR(lrd) is monomeric at a concentration of 100 nM to 2 microM but that the protein readily dimerizes in the presence of its natural ligand ATP. Equilibrium unfolding studies of TyrR(lrd) using guanidinium hydrochloride suggested a two-state model with no detectable stable intermediates. The unfolding transition monitored by CD spectroscopy was responsive to tyrosine and ATP resulting in a shift to higher denaturant concentrations in the presence of these ligands. Differential scanning calorimetry yielded melting temperatures, T(m), of 51.15 and 58.07 degrees C for the unliganded and for the ATP-liganded protein, respectively. ATP is thus proposed to be a major structural cofactor for the molecular architecture of TyrR(lrd).

The influence of ATP on the binding of aromatic amino acids to the ligand response domain of the tyrosine repressor of Haemophilus influenzae.

The binding of aromatic amino acids to the ligand response domain of the tyrosine repressor (TyrR) protein (TyrR(lrd)) of Haemophilus influenzae was investigated using circular dichroism and fluorescence spectroscopy. The induced secondary structural changes were unique for each aromatic amino acid and were further influenced by the presence or absence of ATP. Tyrosine was found to have the highest affinity for TyrR(lrd) in the absence of ATP, whereas the affinity for ATP itself increased in the presence of tyrosine. Binding of tyrosine is therefore the conformational trigger for the activation of TyrR whereas ATP is regarded as a conformational co-activator.

X-ray structure and conformational dynamics of the HIV-1 protease in complex with the inhibitor SDZ283-910: agreement of time-resolved spectroscopy and molecular dynamics simulations.

Based on the X-ray structure of the human immunodeficiency virus type-1 (HIV-1) protease in complex with the statine-derived inhibitor SDZ283-910, a 542 ps molecular dynamics trajectory was computed. For comparison with the 805 ps trajectory obtained for the uncomplexed enzyme, the theoretical fluorescence anisotropy decay of the unliganded protease and the inhibitor complex was calculated from the trajectories of the Trp6A/Trp6B and Trp42A/Trp42B transition dipole moments. This enabled us to directly compare the simulated data with the experimental picosecond time-resolved fluorescence data. Fitting both experimental and simulated data to the Kohlrausch-Williams-Watts (KWW) function exp(-t/tauk)beta revealed a very good agreement for the uncomplexed protease as well as for the SDZ283-910 complex. Binding of the inhibitor induced a faster decay of both the experimental and the computed protease fluorescence anisotropy decay. By this integrative approach, the atomic detail of inhibitor-induced changes in the conformational dynamics of the HIV-1 protease was experimentally verified and will be used for further inhibitor optimisation.

Sensitivity of flavin fluorescence dynamics in neuronal nitric oxide synthase to cofactor-induced conformational changes and dimerization.

The fluorescence intensity of the two flavin prosthetic groups, FMN and FAD, in neuronal nitric oxide synthase (nNOS) was found to decay highly nonexponentially, being best described by four fluorescence lifetimes. This excited state heterogeneity is the result of multiple flavin quenching sites which are due to several flavin microenvironments created mainly by stacking with aromatic amino acids. Investigating nNOS in the absence of one or more of Ca2+/calmodulin, tetrahydrobiopterin, and heme revealed an influence of these cofactors on the microenvironments of the flavin prosthetic groups. Similar effects on the flavin rotational dynamics were found by analyzing the fluorescence anisotropy decay of the holo and of the different apo forms of nNOS. Since the tetrahydrobiopterin and the heme are located in the N-terminal oxygenase domain of nNOS, their effect on the flavins in the C-terminal reductase domain is explained by a folding back of the reductase domain onto the oxygenase domain. Thereby a domain-domain interface is created containing the FAD, FMN, heme, and tetrahydrobiopterin prosthetic groups which allows for efficient electron transfer during catalysis. The heme group, which is known to be essential for homodimerization of nNOS, was also found to be essential for the formation of the domain-domain interface.

Time-resolved fluorescence anisotropy of HIV-1 protease inhibitor complexes correlates with inhibitory activity.

The tryptophan time-resolved fluorescence intensity and anisotropy of the HIV-1 protease dimer is shown to be a quick and efficient method for the conformational characterization of protease inhibitor complexes. Four fluorescence lifetimes were needed to adequately describe the fluorescence decay of the two tryptophan residues, W6 and W42, per protease monomer. As a result of the wavelength dependence of the respective amplitudes, the 2.06 ns and the 4.46 ns decay constants were suggested to be the intrinsic fluorescence lifetimes of the more solvent-exposed W6 and the less exposed W42 residues, respectively. Analysis of the fluorescence anisotropy decay yielded a short correlation time of 250 ps corresponding to local chromophore motions, and a long correlation time of 12.96 ns resulting from overall rotation of the protease enzyme. Fluorescence lifetimes and rotational correlation times changed when inhibitors of the HIV-1 protease were added. The effects of 11 different inhibitors including statine-derived, hydroxyethylamine-derived, and 2 symmetrical inhibitors on the protease fluorescence dynamics were investigated. Inhibitor binding is shown to induce an increase of the mean fluorescence lifetime taumean, an increase of the short rotational correlation time phi1, as well as a decrease of the long rotational correlation time phi2. The mean rotational correlation time phimean was identified as the global dynamic parameter for a given molecular complex, which correlates with the inhibitor dissociation constant Ki, and therefore with the activity of the inhibitor.

Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy.

Specific immunotherapy is an efficient treatment for patients suffering from type I allergy. The mechanisms underlying successful immunotherapy are assumed to operate at the level of T helper cells, leading to a modulation of the immune response to allergens. During immunotherapy, increasing doses of allergens are given on a regular basis, and the beneficial effects for the patient depend on the concentration of allergen used. On the other hand, the risk of IgE-mediated anaphylactic side effects also increase with the amount of allergen applied per injection. Therefore, we have proposed the use of hypoallergenic (low IgE binding activity) forms of allergens for immunotherapy. We evaluated by site-directed mutagenesis the contributions of individual amino acid residues/positions for IgE binding to Bet v 1, the major allergen of birch pollen. We found that IgE binding to Bet v 1 depended on at least six amino acid residues/positions. Immunoblot analyses and inhibition experiments showed that the multiple-point Bet v 1 mutant exhibited extremely low reactivity with serum IgE from birch pollen-allergic patients. In vivo (skin prick) tests showed that the potency of the multiple-point mutant to induce typical urticarial type I reactions in pollen-allergic patients was significantly lower than for wild-type Bet v 1. Proliferation assays of allergen-specific T cell clones demonstrated that these six amino acid exchanges in the Bet v 1 sequence did not influence T cell recognition. Thus, the Bet v 1 six-point mutant displayed significantly reduced IgE binding activity, but conserved T cell activating capacity, which is necessary for immunomodulation. The approach described here may be generally applied to produce allergen variants to be used in a safe therapy form of immediate-type allergies.

Immunological and biological properties of Bet v 4, a novel birch pollen allergen with two EF-hand calcium-binding domains.

We have isolated a cDNA clone coding for a birch pollen allergen, Bet v 4. The deduced amino acid sequence of Bet v 4 contained two typical EF-hand calcium-binding domains. Sequence similarities of Bet v 4 to calmodulin are primarily confined to the calcium-binding domains. However, significant sequence similarities extending outside the Ca2+-binding sites were found with a recently described group of pollen-specific allergens of Brassica and Bermuda grass. Both EF-hand domains of Bet v 4 are able to bind Ca2+, as demonstrated by 45Ca2+ blot overlay of wild type and calcium-binding deficient mutants of Bet v 4. Among pollen-allergic patients, protein-bound Ca2+ was not an absolute requirement for IgE recognition of Bet v 4. However, disruption of the carboxyl-terminal Ca2+-binding domain indicated that most IgE antibodies from allergic patients are directed against this site. IgE inhibition experiments suggested that Bet v 4 represents a highly cross-reactive pollen allergen. Pre-absorption of allergic sera with Bet v 4 drastically reduced IgE binding to proteins of similar molecular weight in pollen extracts from distantly related plant species (e.g. timothy grass, mugwort, lily) but not in extracts from plant-derived foodstuff. To test for a possible biological role in pollen germination and tube growth, we introduced recombinant Bet v 4 protein into growing lily pollen tubes by iontophoresis. As a result, cytoplasmic streaming stopped in the vicinity of the electrode tip, and a slight depolarization of the membrane voltage was measured. These effects were not observed with Ca2+-binding deficient mutants of Bet v 4. Thus, Bet v 4 and homologous proteins represent a new class of pollen-specific Ca2+-binding allergens that may have a physiological role as inhibitors of cytoplasmic streaming in outgrowing pollen tubes.

Intensity-independent fluorometric detection of cellular nitric oxide release.

A new fluorescence method is introduced in which nitric oxide (NO)-derived higher-order oxygen complexes (NO(x)) are quantified at physiological pH. Detecting the fluorescence lifetime shift between 2,3-diaminonaphthalene and the NO(x)-derived protonated 2,3-naphthotriazole allows an intensity independent determination of the NO(x) concentration. The NO release from LPS and IFNgamma-stimulated murine macrophages and iNOS transfected hamster cells was quantified. The lower detection limit for NO2- was found to be 800 pmol/ml. Since the influence of static fluorescence quenching due to cellular components can be neglected, the method is applicable for clear cellular supernatants as well as turbid cellular suspensions.

Evidence for an alpha helical T cell epitope in the C-terminus of the main birch pollen allergen Bet V 1.

Secondary structure prediction of the main birch pollen allergen Bet v 1 was found to be in good agreement with the secondary structural elements found by analysing the Bet v 1 circular dichroism data. According to both experiment and prediction, 32% of 160 amino acids participate in alpha helices, 21% in beta sheets, 24% in turns, and 23% in other structural motifs. The peptide LRAVESYLLAHS which represents one of the major T cell epitopes on Bet v 1 was shown to have a high propensity to form an alpha helix. Time-resolved fluorescence anisotropy measurements of the allergen revealed an overall rotational correlation time of 7.35 ns, which corresponds to a hydrodynamic molecular radius of 19.2 A. This refers to a monomeric Bet v 1 molecule in solution, which is also reflected in the narrow band width of the 1H-NMR spectrum. The results presented here are in good agreement with the recently solved NMR structure of Amb t 5: both allergens are monomers in solution with an extended C-terminal alpha helix containing a major T cell epitope.

Molecular dynamics simulation of the rare amino acid LL-dityrosine and a dityrosine-containing peptide: comparison with time-resolved fluorescence.

The fluorescence of the rare amino acid LL-dityrosine, which is found in insoluble biological materials with structural features, was recently shown to decay non-exponentially (Kungl et al. (1992) J. Fluorescence 2, 63-74). Here we investigated the time-resolved fluorescence of a dityrosine-containing peptide (DCP) to study the influence of side chains on the fluorescence decay of the chromophore. The fluorescence decay of DCP was best fitted by three exponential terms including a sub-nanosecond rise term, the values of which are quite similar to the parameters obtained for the decay of free dityrosine. They were found to depend on the pH of the aqueous solution but not on the temperature. Analysis by an exponential series method revealed broad fluorescence lifetime distributions for DCP. Compared to the corresponding analysis of dityrosine transients, similar lifetime centers were found whereas the widths of the distributions were found broader for DCP. Molecular dyamics (MD) simulations of dityrosine at 300 K show that chi 1 and chi 2 side chain conformers (rotamers) of both tyrosine subunits interconvert on a picosecond timescale. The rates of interconversion were shown to depend critically upon the MD technique applied: in vacuo simulations yielded lower interconversion rates compared to stochastic dynamics (SD) and full MD (water explicitly included). However, MD simulations of the dityrosine-containing peptide revealed no interconversions of the chi 1 and chi 2 side chain rotamers of both tyrosine subunits within a 400 ps trajectory. Interconversions could be induced by raising the temperature of the system (DCP plus solvent) to 340 K. Side chain rotamers of dityrosine are not stable on a fluorescence time scale but are stable when a dityrosine-containing peptide is regarded. Nevertheless both molecules yield similar fluorescence decay patterns. We therefore conclude that the rotamer model proposed for the fluorescence decay of tyrosine and tryptophan cannot be applied to the fluorescence decay of dityrosine and peptides containing this chromophore. This should be of future interest when dityrosine is used as an intrinsic sensor to study complex dityrosine-containing macromolecules by fluorescence spectroscopy.

Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores.

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.

Purification, crystallization and preliminary X-ray diffraction analysis of recombinant human neutrophil-activating peptide 2 (rhNAP-2).

The potent activator and chemoattractant for human neutrophils, neutrophil-activating peptide 2 (NAP-2), has been cloned and expressed in Escherichia coli. The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC. The biological activity of recombinant human NAP-2 (rhNAP-2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP-2. rhNAP-2 has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P222 with unit cell dimensions of a = 30.8 A, b = 39.5 A and c = 95.3 A. A packing density of 3.8 A3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed. The crystals were shown to diffract to beyond 2.0 A on a conventional X-ray source. They are stable to X-rays for several days and are thus suitable for high resolution structure determination.

Characterization of human immunodeficiency virus-1 (HIV-1) rev by (time-resolved) fluorescence spectroscopy.

Fluorescence spectroscopy has been applied to the single tryptophan-containing regulatory protein Rev of human immunodeficiency virus (HIV-1). The fluorescence emission was found to have a maximum at 336 nm which refers to a surrounding of the chromophore of intermediate polarity. Fluorescence transients recorded at the maximum of fluorescence were found to decay nonexponentially. A bimodal lifetime distribution is obtained from exponential series analysis (ESM) with centers at 1.7 and 4.5 ns. Two microenvironments for tryptophan are suggested to be responsible for the two lifetime distributions. No innerfilter effect occurred in a Rev solution up to a concentration of 40 µM. A data quality study of ESM analysis as function of collected counts in the peak channel maximum (CIM) showed that, for reliable reconvolution, at least 15,000 CIM are necessary. The widths of the two distributions are shown to be temperature dependent. The broadening of the lifetime distributions when the temperature is raised to 50°C is interpreted as extension of the number of conformational substates which do not interconvert on the fluorescence time scale. The thermal deactivation (temperature quenching) is reflected in a constant decrease in the center of the short-lived lifetime distribution.

Rotamer interconversion and its influence on the fluorescence decay of tyrosine: a molecular dynamics study.

To test the hypothesis of charge-transfer quenching between an electrophile in the alanyl sidechain (carbonyl carbon or protonated amino group) and the excited aromatic phenol-subunit, which leads to a bi-exponential fluorescence decay of tyrosine in acidic aqueous solution, we investigated the dynamics of this amino acid and of the peptide Gly-Tyr-Gly in vacuo and in water with classical molecular dynamics (MD) and with stochastic dynamics (SD) computer simulation. The proposed low-frequency of interconversions between sidechain rotamers on a fluorescence time-scale could not be confirmed. Instead, frequent transitions for both, chi 1 and chi 2, dihedrals were observed. Simulating a low pH situation (protonated carboxylate group) did not significantly affect the transition frequency. Rotamer interconversions in the peptide Gly-Tyr-Gly, though significantly less, were also observed although the fluorescence decay of this compound could be described by a uni-modal lifetime distribution centered at 0.8 ns. The results obtained from simulations in vacuo and in solution were critically compared with those of stochastic simulations. We found the stochastic simulation in a better agreement to full MD (water explicitly included), which is highly time consuming, whereas the in vacuo simulations clearly deviated from both. We conclude from our results that, since the rotamers do frequently interconvert within the fluorescence lifetime of tyrosine, their contribution to the non-exponential fluorescence decay should be negligible.

L-Dityrosine: A time-resolved fluorescence investigation.

We have investigated the time-resolved fluorescence properties ofLL-dityrosine in aqueous solution. Typically, three exponential components were needed to fit the fluorescence pattern adequately, with pure decay terms for the low-intensity, high-energy state (λem = nm) but with a pronounced subnanosecond rise phase for the predominant red-edge fluorescence (λem > 380). Dual fluorescence behavior is indicative of an intramolecular precursorsuccessor pair, i.e., a consecutive intramolecular excited-state reaction. We suggest that this reaction is a torsional motion of the (deprotonated) monoanion along the biphenolic bond. Analysis of the fluorescence anisotropy decay of dityrosine yielded two rotational correlation times, the longer of which is associated with a negative preexponential term. The increase with time in the horizontally polarized component of the intensity decay is interpreted as the result of an electronic rearrangement in the excited state when the successor form of dityrosine is generated. Lifetime distributions of experimental data were probed by an unbiased exponential series method which uses a Tikhonov-type regularization function. The procedure revealed three well-separated groups of lifetimes, the short-lived ensemble forming a formally negative distribution. A photophysical model is introduced which interprets the biexponential decay of dityrosine in terms of overlapping emission signals from the precursor and the successor molecule.