bluuebunny/crossref_metadata_2025 huggingface dataset:
α3‐Galactosylated glycoproteins can bind to the hepaticasialoglycoprotein receptor
Authors: Joziasse David H. , Lee Reiko T. , Lee Yuan C. , Biessen Erik A. L. , Schiphorst Wietske E. C. M. , Koeleman Carolien A. M. , van den Eijnden Dirk H.
Published: March 2003

In mammals, clearance of desialylated serum glycoproteins to the liver is mediated by a galactose‐specific hepatic lectin, the ‘asialoglycoprotein receptor’. In humans, serum glycoprotein glycans are usually capped with sialic acid, which protects these proteins against hepatic uptake. However, in most other species, an additional noncharged terminal element with the structure Galα1→3Galβ1→4R is present on glycoprotein glycans. To investigate if α3‐galactosylated glycoproteins, just like desialylated glycoproteins, could be cleared by the hepatic lectin, the affinities of α3‐galactosylated compounds towards this lectin were determined using an in vitro inhibition assay, and were compared with those of the parent compounds terminating in Galβ1→4R. Diantennary, triantennary and tetraantennary oligosaccharides that form part of N‐glycans were α3‐galactosylated to completion by use of recombinant bovine α3‐galactosyltransferase. Similarly, desialylated α1‐acid glycoprotein (orosomucoid) was α3‐galactosylated in vitro. The α3‐galactosylation of a branched, Galβ1→4‐terminated oligosaccharide lowered its affinity for the membrane‐bound lectin on whole rat hepatocytes 50–250‐fold, and for the detergent‐solubilized hepatic lectin 7–50‐fold. In contrast, α3‐galactosylation of asialo‐α1‐acid glycoprotein caused only a minor decrease in affinity, increasing the IC50 from 5 to 15 nm.

Fully α3‐galactosylated α1‐acid glycoprotein, intravenously injected into the mouse, was rapidly cleared from the circulation, with a clearance rate close to that of asialo‐α1‐acid glycoprotein (t1/2 of 0.42 min vs. 0.95 min). Its uptake was efficiently inhibited by pre‐injection of an excess asialo‐fetuin. Organ distribution analysis showed that the injected α1‐acid glycoprotein accumulated predominantly in the liver. Taken together, these observations suggest that serum glycoproteins that are heavily α3‐galactosylated will be rapidly cleared from the bloodstream via the hepatic lectin. It is suggested that glycosyltransferase expression in murine hepatocytes is tightly regulated in order to prevent undesired uptake of hepatocyte‐derived, circulating glycoproteins.
Effects of proline mutations in the major house dust mite allergen Der f 2 on IgE‐binding and histamine‐releasing activity
Authors: Takai Toshiro , Ichikawa Saori , Hatanaka Hideki , Inagaki Fuyuhiko , Okumura Yasushi
Published: March 2003

Der f 2 is the major group 2 allergen from house dust mite Dermatophagoides farinae and is composed of 129 amino‐acid residues. Wild‐type and six proline mutants of Der f 2 (P26A, P34A, P66A, P79A, P95A, and P99A) expressed in Escherichia coli were refolded and purified. Formations of intramolecular disulfide bonds in the purified proteins were confirmed correct. The apparent molecular masses analyzed by gel‐filtration were 14–15 kDa. The IgE‐binding capacity in the sera of seven mite‐allergic patients, inhibitory activity for IgE‐binding to immobilized wild‐type Der f 2, and activity to stimulate peripheral blood basophils to release histamine in two volunteers were analyzed. P95A and P99A, which slightly differed from the wild‐type Der f 2 in their CD spectrum, showed reduced IgE‐binding, reduced inhibitory activity, and less histamine‐releasing activity than the wild‐type. P34A also showed reduced allergenicity. Considering that Pro95, Pro99 and Pro34 are closely located in loops at one end of the tertiary structure of Der f 2, we concluded that these loop regions included an IgE‐binding site common to all tested patients. P66A showed reduced IgE‐binding in two sera out of seven. P26A and P79A showed no reduced allergenicity. However, in immunoblot analysis after SDS/PAGE under reduced conditions, P79A showed no or markedly reduced IgE‐binding while the other mutants showed IgE‐binding corresponding to that in the assay using correctly refolded proteins. This suggests that Pro79 is involved in refolding of Der f 2. The findings in this study are important for the understanding of the antigenic structure of mite group 2 allergens and for manipulation of the allergens for specific immunotherapy.
Conformations of the regulatory domain of cardiac troponin C examined by residual dipolar couplings
Authors: Pääkkönen Kimmo , Sorsa Tia , Drakenberg Torbjörn , Pollesello Piero , Tilgmann Carola , Permi Perttu , Heikkinen Sami , Kilpeläinen Ilkka , Annila Arto
Published: March 2003

Conformations of the regulatory domain of cardiac troponin C (cNTnC) were studied by means of residual dipolar couplings measured from samples dissolved in dilute liquid crystals. Changes in the main chain HN residual dipolar couplings revealed a conformational change in cNTnC due to the complexation with the second binding region (amino acids 148–163) of cardiac troponin I (cTnI). Formation of the complex is accompanied with a molecular realignment in the liquid crystal. The residual dipolar couplings measured for apo‐cNTnC and the complex with TnI were in agreement with the values computed from the corresponding closed and open solution structures, whereas for the calcium‐loaded conformation the correlation and quality factor were only modest. Ca2+‐cNTnC may be subject to conformational exchange. The data support the model that cardiac troponin C functions as a calcium‐dependent open–closed switch, such as the skeletal troponin C.
Characterization of active‐site mutants of Schizosaccharomyces pombe phosphoglycerate mutase
Authors: Nairn Jacqueline , Duncan Doris , Price Naomi E. , Kelly Sharon M. , Fothergill‐Gilmore Linda A. , Uhrinova Stanislava , Barlow Paul N. , Rigden Daniel J. , Price Nicholas C.
Published: March 2003

The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site‐directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site‐directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150‐gpm::HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3‐phosphoglycerate) to wild‐type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques.
Negative regulation of the platelet Na+/H+ exchanger by trimeric G‐proteins
Authors: van Willigen Gijsbert , Nieuwland Rienk , Nürnberg Bernd , Gorter Gertie , Akkerman Jan‐Willem N.
Published: March 2003

Human platelets contain a Na+/H+ exchanger (NHE) that regulates the cytosolic pH. The role of trimeric G‐proteins in NHE control was investigated in plasma membrane vesicles by measuring exchange of intravesicular protons for extravesicular Na+. Exchange was saturable, independent of membrane potential and inhibited by ethylisopropyl amiloride (Ki 0.05 µmol·L−1), demonstrating the involvement of NHE‐1. The G‐protein activators AlF4− and GMP‐P(NH)P reduced exchange by increasing the Km for Na+ from 11.3 ± 2.1 m
m to 21.6 ± 1.4 m
m (AlF4−) and 19.8 ± 1.1 m
m (GMP‐P(NH)P), leaving Vmax and the Hill coefficient unchanged. This effect was abolished by inhibitors of Gi‐proteins (N‐ethylmaleimide, holoenzyme‐ and A‐protomer of pertussis toxin) and by an anti‐Gα Ig and GDP(β)S. Activation of Gi‐proteins by mastoparan and its synthetic analogue Mas7 also strongly reduced NHE activity. These data show that in platelets NHE‐1 is under negative control of the Gi‐family of trimeric G‐proteins.
Identification of two highly divergent catalase genes in the fungal tomato pathogen, Cladosporium fulvum
Authors: Bussink Henk‐Jan , Oliver Richard
Published: March 2003

Catalases of pathogenic micro‐organisms have attracted attention as potential virulence factors. Homology‐based screens were performed to identify catalase genes in the fungal tomato pathogen Cladosporium fulvum. Two highly divergent genes, Cat1 and Cat2, were isolated and characterized. Cat1 codes for a putative 566‐amino‐acid catalase subunit and belongs to the gene family that also encodes the mainly peroxisome‐localized catalases of animal and yeast species. Cat2 codes for a putative catalase subunit of 745 amino acids and belongs to a different gene family coding for the large‐subunit catalases similar to ones found in bacteria and filamentous fungi. Neither catalase had an obvious secretory signal sequence. A search for an extracellular catalase was unproductive. The Cat1 and Cat2 genes showed differential expression, with the Cat1 mRNA preferentially accumulating in spores and the Cat2 mRNA preferentially accumulating in response to external H2O2. With Cat2‐deleted strains, activity of the Cat2 gene product (CAT2) was identified among four proteins with catalase activity separated on non‐denaturing gels. The CAT2 activity represented a minor fraction of the catalase activity in spores and H2O2‐stressed mycelium, and no phenotype was observed for Cat2‐deleted strains, which showed a normal response to H2O2 treatment. These results indicate the existence of a complex catalase system in C. fulvum, with regard to both the structure and regulation of the genes involved. In addition, efficient C. fulvum gene‐replacement technology has been established.
Increasing the thermal stability of euphauserase
Authors: Benjamin David C. , Kristjánsdóttir Sigrídur , Gudmundsdóttir Ágústa
Published: March 2003

A molecular model of Antarctic krill euphauserase based on the known crystal structure of its fiddler crab analog, collagenase I, indicates that the core structure of these enzymes is almost identical. Euphauserase is a cold‐active and thermally sensitive enzyme with a high affinity for Lys, Arg and large hydrophobic amino acids. Residue Phe137 in euphauserase, localized in loop D (autolysis loop), is highly exposed on the surface of the molecule. Therefore, it appeared to be an easy target for autolysis. The broadly specific euphauserase has a low affinity for negatively charged residues. In order to increase the stability of the enzyme, two mutants were created in which residue Phe137 was replaced by a Glu and an Asp residue. Both mutations resulted in increased stability of the recombinant euphauserase towards thermal inactivation.
Effects of pressure on the activity and spectroscopic properties of carboxyl proteinases
Authors: Fujiwara Satoshi , Kunugi Shigeru , Oyama Hiroshi , Oda Kohei
Published: March 2003

The pressure dependence of the activity and spectroscopic properties of four carboxyl proteinases were investigated. Two were pepstatin‐sensitive carboxyl proteinases (porcine pepsin and proteinase A from baker's yeast) and two were pepstatin‐insensitive carboxyl proteinases (from Pseudomonas sp. 101 (pseudomonapepsin; PCP) and Xanthomonas sp. T‐22 (xanthomonapepsin; XCP)). The specificity constant [kcat/Km(app)] of PCP and XCP for a synthetic peptide substrate showed only a slight decrease with increasing pressure, whereas pepsin and proteinase A showed substantial disactivation at higher pressures. The calculated apparent activation volume (ΔVkcat/Km) was about 1, 3, 13, and 14 mL·mol−1 for PCP, XCP, pepsin, and proteinase A, respectively. The hydrolysis of acid‐denatured myoglobin by the four carboxyl proteinases was only slightly affected by high pressure (except for proteinase A at 400 MPa), in contrast to the results for the peptide hydrolysis. In fact, PCP, XCP, and proteinase A actually showed slightly higher degradations of acid‐denatured myoglobin at higher pressures. The residual activities of these enzymes after the incubation at high pressures implied a pressure‐induced stabilization towards autolysis. The changes in the fourth derivative near‐UV absorbance spectrum of the four enzymes in aqueous solution were measured at various pressures from 0.1 to 400 MPa. Upon an increase in pressure, the peaks from PCP and XCP red‐shifted slightly, whereas pepsin and proteinase A blue‐shifted substantially, thus indicating a more polar environment. The intrinsic fluorescence also decreased upon increasing pressure. However, the change for XCP was rather small, but the change for the other three was very large. The changes in the peak wavelength for pepsin and proteinase A were characteristic, and also indicated a more polar environment under high pressure. An analysis by the center of spectra mass (CSM) gave the ΔG and ΔV of transition as 9.8 kJ·mol−1 and −24 mL·mol−1 (pepsin) and 11.7 kJ·mol−1 and −43 mL·mol−1 (proteinase A), respectively, by assuming a simple two‐state transition. The circular dichroism (CD) showed relatively small changes after 1‐h incubations at 400 MPa, indicating that the secondary structures were largely maintained.
Targeting neighbouring poly(purine·pyrimidine) sequences located in the human bcr promoter by triplex‐forming oligonucleotides
Authors: Xodo Luigi E. , Rathinavelan Thenmalarchelvi , Quadrifoglio Franco , Manzini Giorgio , Yathindra Narayanarao
Published: March 2003

Most poly(purine·pyrimidine) [poly(R·Y)] sequences in eukaryotic genomes are interrupted by one or more base pair inversions. When the inversions are centrally located, the poly(R·Y) sequences can be regarded as the sum of two abutting sites, each potentially capable of forming a triple helix. Employing band‐shift, footprinting and modeling methods we examined the formation of triple helices at a critical 27 bp poly(R·Y) sequence interrupted by two adjacent CG inversions, and located in the promoter of the human bcr gene at transcription initiation. We designed several 13‐mer and 14‐mer triplex‐forming oligonucleotides (TFOs) capable of binding the bcr abutting sites, thereby generating different base juxtapositions at the triple helical junction, to examine whether triplex formation occurs in a cooperative manner. It is found that in 50 mm Tris/HCl, pH 7.4, 10 mm MgCl2, 2 mm spermine, 37 °C, the 13‐and the 14‐mer TFOs bind to one half of the bcr site with ΔG between −30 and −35 kJ·mol−1. However, when different 13‐mer/14‐mer combinations of TFOs were directed against the abutting poly(R·Y) sites, triplex formation has been found to be enhanced only for the triple helical junction formed by the 5′‐A‐T‐3′ base juxtaposition, in keeping with a partial stacking suggested from modeling analysis. On the other hand, a longer 24‐mer TFO, binding noncooperatively to the same abutting sites, forms a much more stable triplex (ΔG = −51 kJ·mol−1), notwithstanding the two T·CG triads in the middle. Modeling investigations reveal that there is no continuity or propagation of base stacking involving adjacent bases of the third strand at the site of base inversion as well as on the 5′ side. The data indicate that the entropy penalty of forming a triplex with two oligonucleotides is much higher than the energy gained from base stacking interactions at the triplex junction formed between the two TFOs.
Comparison of the three rat GDP‐L‐fucose:β‐D‐galactoside 2‐α‐L‐fucosyltransferases FTA, FTB and FTC
Authors: Bureau Valérie , Marionneau Séverine , Cailleau‐Thomas Anne , Le Moullac‐Vaidye Béatrice , Liehr Thomas , Le Pendu Jacques
Published: March 2003

The complete coding sequences of three rat α1,2fucosyltransferase genes were obtained. Sequence analysis revealed that these genes, called FTA, FTB and FTC, were homologous to human FUT1, FUT2 and Sec1, respectively. A distance analysis between all α1,2fucosyltransferase sequences available showed that the two domains of the catalytic region evolved differently with little divergence between the FUT2 and Sec1 N‐terminal domains, quite distant from that of FUT1. At variance, FUT1 and FUT2 C‐terminal domains were less distant while a high evolutionary rate was noted for Sec1 C‐terminal domain. Whereas FTA and FTB encode typical glycosyltransferases, FTC lacks the homologous start codon and encodes a protein devoid of intracellular and transmembrane domains. It is located on rat chromosome 1q34. Transfection experiments revealed that unlike FTA and FTB, FTC does not generate enzyme activity. Analysis by flow cytometry showed that H type 2 epitopes were synthesized in Chinese hamster ovary cells transfected by both FTA and FTB cDNA, but only FTB transfectants possessed H type 3 determinants. In REG rat carcinoma cells, both FTA and FTB allowed synthesis of H type 2 and H type 3 at the cell surface. Western blots showed that, in both cell types, FTA was able to synthesize H type 2 epitopes on a larger set of glycoproteins than FTB. Analysis of the kinetic parameters obtained using small oligosaccharides revealed only a slight preference of FTA for type 2 over other types of acceptor substrates, whereas FTB was barely able to fucosylate this substrate.
NMR 15N relaxation of the insulin‐like growth factor (IGF)‐binding domain of IGF binding protein‐5 (IGFBP‐5) determined free in solution and in complex with IGF‐II
Authors: Renner Christian , Holak Tad A.
Published: March 2003

15N NMR relaxation rates of mini‐IGFBP‐5, an N‐terminal insulin‐like growth factor binding domain of the insulin‐like growth factor binding protein 5 (IGFBP‐5), were analysed at three field strengths using the Lipari–Szabo procedure (see below) and reduced spectral density methods. Isotropic and anisotropic Lipari–Szabo models were analysed and an analytical formula for the overall correlation time for anisotropic molecules is presented. Mini‐IGFBP‐5 was found to be mainly rigid on fast ps time scales except for 11 unstructured flexible residues at the C‐terminus. The insulin‐like growth factor binding loop in the apo‐protein exhibits small amounts of flexibility on fast time scales (ps to ns) but several loop residues show significant exchange broadening. These loop residues display no exchange broadening in the complex of IGF‐II/mini‐IGFBP‐5. The isotropic overall tumbling time in solution at 31 °C of mini‐IGFBP‐5 complexed to IGF‐II is τc = 18.4 ± 0.2 ns indicating a strong tendency for aggregation.
A polymorphic GT repeat from the human cardiac Na+Ca2+ exchanger intron 2 activates splicing
Authors: Gabellini Nadia
Published: March 2003

The sequence analysis of the human intron 2 from the Na+/Ca2+ exchanger 1 (NCX1) gene has revealed a GT repeat of variable length (10–16). The 5′ sequence of intron 2 exhibited significant homology (65–70%) with other minisatellite sequences. DNA segments at the 5′ end of intron 2 were inserted in the NCX1 cDNA (3.7 kb) to reconstruct the exon 2/intron 2 junction. Transient expression of these constructs in HEK293 cells generated shortened mRNAs (≈ 2.5 kb). RT‐PCR and ribonuclease protection analysis of the 3′ end of the short transcripts indicated a splicing event at the intron 2/exon 2 junction (5′ site) and in the vector sequence downstream of the NCX1 insert (3′ site). Molecular dissection of the 5′‐intron 2 sequence showed that the GT repeat was required for splicing activation, whereas the remainder of the 5′‐intron 2 segment was completely inactive. The results indicate that the GT repeat is a strong intronic splicing enhancer that could be involved in the regulation of NCX1 expression, possibly mediating tissue‐specific alternative splicing of the mutually exclusive exons 3 and 4, and/or exon‐2 circularization.
Structure–activity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants
Authors: De Wilde Gert , Murray‐Rust Judith , Boone Elke , Olerenshaw Dionne , McDonald Neil Q. , Ibanez Carlos , Haegeman Guy , Wollmer Axel , Federwisch Matthias
Published: March 2003

Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells. Clustering of the intracellular domain of the receptor, the so‐called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli. Circular dichroism, steady‐state and time‐resolved fluorescence spectroscopy were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas‐associating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3‐dimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution. This prompted a homology modeling of the TNFR DD 3‐D structure using FADD as a template. In vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations. Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55 DD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference.
Regulation of the activity of matriptase on epithelial cell surfaces by a blood‐derived factor
Authors: Benaud Christelle , Dickson Robert B. , Lin Chen‐Yong
Published: March 2003

Matriptase is an epithelial‐derived, integral membrane, trypsin‐like serine protease. We have shown previously that matriptase exists both in complexed and noncomplexed forms. We now show that the complexed matriptase is an activated, two‐chain form, which is inhibited in an acid‐sensitive, reversible manner through binding to its cognate, Kunitz‐type inhibitor, HAI‐1 (hepatocyte growth factor activator inhibitor‐1). Conversely, the majority of the noncomplexed matriptase is a single‐chain zymogen, which lacks binding affinity to HAI‐1, suggesting that matriptase, similar to most other serine proteases, is activated by proteolytic cleavage at a canonical activation motif. We have now generated mAbs specific for the conformational changes associated with the proteolytic activation of matriptase. Using these mAbs, which specifically recognize the two‐chain form of matriptase, we demonstrate that matriptase is transiently activated on 184A1N4 human mammary epithelial cell surfaces following their exposure to serum. The ability of serum to activate matriptase is highly conserved across reptilian, avian, and mammalian species. This serum‐dependent activation of matriptase on epithelial cell surfaces is followed by ectodomain shedding of both matriptase and its Kunitz‐type inhibitor.
Solution structure of cytochrome b5 mutant (E44/48/56A/D60A) and its interaction with cytochrome c
Authors: Wu Yibing , Wang Yunhua , Qian Chengmin , Lu Jun , Li Ercheng , Wang Wenhu , Lu Junxia , Xie Yi , Wang Jinfeng , Zhu Dexi , Huang Zhongxian , Tang Wenxia
Published: March 2003

Using 1617 meaningful NOEs with 188 pseudocontact shifts, a family of 35 conformers of oxidized bovine microsomal cytochrome b5 mutant (E44/48/56A/D60A) has been obtained and is characterized by good resolution (rmsd to the mean structure are 0.047 ± 0.007 nm and 0.095 ± 0.008 nm for backbone and heavy atoms, respectively). The solution structure of the mutant, when compared with the X‐ray structure of wild‐type cytochrome b5, has no significant changes in the whole folding and secondary structure. The binding between cytochrome b5 and cytochrome c shows that the association constant of the mutant–cytochrome c complex is much lower than the one for wild‐type complex (2.2 × 104 m−1 vs. 5.1 × 103 m−1). The result suggests the four acidic residues have substantial effects on the formation of the complex between cytochrome b5 and cytochrome c, and therefore it is concluded reasonably that the electrostatic interaction plays an important role in maintaining the stability and specificity of the complex formed. The competition between the ferricytochrome b5 mutant and [Cr(oxalate)3]3– for ferricytochrome c shows that site III of cytochrome c, which is a strong binding site to wild‐type cytochrome b5, still binds to the mutant with relatively weaker strength. Our results indicate that certain bonding geometries do occur in the interaction between the present mutant and cytochrome c and these geometries, which should be quite different from the ones of the Salemme and Northrup models.
Structural and functional properties of Escherichia coli‐derived nucleoplasmin
Authors: Hierro Aitor , Arizmendi Jesús M. , De Las Rivas Javier , Urbaneja M. Angeles , Prado Adelina , Muga Arturo
Published: March 2003

Fourier transform infrared spectroscopy, circular dichroism and prediction techniques have been used to investigate the conformational properties of nucleoplasmin isolated from oocytes and eggs of Xenopus. laevis and overexpressed in Escherichia coli. A simple and fast method allows purification of recombinant nucleoplasmin free of truncated and/or aggregated forms, and therefore provides a suitable sample to carry out the structural and functional comparison between these proteins. The secondary structure of the three proteins estimated from both spectroscopic techniques was very similar, and was found to be 31–33% loops, 27–34% β structure, 22–26% turns and 9–14% α helix. Prediction studies, in good agreement with experimental data, also suggest that β structure is the major regular conformation, and that loops and turns are the most abundant conformational features within the secondary structure of nucleoplasmin. Furthermore, the spectroscopic characterization of a truncated version of the protein, lacking 80 residues at the C‐terminus, and the prediction data indicate that the secondary structure elements of the protein are segregated into two regions. The N‐terminal fragment (comprising residues 1–120) which holds all the putative β strands, and the solvent‐exposed C‐terminal region, that is suggested to be enriched in turn and loop structures. The phosphate/protein monomer molar ratios, obtained from chemical analysis and mass spectrometry, are 0, 3 and 7–10 for recombinant, oocyte and egg nucleoplasmin, respectively. Phosphorylation does not significantly affect the secondary structure of the protein, but clearly modulates its ability to decondense sperm nuclei and to remove basic proteins from DNA.
Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc
Authors: Müller Eva‐Christina , Lapko Anna , Otto Albrecht , Müller Jürgen J. , Ruckpaul Klaus , Heinemann Udo
Published: March 2003

NADPH‐dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero‐length crosslinks mediated by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X‐ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin −P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin−P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.
FeMo cofactor biosynthesis in a nifE − mutant of Rhodobacter capsulatus
Authors: Siemann Stefan , Schneider Klaus , Behrens Kai , Knöchel Arndt , Klipp Werner , Müller A.
Published: March 2003

In all diazotrophic micro‐organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non‐sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild‐type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene‐reduction assay, was strictly Mo‐dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20–50 µm). Moreover, in these strains a high proportion of ethane was produced from acetylene (≈ 10% of ethylene) in vivo. However, in in vitro measurements with cell‐free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q‐Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco‐containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.
Protein kinase C regulates transcription of the human guanylate cyclase C gene
Authors: Roy Nivedita , Guruprasad Medigeshi R. , Kondaiah Paturu , Mann Elizabeth A. , Giannella Ralph A. , Visweswariah Sandhya S.
Published: March 2003

Guanylate cyclase C is the receptor for the bacterial heat‐stable enterotoxins and guanylin family of peptides, and mediates its action by elevating intracellular cGMP levels. Potentiation of ligand‐stimulated activity of guanylate cyclase C in human colonic T84 cells is observed following activation of protein kinase C as a result of direct phosphorylation of guanylate cyclase C. Here, we show that prolonged exposure of cells to phorbol esters results in a decrease in guanylate cyclase C content in 4β‐phorbol 12‐myristate 13‐acetate‐treated cells, as a consequence of a decrease in guanylate cyclase C mRNA levels. The reduction in guanylate cyclase C mRNA was inhibited when cells were treated with 4β‐phorbol 12‐myristate 13‐acetate (PMA) in the presence of staurosporine, indicating that a primary phosphorylation event by protein kinase C triggered the reduction in RNA levels. The reduction in guanylate cyclase C mRNA levels was not due to alterations in the half‐life of guanylate cyclase C mRNA, but regulation occurred at the level of transcription of guanylate cyclase C mRNA. Expression in T84 cells of a guanylate cyclase C promoter‐luciferase reporter plasmid, containing 1973 bp of promoter sequence of the guanylate cyclase C gene, indicated that luciferase activity was reduced markedly on PMA treatment of cells, and the protein kinase C‐responsive element was present in a 129‐bp region of the promoter, containing a HNF4 binding element. Electrophoretic mobility shift assays using an oligonucleotide corresponding to the HNF4 binding site, indicated a decrease in binding of the factor to its cognate sequence in nuclear extracts prepared from PMA‐treated cells. We therefore show for the first time that regulation of guanylate cyclase C activity can be controlled at the transcriptional level by cross‐talk with signaling pathways that modulate protein kinase C activity. We also suggest a novel regulation of the HNF4 transcription factor by protein kinase C.
Solution structure of the main α‐amylase inhibitor from amaranth seeds
Authors: Martins José C. , Enassar Mohammed , Willem Rudolph , Wieruzeski Jean‐Michel , Lippens Guy , Wodak Shoshana J.
Published: March 2003

The most abundant α‐amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit α‐amylase activity of insect larvae while leaving that of mammals unaffected. In solution, 1H NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis–trans isomerization of the Val15–Pro16 peptide bond. Both solution structures have been determined using 2D 1H‐NMR spectroscopy and xplor followed by restrained energy refinement in the consistent‐valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium‐range and 173 long‐range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine‐knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10–Pro20 segment, while preserving the overall global fold. The energy‐refined ensemble of the major isomer, consisting of 20 low‐energy conformers with an average backbone rmsd of 0.29 ± 0.19 Å and no violations larger than 0.4 Å, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid‐phase synthesis, which was determined with only half the number of medium‐range and long‐range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X‐ray structure of the inhibitor when bound to its α‐amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein–protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target α‐amylase, confirming the view that it acts as a lid on top of the α‐amylase active site.
Activation of the Sarcophaga lectin gene promoter by (A + T)‐stretch binding protein
Authors: Aozasa Naohiko , Shiraishi Hirohisa , Nakanishi‐Matsui Mayumi , Kobayashi Ayako , Sekimizu Kazuhisa , Kubo Takeo , Natori Shunji
Published: March 2003

Previously, we purified and isolated a cDNA for (A + T)‐stretch binding protein (ATBP) that binds to (A + T)‐stretches in the 5′ upstream region of the Sarcophaga lectin gene [Nakanishi‐Matsui, M., Kubo, T. & Natori, S. (1995) Eur. J. Biochem.230, 396–400]. Here, we used a luciferase reporter to examine the effect of ATBP on transcription of the Sarcophaga lectin gene. Deletion experiments revealed that ATBP activates the Sarcophaga lectin gene in a 5′ upstream sequence‐dependent manner, and that at least the N‐terminal 25 residues, the three Zn‐finger domains, an acidic domain and the third hydrophobic domain of ATBP are indispensable for its function. Furthermore, a synergistic effect was detected between ATBP and Dif, suggesting that ATBP is involved in the activation of insect immunity genes.
ATP analogue binding to the A subunit induces conformational changes in the E subunit that involves a disulfide bond formation in plant V‐ATPase
Authors: Kawamura Yukio , Arakawa Keita , Maeshima Masayoshi , Yoshida Shizuo
Published: March 2003

Vacuolar H+‐ATPase (V‐ATPase) consists of a catalytic head, a stalk part and a membrane domain. We indirectly investigated the interaction between the A subunit (catalytic head) and the E subunit (stalk part) using an ATP analogue, adenosine 5′‐[β,γ‐imino]triphosphate (AMP‐PNP), which holds the enzyme in the substrate‐binding state. AMP–PNP treatment caused a mobility shift of the E subunit with a faster migration in SDS/polyacrylamide gel electrophoresis without a reductant, while ATP treatment did not. A mobility shift of the E subunit has been detected in several plants. As polypeptides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intramolecular disulfide bond by two cysteine residues conserved among several plant species. The mobility shift may be involved in the binding of AMP–PNP to the ATP‐binding site, which exists in the A and B subunits, as it was inhibited by the addition of ATP. Pretreatment with 2′‐3′‐O‐(4‐benzoylbenzoyl)‐ATP (Bz‐ATP), which modifies the ATP‐binding site of the B subunit under UV illumination, did not inhibit the mobility shift of the E subunit caused by AMP–PNP treatment. The response of V‐ATPase following the AMP–PNP binding may cause a conformational change in the E subunit into a form that is susceptible to oxidation of cysteine residues. This is the first demonstration of interaction between the A and E subunits in the substrate‐binding state of a plant V‐ATPase.
Differential recognition of epitopes present on monomeric and oligomeric forms of gp160 glycoprotein of human immunodeficiency virus type 1 by human monoclonal antibodies
Authors: Zeder‐Lutz Gabrielle , Hoebeke Johan , Van Regenmortel Marc H. V.
Published: March 2003

The mechanism of infectivity neutralization of human immunodeficiency virus type 1 (HIV‐1) by Ig is poorly understood. Three human monoclonal antibodies (mAbs 1b12, 2G12 and 2F5) that are able to neutralize primary isolates of HIV‐1 in vitro have been shown to act synergistically. In the present study this synergy was analyzed by measuring the epitope accessibility and binding kinetics for these three mAbs with respect to monomeric and oligomeric env protein gp160 IIIB using surface plasmon resonance. The results indicate that oligomerization of gp160 affects the accessibility of some of the epitopes recognized by the mAbs and provide some insight into the mechanism of synergy between different anti‐(HIV‐1) mAbs.
Cα methylation in molecular recognition
Authors: Sagan Sandrine , Lequin Olivier , Frank Fabrice , Convert Odile , Ayoub Mimoun , Lavielle Solange , Chassaing Gérard
Published: March 2003

Two binding sites NK‐1M (major, more abundant) and NK‐1m (minor) are associated with the neurokinin‐1 receptor. For the first time with a bioactive peptide, the Cα methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK‐1M and NK‐1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Cα methylated analogues of the undecapeptide SP (from position 5–11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Cα methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Cα methylated SP analogues do not allow the identification of any clear‐cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [αMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK‐1m binding site. The conformational analyses show that the structural consequences associated with Cα methylation of SP are sequence dependent. Moreover, a single Cα methylation is not sufficient by itself to drastically stabilize a helical structure even pre‐existing in solution, except when Gly9 is substituted by an α‐aminoisobutyric acid. Furthermore, Cα methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4–8 domain of other potent SP analogues.
Phosphorylation of proteins in the light‐dependent signalling pathway of a filamentous cyanobacterium
Authors: Hübschmann Thomas , Jorissen Helena J. M. M. , Börner Thomas , Gärtner Wolfgang , de Marsac Nicole Tandeau
Published: March 2003

The genome of the filamentous cyanobacterium Calothrix sp. PCC7601 contains two genes, cphA and cphB, encoding proteins with similarity to plant phytochromes and bacterial histidine kinases. In vitro, CphA and CphB readily attach a tetrapyrrole chromophore to develop spectrally active holoproteins that are photointerconvertible between a red light‐absorbing and a far‐red light‐absorbing form. Together with the putative response regulators, RcpA and RcpB, the putative histidine kinases, CphA and CphB, are suggested to constitute two two‐component systems of light‐dependent signal transduction. In this report, we demonstrate the kinase activity of both CphA and CphB. In vitro experiments carried out on the purified proteins show that CphA and CphB are autophosphorylated in the presence of ATP and that phospho‐CphA is capable of efficient phosphotransfer to RcpA as is phospho‐CphB towards RcpB. The autophosphorylation and the phosphorelay are dependent on light. Both activities are reduced under red light vs. far‐red light irradiation. No phosphoryl transfer occurred between phospho‐CphA and RcpB or between phospho‐CphB and RcpA. The response regulators RcpA and RcpB can receive a phosphoryl moiety also from the small phospho‐donor acetyl phosphate. The stability of the phosphorylated regulators is not affected by CphA and CphB or light.
Characterization of human muscle type cofilin (CFL2) in normal and regenerating muscle
Authors: Thirion Christian , Stucka Rolf , Mendel Bettina , Gruhler Albrecht , Jaksch Michaela , Nowak Kristen J. , Binz Nicolette , Laing Nigel G. , Lochmüller Hanns
Published: March 2003

Cofilins are actin binding proteins and regulate actin assembly in vivo. Numerous cofilin homologues have been characterized in various organisms including mammals. In mice, a ubiquitously expressed cofilin (CFL1) and a skeletal muscle specific cofilin (CFL2) have been described. In the present study, we identified and characterized a human CFL2 gene localized on chromosome 14, with high homology to murine CFL2. Furthermore, we provide evidence for differentially spliced CFL2 transcripts (CFL2a and CFL2b). CFL2b is expressed predominantly in human skeletal muscle and heart, while CFL2a is expressed in various tissues. Genetic defects of CFL2 were excluded for one human muscle disorder, the chromosome 14 linked distal myopathy MPD1, and shown to be only possible to be a rare cause of another, nemaline myopathy. In a mouse model of mechanically induced muscle damage the changes of cofilin expression were monitored during the first 10 days of regeneration, with dephosphorylated CFL2 being the major isoform at later stages of muscle regeneration. A similar predominance of dephosphorylated CFL2 was observed in chronically regenerating dystrophin‐deficient muscles of Duchenne muscular dystrophy patients. Therefore, the CFL2 isoform may play an important role in normal muscle function and muscle regeneration.
Rapid and cooperative binding of factor XII to human umbilical vein endothelial cells
Authors: Schousboe Inger
Published: March 2003

When activated, factor XII (FXII) has been shown to play a role in a series of proteolytic cascades including systems as the fibrinolytic, the coagulation, the kallikrein–kinin and the complement. How FXII is activated in vivo remains poorly understood as the concentration and density of surface bound negative charges known to trigger the activation in vitro is far from sufficient in vivo. Specific binding of FXII to cellular receptors in the blood stream may, however, solve this problem which may be a question of inter molecular vicinity enhanced by binding to any surface. Here we report that the Zn2+‐dependent binding of FXII to endothelial cells is rapid, saturable, specific and cooperative. Each endothelial cell from the human umbilical veins was found to bind (417 ± 202) × 103 molecules of FXII with a Kd of (65 ± 23) nM and a Hill coefficient of 2.1. The binding was inhibited by α‐FXIIa but not by β‐FXIIa. The Kd for binding α‐FXIIa was (50 ± 27) nM. The rate of association was found to be 1.9 × 105m−1· min−1. A confirmed inhibition by HK increased the Kd without affecting the maximal number of binding sites and the Hill coefficient. The concentration of HK in serum did not prevent binding of FXII/FXIIa to cells incubated with serum supplemented with Zn2+. The optimal concentration of Zn2+ was 15 µm for binding factor XII/FXIIa whether purified or in serum.
Dichloromethane mediated in vivo selection and functional characterization of rat glutathione S‐transferase theta 1‐1 variants
Authors: Gisi Daniel , Maillard Julien , Flanagan Jack U. , Rossjohn Jamie , Chelvanayagam Gareth , Board Philip G. , Parker Michael W. , Leisinger Thomas , Vuilleumier Stéphane
Published: March 2003

Methylobacterium dichloromethanicum DM4 is able to grow with dichloromethane as the sole carbon and energy source by using a dichloromethane dehalogenase/glutathione S‐transferase (GST) for the conversion of dichloromethane to formaldehyde. Mammalian homologs of this bacterial enzyme are also known to catalyze this reaction. However, the dehalogenation of dichloromethane by GST T1‐1 from rat was highly mutagenic and toxic to methylotrophic bacteria. Plasmid‐driven expression of rat GST T1‐1 in strain DM4‐2cr, a mutant of strain DM4 lacking dichloromethane dehalogenase, reduced cell viability 105‐fold in the presence of dichloromethane. This effect was exploited to select dichloromethane‐resistant transconjugants of strain DM4‐2cr carrying a plasmid‐encoded rGSTT1 gene. Transconjugants that still expressed the GST T1 protein after dichloromethane treatment included rGSTT1 mutants encoding protein variants with sequence changes from the wild‐type ranging from single residue exchanges to large insertions and deletions. A structural model of rat GST T1‐1 suggested that sequence variation was clustered around the glutathione activation site and at the protein C‐terminus believed to cap the active site. The enzymatic activity of purified His‐tagged GST T1‐1 variants expressed in Escherichia coli was markedly reduced with both dichloromethane and the alternative substrate 1,2‐epoxy‐3‐(4′‐nitrophenoxy)propane. These results provide the first experimental evidence for the involvement of Gln102 and Arg107 in catalysis, and illustrate the potential of in vivo approaches to identify catalytic residues in GSTs whose activity leads to toxic effects.