bluuebunny/crossref_metadata_2025 huggingface dataset:
Definition of receptor binding sites on human interleukin‐11 by molecular modeling‐guided mutagenesis
Authors: Tacken Ingrid , Dahmen Heike , Boisteau Olivier , Minvielle Stéphane , Jacques Yannick , Grötzinger Joachim , Küster Andrea , Horsten Ursula , Blanc Chrystel , A. Montero‐Julian Felix , Heinrich Peter C. , Müller‐Newen Gerhard
Published: March 2003

Interleukin‐11 (IL‐11) belongs to the interleukin‐6 (IL‐6)‐type subfamily of long‐chain helical cytokines including IL‐6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, and cardiotrophin‐1, which all share the glycoprotein gp130 as a signal transducing receptor component. IL‐11 acts on cells expressing gp130 and the IL‐11 receptor (IL‐11R) α‐subunit (IL‐11Rα). The structural epitopes of IL‐11 required for the recruitment of the individual receptor subunits have not yet been defined. Based on the structure of CNTF, a three‐dimensional model of human IL‐11 was built. Using this model, 10 surface exposed amino acid residues of IL‐11 were selected for mutagenesis using analogies to the well‐characterized receptor recruitment sites of IL‐6, CNTF, and LIF. The respective mutants of human IL‐11 were expressed as soluble fusion proteins in bacteria. Their biological activities were determined on HepG2 and Ba/F3‐130‐11α cells. Several mutants with substantially decreased bioactivity and one hyperagonistic mutant were identified and further analyzed with regard to recruitment of IL‐11Rα and gp130. The low‐activity mutant I171D still binds IL‐11Rα but fails to recruit gp130, whereas the hyperagonistic variant R135E more efficiently engages the IL‐11R subunits. The low‐activity mutants R190E and L194D failed to bind to IL‐11Rα. These findings reveal a common mechanism of receptor recruitment in the family of IL‐6‐type cytokines and offer considerable perspectives for the rational design of IL‐11 antagonists and hyperagonists.
Mapping of the interaction between the immunodominant loop of the ectodomain of HIV‐1 gp41 and human complement protein C1q
Authors: Quinkal Isabelle , Hernandez Jean‐François , Chevallier Sylvie , Arlaud Gérard J. , Vernet Thierry
Published: March 2003

The human immunodeficiency virus type 1 transmembrane envelope glycoprotein gp41 has been previously shown to activate the C1 complex of human complement through direct interaction with its C1q subunit. The major interaction site has been located within the gp41 immunodominant region (residues 590–620), and a synthetic peptide overlapping residues 601–613 of gp41 (sequence GIWGCSGKLICTT) was shown to inhibit binding of gp41 to C1q in vitro (Thielens, N.M., Bally, I.M., Ebenbichler, C_f., Dierich, M.P. & Arlaud, G.J. (1993) J. Immunol.151, 6583–6592). The ectodomain of gp41 (s‐gp41) was secreted from the methylotrophic yeast Pichia pastoris and purified by immunoaffinity chromatography. Enzymatic deglycosylation of the recombinant s‐gp41 was necessary to allow its in vitro interaction with C1q. A solid‐phase competition assay was used to monitor the effect of mutant peptides derived from segment 601–613 of gp41 on the binding of deglycosylated s‐gp41 to C1q. Whereas mutation of Ser606 had no effect, replacement of Ile602, Trp603, Lys608, Leu609 and Ile610 by Ala abolished the ability of the resulting peptides to inhibit binding of s‐gp41 to C1q, suggesting that these residues participate in the interaction between gp41 and C1q. These findings are discussed in the light of a structural model of the immunodominant loop of gp41. It is proposed that the recognition of gp41 by C1q is driven by hydrophobic interactions, and that the sites of gp41 responsible for interaction with gp120 and C1q partly overlap.
Structural characterization of the oligosaccharide chains of native and crystallized boar seminal plasma spermadhesin PSP‐I and PSP‐II glycoforms
Authors: Nimtz Manfred , Grabenhorst Eckart , Conradt Harald S. , Sanz Libia , Calvete Juan J.
Published: March 2003

The PSP‐I/PSP‐II heterodimer is the major protein of boar seminal plasma. Both subunits are glycoproteins of the spermadhesin family and each contains a single N‐glycosylation site. After enzymatic release of the oligosaccharides from isolated PSP‐I and PSP‐II, mainly neutral and monosialylated oligosaccharides, and small amounts of disialylated oligosaccharides, were recovered from both proteins. Twenty‐two neutral oligosaccharides, 11 monosialylated glycans and three disialylated carbohydrate chains were characterized using mass spectrometric and NMR techniques. PSP‐I and PSP‐II share the same glycans but differ in their relative molar ratios. Most glycan structures are proximally α1‐6‐fucosylated, diantennary complex‐type bearing nonsialylated or α2‐6‐sialylatedN‐acetyllactosamine or di‐N‐acetyllactosamine antennae. The majority of nonsialylatedN‐acetyllactosamine antennae bear terminal α1‐3‐linked Gal residues. In addition, theN‐acetylglucosamine residue of nonsialylatedN‐acetyl and di‐N‐acetyllactosamine antennae can be modified by an α1‐3‐linked fucose residue. Structures of higher antennarity, as well as structures 3,6‐branched at galactose residues, were found in smaller amounts. In one oligosaccharide,N‐acetylneuraminic acid is substituted byN‐glycolylneuraminic acid. Mass spectrometric analysis of PSP‐I and PSP‐II glycoforms isolated from crystallized PSP‐I/PSP‐II heterodimer showed the coexistence of major PSP‐I and PSP‐II glycoforms in the hexagonal crystals. Oligosaccharides with the NeuNAcα2‐6GalNAcβ1‐4GlcNAc‐R motif block adhesive and activation‐related events mediated by CD22, suggesting a possible immunoregulatory activity for PSP‐I/PSP‐II.
A Dictyostelium protein binds to distinct oligo(dA)·oligo(dT) DNA sequences in the C‐module of the retrotransposable element DRE
Authors: Horn Jens , Dietz‐Schmidt Anja , Zündorf Ilse , Garin Jérôme , Dingermann Theodor , Winckler Thomas
Published: March 2003

The genome of the eukaryotic microbe Dictyostelium discoideum contains some 200 copies of the nonlong‐terminal repeat retrotransposon DRE. Among several unique features of this retroelement, DRE is transcribed in both directions leading to the formation of partially overlapping plus strand and minus strand RNAs. The synthesis of minus strand RNAs is controlled by the C‐module, a 134‐bp DNA sequence located at the 3′‐end of DRE. A nuclear protein (CMBF) binds to the C‐module via interaction with two almost homopolymeric 24 bp oligo(dA)·oligo(dT) sequences. The DNA‐binding drugs distamycin and netropsin, which bind to A·T‐rich DNA sequences in the minor groove, competed efficiently for the binding of CMBF to the C‐module. The CMBF‐encoding gene, cbfA, was isolated and a DNA‐binding domain was mapped to a 25‐kDa C‐terminal region of the protein. A peptide motif involved in the binding of A·T‐rich DNA by high mobility group‐I proteins (‘GRP’ box) was identified in the deduced CMBF protein sequence, and exchange of a consensus arginine residue for alanine within the CMBF GRP box abolished the interaction of CMBF with the C‐module. The current data support the theory that CMBF binds to the C‐module by detecting its long‐range DNA conformation and interacting with A·T base pairs in the minor groove of oligo(dA)·oligo(dT) stretches.
Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2α kinase
Authors: Berlanga Juan J. , Santoyo Javier , de Haro César
Published: March 2003

In eukaryotic cells, protein synthesis is regulated in response to various environmental stresses by phosphorylating the α subunit of the eukaryotic initiation factor 2 (eIF2α). Three different eIF2α kinases have been identified in mammalian cells, the heme‐regulated inhibitor (HRI), the interferon‐inducible RNA‐dependent kinase (PKR) and the endoplasmic reticulum‐resident kinase (PERK). A fourth eIF2α kinase, termed GCN2, was previously characterized from Saccharomyces cerevisiae, Drosophila melanogaster and Neurospora crassa. Here we describe the cloning of a mouse GCN2 cDNA (MGCN2), which represents the first mammalian GCN2 homolog. MGCN2 has a conserved motif, N‐terminal to the kinase subdomain V, and a large insert of 139 amino acids located between subdomains IV and V that are characteristic of the known eIF2α kinases. Furthermore, MGCN2 contains a class II aminoacyl‐tRNA synthetase domain and a degenerate kinase segment, downstream and upstream of the eIF2α kinase domain, respectively, and both are singular features of GCN2 protein kinases. MGCN2 mRNA is expressed as a single message of ≈ 5.5 kb in a wide range of different tissues, with the highest levels in the liver and the brain. Specific polyclonal anti‐(MGCN2) immunoprecipitated an eIF2α kinase activity and recognized a 190 kDa phosphoprotein in Western blots from either mouse liver or MGCN2‐transfected 293 cell extracts. Interestingly, serum starvation increased eIF2α phosphorylation in MGCN2‐transfected human 293T cells. This finding provides evidence that GCN2 is the unique eIF2α kinase present in all eukaryotes from yeast to mammals and underscores the role of MGCN2 kinase in translational control and its potential physiological significance.
Characterization of recombinant and plant‐derived mistletoe lectin and their B‐chains
Authors: Eck Jürgen , Langer Martin , Möckel Babette , Witthohn Klaus , Zinke Holger , Lentzen Hans
Published: March 2003

Mistletoe lectin I (pML) and its isoforms ML II and III constitute the active principle in extract preparations from mistletoe, commonly used as immunomodulator in adjuvant tumour therapy. The heterodimeric disulfide‐linked cytotoxic protein is classified as type II ribosome inactivating protein (RIP). Recently, the sequence coding for the mistletoe lectin prepro‐protein was identified and the existence of a single intron‐free gene was shown [Eck, J., Langer, M., Möckel, B., Baur, A., Rothe, M., Zinke, H. & Lentzen, H. (1999) Eur. J. Biochem.264, 775–784].

 The aim of this study was to prepare pure and homogeneous rMLB‐chain as well as rML heterodimer for studying the carbohydrate binding specificity of recombinant versus natural protein and its contribution to the observed cytotoxic effect. Expression in E. coli resulted in the production of insoluble protein (inclusion bodies). A procedure for generating correctly folded, biochemically and biologically active rMLB was established starting from the insoluble single chain. Carbohydrate binding and specificity of pMLB and rMLB were analysed by a competitive enzyme linked lectin assay (ELLA). Asialofetuin was able to compete with binding of both chains (50% at 0.8 µm). The specificity of the B‐chains to lactose was more distinct with halfmaximal competition at 4.9 mm (pMLB) and > 90 mm (rMLB), respectively.

 Furthermore, in a coassociation process rMLA‐ and rMLB inclusion bodies were associated in one step by defined dilution yielding active rML‐heterodimer. The activities of recombinant (rML) and plant derived mistletoe lectin (pML) were compared. Cytotoxicity was determined using MOLT‐4 cells and enzymatic rRNA N‐glycosidase activity was measured in a coupled transcription/translation assay. The IC50 values of the two heterodimers were similar in both assays; rMLB‐chain did not show any cytotoxic effect. In the ELLA with lactose as a competitor 50% competition of binding to asialofetuin was achieved at 1.6 mm (rML) and 1.8 mm (pML). Hence, using three different assays we found no significant differences between the recombinant protein and the glycosylated form of ML. Comparing the biological activities of the single chains with those of the heterodimer we conclude, that both, lectin activity and the rRNA N‐glycosidase activity, are prerequisites for the cytotoxic effects on target cells.
Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70 subunit of DNA polymerase α
Authors: Kukimoto Iwao , Igaki Hiroshi , Kanda Tadahito
Published: March 2003

Budding yeast CDC45 encodes Cdc45p, an essential protein required to trigger initiation of DNA replication in late G1 phase. We cloned four and one species of the human Cdc45p homolog cDNA, resulting from different splicing patterns, from HeLa cell and human placenta cDNA libraries, respectively. A comparison of the cDNAs and the genomic sequence showed that the longest encoding a 610‐amino acid protein was comprised of 20 exons. One species, which lacks exon 7 and contains the shorter of two exons 18, was identical with the previously reported CDC45L cDNA and constituted 24 out of 28 clones from HeLa cells. Splicing was different in HeLa cells and TIG‐1 cells, a human diploid cell line. Human CDC45 protein was found to bind directly in vitro to human minichromosome maintenance 7 protein (hMCM7) and to the p70 subunit of DNA polymerase α. The data support a thesis that human CDC45 acts as a molecular tether to mediate loading of the DNA polymerase α on to the DNA replication complex through binding to hMCM7.
Enzymatic properties of human 25‐hydroxyvitamin D3 1α‐hydroxylase
Authors: Sawada Natsumi , Sakaki Toshiyuki , Kitanaka Sachiko , Takeyama Ken‐ichi , Kato Shigeaki , Inouye Kuniyo
Published: March 2003

We have cloned human 25‐hydroxyvitamin D3 1α‐hydroxylase cDNAs from normal subjects and patients with pseudovitamin D‐deficient rickets (PDDR), and expressed the cDNAs in Escherichia coli JM109 cells. Kinetic analysis of normal 1α‐hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R),25(OH)2D3 were 2.7 and 1.1 µm, respectively. The lower Km value and higher Vmax/Km value for (24R),25(OH)2D3 indicated that it is a better substrate than 25(OH)D3 for 1α‐hydroxylase. These results are quite similar to those of mouse 1α‐hydroxylase. To establish a highly sensitive in vivo system, 1α‐hydroxylase, adrenodoxin and NADPH–adrenodoxin reductase were coexpressed in E. coli cells. The recombinant E. coli cells showed remarkably high 1α‐hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH–adrenodoxin reductase through adrenodoxin to 1α‐hydroxylase in E. coli cells. Using this system, the activities of four mutants of 1α‐hydroxylase, R107H, G125E, R335P and P382S, derived from patients with PDDR were examined. Although no significant reduction in expression of these mutants was observed, none showed detectable activity. These results strongly suggest that the mutations found in the patients with PDDR completely abolished 1α‐hydroxylase activity by replacement of one amino acid residue.
Critical residues of the homeodomain involved in contacting DNA bases also specify the nuclear accumulation of thyroid transcription factor‐1
Authors: Christophe‐Hobertus Christiane , Duquesne Virginie , Pichon Bruno , Roger Pierre P. , Christophe Daniel
Published: March 2003

The N‐terminal end of thyroid transcription factor‐1 (TTF‐1) homeodomain is composed of a stretch of five basic amino‐acids that is conserved in both POU‐ and NK2‐class homeodomains and constitutes a functional nuclear localization signal. By analyzing the cellular distribution of fusion proteins, composed of a jellyfish green fluorescent variant and different parts of TTF‐1, we show here that the presence of this basic sequence is not sufficient by itself to confer complete nuclear accumulation. By mutagenesis, we identified a second region located in the center of the DNA recognition helix of the homeodomain that is also able to specify a predominantly nuclear localization of the chimeric proteins, independently of the presence of the basic NLS. The destruction, by mutagenesis, of both the basic stretch and the motif in the DNA recognition helix led to the total loss of nuclear accumulation, indicating that complete nuclear accumulation of TTF‐1 results from the concerted action of these two proteic signals. Both of the regions of the homeodomain that are involved in nuclear targeting also encompass critical amino‐acids responsible for DNA binding site recognition, as evidenced by the loss of DNA binding activityin vitroupon mutagenesis. Specifically, residues in the central part of the DNA recognition helix are involved in contacting bases in the major groove of DNA and are the most conserved in homeodomain proteins, suggesting that this part of the homeodomain could play a general role in the nuclear localization of members of this family of proteins.
Nitration of angiotensin II by ·NO2 radicals and peroxynitrite
Authors: Cudic Mare , Dendane Maryvonne , Houé‐Levin Chantal , Ducrocq Claire
Published: March 2003

To react with peptides, nitric oxide ·NO has to be activated by oxidation, or by coupling with superoxide (O·−2) thereby producing peroxynitrite. In the course of ·NO oxidation, ·NO2 free radicals and N2O3 may be formed. Using γ‐irradiation methods, we characterized the products formed by these nitrogen oxides with angiotensin II. Angiotensin II is specifically nitrated at its tyrosinyl residue by ·NO2 or peroxynitrite. Equimolecular amounts of each reagent in K+/Pi solutions at pH 7.4 led to 56% and 5% nitration yields, respectively. Nitrogen oxides produced by autoxidation of ·NO, as well as ·NO2 under ·NO, reacted only with the arginine residue, giving a mixture of peptides containing citrulline, a N‐(hydroxylamino‐cyanamido‐) instead of guanido group, and a conjugated diene derived from an arginine side‐chain. However, nitrosation reactions by N2O3 occurred only when the initial concentration of ·NO2 was 10 times that able to react with angiotensin II. Thus, in this case ·NO appears to protect against ·NO2 action.
Nonhomologous DNA end joining in cell‐free systems
Authors: Labhart Paul
Published: March 2003

Double strand DNA breaks are usually caused by ionizing radiation and radiomimetic drugs, but can also occur under normal physiological conditions during double strand break‐induced recombination, such as the rearrangement of T‐cell receptor and immunoglobulin genes during lymphoid development or the mating type switching in yeast. The main repair mechanism for double strand breaks in higher eukaryotes is nonhomologous DNA end joining (NHEJ), which modifies and ligates the two DNA ends without the help of extensive base–pairing interactions for alignment. Defects in double strand break repair are associated with radiosensitivity, predisposition to cancer and immunodeficiency syndromes, and the analysis of the underlying mutations has lead to the identification of several proteins involved in NHEJ. However, these genetic studies have yielded little information on the mechanism of NHEJ, and while some of the protein factors identified possess the expected enzymatic or DNA‐binding activities, the precise role of others remains unclear. Systems for cell‐free NHEJ have been available for over 10 years, but the biochemical analysis of NHEJ has lagged behind the genetic analysis, and not a single protein factor required for NHEJ has been identified by biochemical purification and reconstitution of NHEJ activity. Here I review the current status of in vitro systems for NHEJ, summarize the results obtained and information gained, and discuss the outlook for biochemical approaches to study NHEJ.
Stability and physicochemical properties of the bovine brain phosphatidylethanolamine‐binding protein
Authors: Vallée Béatrice , Teyssier Caroline , Maget‐Dana Régine , Ramstein Jean , Bureaud Nicole , Schoentgen Françoise
Published: March 2003

The equilibrium behaviour of the bovine phosphatidylethanolamine‐binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far‐UV and near‐UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of β‐sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three‐dimensional structure determined by X‐ray crystallography. The pH‐induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two‐state model for the equilibrium and display coincident values with a melting temperature Tm = 54 °C, an enthalpy change ΔH = 119 kcal·mol−1 and a free energy change ΔG(H2O, 25 °C) = 5 kcal·mol−1. The urea‐induced unfolding profiles of PEBP show a midpoint of the two‐state unfolding transition at 4.8 m denaturant, and the stability of PEBP is 4.5 kcal·mol−1 at 25 °C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well‐structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.
Human monocyte activation by cleaved form of alpha‐1‐antitrypsin
Authors: Janciauskiene Sabina , Lindgren Stefan
Published: March 2003

Production of alpha‐1‐antitrypsin (AAT) by human monocytes is an important factor in controlling tissue damage by proteases in the microenvironment of inflammation. Increases, of four‐ to eightfold, in numbers of macrophages and levels of AAT and its cleavage fragments have been found in various inflammatory loci. We have found that the C‐terminal peptide (C‐36) of AAT, produced by specific proteinase cleavage when added in its fibrillar form at concentrations ≥5 µm to monocytes in culture for 24 h, significantly increases low density lipoprotein (LDL) binding and uptake, up‐regulates levels of LDL receptors and also induces proinflammatory cytokine (interleukin‐1, interleukin‐6 and tumour necrosis factor α) production and glutathione reductase activity. Because it is known that various cells selectively internalize surface receptors and their ligands through receptor‐mediated endocytosis via clathrin‐coated pits, we tested whether antibodies raised against the clathrin heavy chain would block the effects of the fibrillar form of C‐36 on human monocytes in culture. Addition of excess anti‐(clathrin HC) with 10 µm fibrillar C‐36 diminished the stimulatory effects of the latter on LDL binding, uptake and LDL receptor levels. In contrast, however, in the presence of anti‐(clathrin HC), the potentially cytotoxic effects of fibrils, such as induction of cytokines, free radicals and cytosolic activity of cathepsin D, were much greater than those observed when cells were treated with fibrils alone. These results suggest that endocytosis is the pathway by which C‐36 fibrils upregulate LDL receptors, and may be the natural mechanism for fibril clearance. We infer that human monocytes clear C‐36 fibrils by a clathrin‐dependent pathway, presumably endocytotic, and that loss of this pathway amplifies the cytotoxic effects of the fibrils by increasing their availability to other specific or nonspecific sites through which they exert their cytotoxic effects.
Differential effects of mutations in the chromophore pocket of recombinant phytochrome on chromoprotein assembly and Pr‐to‐Pfr photoconversion
Authors: Remberg Anja , Schmidt Peter , Braslavsky Silvia E. , Gärtner Wolfgang , Schaffner Kurt
Published: March 2003

Site‐directed mutagenesis was performed with the chromophore‐bearing N‐terminal domain of oat phytochrome A apoprotein (amino acid residues 1–595). Except for Trp366, which was replaced by Phe (W366F), all the residues exchanged are in close proximity to the chromophore‐binding Cys321 (i.e. P318A, P318K, H319L, S320K, H322L and the double mutant L323R/Q324D). The mutants were characterized by their absorption maxima, and the kinetics of chromophore‐binding and the Pr→Pfr conversion. The strongest effect of mutation on the chromoprotein assembly, leading to an almost complete loss of the chromophore binding capability, was found for the exchanges of His322 by Leu (H322L) and Pro318 by Lys (P318K), whereas a corresponding alanine mutant (P318A) showed wild‐type behavior. The second histidine (H319) is also involved in chromophore fixation, as indicated by a slower assembly rate upon mutation (H319L). For the other mutants, an assembly process very similar to that of the wild‐type protein was found. The light‐induced Pr→Pfr conversion kinetics is altered in the mutations H319L and S320K and in the double mutant L323R/Q324D, all of which exhibited a significantly faster I700 decay and accelerated Pfr formation. P318 is also involved in the Pr→Pfr conversion, the millisecond steps (formation of Pfr) being significantly slower for P318A. Lacking sufficient amounts of W366F, assembly kinetics could not be determined in this case, while the fully assembled mutant underwent the Pr→Pfr conversion with kinetics similar to wild‐type protein.
Transcriptional control is relevant in the modulation of mosquito ferritin synthesis by iron
Authors: Pham Daphne Q.‐D. , Winzerling Joy J. , Dodson Mark S. , Law John H.
Published: March 2003

In yellow fever mosquito cells (Aag2 clone), iron treatment induces a threefold increase in ferritin message (fer mRNA) and protein (ferritin) by 16 h. These data contrast with work in mammalian hepatocytes and fibroblasts in which fer mRNA levels do not change with iron stimulation, but ferritin levels increase 50‐fold. Pretreatment of the Aag2 cells with actinomycin D blocks induction of fer mRNA and reduces the ferritin subunit synthesis, suggesting that iron induction of ferritin subunit synthesis is subjected to transcriptional control. A putative iron‐regulatory protein has also been identified in cytoplasmic extracts from Aag2 cells.
Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and endoglucanase I
Authors: Väljamäe Priit , Sild Veljo , Nutt Anu , Pettersson Göran , Johansson Gunnar
Published: March 2003

Intact and partially acid hydrolyzed cellulose from Acetobacter xylinum were used as model substrates for cellulose hydrolysis by 1,4‐β‐d‐glucan‐cellobiohydrolase I (CBH I) and 1,4‐β‐d‐endoglucanase I (EG I) from Trichoderma reesei. A high synergy between CBH I and EG I in simultaneous action was observed with intact bacterial cellulose (BC), but this synergistic effect was rapidly reduced by acid pretreatment of the cellulose. Moreover, a distinct synergistic effect was observed upon sequential endo–exo action on BC, but not on bacterial microcrystalline cellulose (BMCC). A mechanism for endo–exo synergism on crystalline cellulose is proposed where the simultaneous action of the enzymes counteract the decrease of activity caused by undesirable changes in the cellulose surface microstructure.
Structure and chromosomal location of the rat gene encoding the heart fatty acid‐binding protein
Authors: Zhang Jun , Rickers‐Haunerland Jutta , Dawe Ian , Haunerland Norbert H.
Published: March 2003

The gene coding for rat heart fatty acid‐binding protein (FABP), along with 1.2 kb of its 5′‐untranscribed region, was amplified by PCR, cloned and sequenced. As in other FABP genes, the coding sequence is interrupted by three introns of 3.4, 1.4 and 1.1 kb, respectively. Fluorescence in situ hybridization mapping revealed that the gene is located on chromosome 5q36. Using intron‐specific primers flanking exon 2, unspliced primary transcript RNA of the FABP gene was detected in a preparation of total RNA isolated from rat heart, proving that the cloned gene is expressed in adult cardiac tissue.
Lectinochemical characterization of a GalNAc and multi‐Galβ1→4GlcNAc reactive lectin from Wistaria sinensis seeds
Authors: Song Shuh‐chyung , Chen Yuen‐yuen , Herp Anthony , Tsai Ming‐sung , Wu Albert M.
Published: March 2003

An agglutinin that has high affinity for GalNAcβ1→, was isolated from seeds of Wistaria sinensis by adsorption to immobilized mild acid‐treated hog gastric mucin on Sepharose 4B matrix and elution with aqueous 0.2 m lactose. The binding property of this lectin was characterized by quantitative precipitin assay (QPA) and by inhibition of biotinylated lectin–glycan interaction. Of the 37 glycoforms tested by QPA, this agglutinin reacted best with a GalNAcβ1→4 containing glycoprotein (GP) [Tamm–Horsfall Sd(a+) GP]; a Galβ1→4GlcNAc containing GP (human blood group precursor glycoprotein from ovarian cyst fluid and asialo human α1‐acid GP) and a GalNAcα1→3GalNAc containing GP (asialo bird nest GP), but poorly or not at all with most sialic acid containing glycoproteins.

Among the oligosaccharides tested, GalNAcα1→3GalNAcβ1→3Galα1→4Galβ1→4Glc (Fp) was the most active ligand. It was as active as GalNAc and two to 11 times more active than Tn cluster mixtures, Galβ1→ 3/4GlcNAc (I/II), GalNAcα1→3(l‐Fucα1→2)Gal (Ah), Galβ1→4Glc (L), Galβ1→3GalNAc (T) and Galα1→ 3Galα→methyl (B). Of the monosaccharides and their glycosides tested, p‐nitrophenyl βGalNAc was the best inhibitor; it was approximately 1.7 and 2.5 times more potent than its corresponding α anomer and GalNAc (or Fp), respectively. GalNAc was 53.3 times more active than Gal.

From the present observations, it can be concluded that the Wistaria agglutinin (WSA) binds to the C‐3, C‐4 and C‐6 positions of GalNAc and Gal residues; the N‐acetyl group at C‐2 enhances its binding dramatically. The combining site of WSA for GalNAc related ligands is most likely of a shallow type, able to recognize both α and β anomers of GalNAc. Gal ligands must be Galβ1→3/4GlcNAc related, in which subterminal β1→3/4 GlcNAc contributes significantly to binding; hydrophobicity is important for binding of the β anomer of Gal. The decreasing order of the affinity of WSA for mammalian structural carbohydrate units is Fp ≥ multi‐II > monomeric II ≥ Tn, I and Ah ≥ E and L > T > Gal.
Enovin, a member of the glial cell‐line‐derived neurotrophic factor (GDNF) family with growth promoting activity on neuronal cells
Authors: Masure Stefan , Geerts Hugo , Cik Miroslav , Hoefnagel Evert , Van Den Kieboom Gerd , Tuytelaars An , Harris Sarah , Lesage Anne S. J. , Leysen Josee E. , van der Helm Liesbet , Verhasselt Peter , Yon Jeff , Gordon Robert D.
Published: March 2003

Glial cell‐line‐derived neurotrophic factor (GDNF), neurturin and persephin are neurotrophic factors involved in neuroneal differentiation, development and maintenance. They act on different types of neuroneal cells and signal through a receptor complex composed of a specific ligand‐binding subunit of the GDNF family receptor α (GFRα) family together with a common signaling partner, the cRET protein tyrosine kinase. We describe the molecular cloning, expression, chromosomal localization and functional characterization of enovin, a fourth GDNF family member almost identical to the recently described artemin. We show the occurence in most tissues of several differently spliced mRNA variants for enovin, of which only two are able to translate into functional enovin protein. Some tissues seem to express only nonfunctional transcripts. These observations may underlie a complex transcriptional regulation pattern. Enovin mRNA expression is detectable in all adult and fetal human tissues examined, but expression levels are highest in peripheral tissues including prostate, placenta, pancreas, heart and kidney. This tissue distribution pattern is in accordance with that of GFRα‐3, which here is shown to be the preferred ligand‐binding receptor for enovin (Kd = 3.1 nm). The human enovin gene is localized on chromosome 1, region p31.3‐p32. In vitro, enovin stimulates neurite outgrowth and counteracts taxol‐induced neurotoxicity in staurosporine‐differentiated SH‐SY5Y human neuroblastoma cells. The peripheral expression pattern of enovin and its receptor together with its effects on neuroneal cells suggest that enovin might be useful for the treatment of neurodegenerative diseases in general and peripheral neuropathies in particular.
Proteome mapping, mass spectrometric sequencing and reverse transcription‐PCR for characterization of the sulfate starvation‐induced response in Pseudomonas aeruginosa PAO1
Authors: Quadroni Manfredo , James Peter , Dainese‐Hatt Paola , Kertesz Michael A.
Published: March 2003

A set of proteins induced in Pseudomonas aeruginosa PAO1 during growth in the absence of sulfate was characterized by differential two‐dimensional electrophoresis and MS. Thirteen proteins were found to be induced de novo or upregulated in P. aeruginosa grown in a succinate/salts medium with sodium cyclohexylsulfamate as the sole sulfur source. Protein spots excised from the two‐dimensional gels were analysed by N‐terminal Edman sequencing and MS sequencing (MS/MS) of internal protein fragments. The coding sequences for 11 of these proteins were unambiguously identified in the P. aeruginosa genome sequence. Expression of these genes was investigated by reverse transcription‐PCR, which confirmed that repression in the presence of sulfate was acting at a transcriptional level. Three classes of sulfur‐regulated proteins were found. The first class (five proteins) were high‐affinity periplasmic solute‐binding proteins with apparent specificity for sulfate and sulfonates. A second class included enzymes involved in sulfonate and sulfate ester metabolism (three proteins). The remaining three proteins appeared to be part of a more general stress response, and included two antioxidant proteins and a putative lipoprotein. This study demonstrates the power of the proteomics approach for direct correlation of the responses of an organism to an environmental stimulus with the genetic structures responsible for that response, and the application of reverse transcription‐PCR significantly increases the conclusions that can be drawn from the proteomic study.
Structure and interaction of VacA of Helicobacter pylori with a lipid membrane
Authors: Pagliaccia Cristina , Wang Xiao‐Ming , Tardy Florence , Telford John L. , Ruysschaert Jean‐Marie , Cabiaux Véronique
Published: March 2003

In its mature form, the VacA toxin of Helicobacter pylori is a 95‐kDa protein which is released from the bacteria as a low‐activity complex. This complex can be activated by low‐pH treatment that parallels the activity of the toxin on target cells. VacA has been previously shown to insert itself into lipid membranes and to induce anion‐selective channels in planar lipid bilayers. Binding of VacA to lipid vesicles and its ability to induce calcein release from these vesicles were systematically compared as a function of pH. These two phenomena show a different pH‐dependence, suggesting that the association with the lipid membrane may be a two‐step mechanism. The secondary and tertiary structure of VacA as a function of pH and the presence of lipid vesicles were investigated by Fourier‐transform infrared spectroscopy. The secondary structure of VacA is identical whatever the pH and the presence of a lipid membrane, but the tertiary structure in the presence of a lipid membrane is dependent on pH, as evidenced by H/D exchange.
Homologous desensitization of the human guanylate cyclase C receptor
Authors: Bakre Manjiri M. , Ghanekar Yashoda , Visweswariah Sandhya S.
Published: March 2003

Guanylate Cyclase C (GCC) serves as a receptor for the endogenous ligands, guanylin and uroguanylin, as well as the family of bacterial heat‐stable enterotoxins (ST), which are one of the major causes of diarrhoea the world over. We had earlier provided evidence that GCC, present in the human colonic T84 cell line, is desensitized on prolonged exposure to ST, and this desensitization was reflected in a reduced ST‐stimulated guanylate cyclase activity of GCC [Bakre, M.M. & Visweswariah, S.S. (1997) FEBS Lett.408, 345–349]. In this study, we have investigated the mechanisms that underlie this cellular desensitization process. Desensitization of T84 cells was not a result of reduction in GCC present in membranes prepared from desensitized T84 cells, nor due to increased cGMP‐phosphodiesterase activity associated with the membrane fraction. The decrease in ST‐stimulatable guanylate cyclase activity of GCC was due to a dramatic reduction in the Vmax of the cyclase, which was also seen when MnGTP was used as the substrate. GCC undergoes ligand‐induced inactivation in vitro, which is alleviated in the presence of ATP. In vivo desensitized GCC could be further inactivated in vitro when preincubated with ST, indicating that the two mechanisms of GCC inactivation are distinct. Cellular refractoriness as reflected by a reduced responsiveness to further ST‐stimulation following prior exposure to IST, coupled with GCC desensitization was also observed in another colonic cell line, Caco2. However, HEK293 cells, stably transfected with GCC cDNA, when exposed to ST for prolonged periods, did not result in GCC desensitization, indicating that desensitization of GCC appeared to be a cell specific phenomenon. GCC expressed in HEK293‐GCC cells, however, showed in vitro ligand induced inactivation, suggesting that there are two independent means of ligand‐induced desensitization of GCC, perhaps distinct from the mechanisms that have been described earlier for other members of the guanylate cyclase receptor family.
Characterization of Streptococcus pneumoniae 5‐enolpyruvylshikimate 3‐phosphate synthase and its activation by univalent cations
Authors: Du Wensheng , Wallis Nicola G. , Mazzulla Marie J. , Chalker Alison F. , Zhang Lily , Liu Wu‐Schyong , Kallender Howard , Payne David J.
Published: March 2003

The aroA gene (Escherichia coli nomenclature) encoding 5‐enolpyruvylshikimate‐3‐phosphate (EPSP) synthase from the gram‐positive pathogen Streptococcus pneumoniae has been identified, cloned and overexpressed in E. coli, and the enzyme purified to homogeneity. It was shown to catalyze a reversible conversion of shikimate 3‐phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate. Activation by univalent cations was observed in the forward reaction, with NH+4, Rb+ and K+ exerting the greatest effects. Km(PEP) was lowered by increasing [NH+4] and [K+], whereas Km(S3P) rose with increasing [K+], but fell with increasing [NH+4]. Increasing [NH+4] and [K+] resulted in an overall increase in kcat. Glyphosate (GLP) was found to be a competitive inhibitor with PEP, but the potency of inhibition was profoundly affected by [NH+4] and [K+]. For example, increasing [NH+4] and [K+] reduced Ki(GLP versus PEP) up to 600‐fold. In the reverse reaction, the enzyme catalysis was less sensitive to univalent cations. Our analysis included univalent cation concentrations comparable with those found in bacterial cells. Therefore, the observed effects of these metal ions are more likely to reflect the physiological behavior of EPSP synthase and also add to our understanding of how to inhibit this enzyme in the host organism. As there is a much evidence to suggest that EPSP synthase is essential for bacterial survival, its discovery in the serious gram‐positive pathogen S. pneumoniae and its inhibition by GLP indicate its potential as a broad‐spectrum antibacterial target.
Sequence analysis and modular organization of threonyl‐tRNA synthetase from Thermus thermophilus and its interrelation with threonyl‐tRNA synthetases of other origins
Authors: Cura Vincent , Moras Dino , Kern Daniel
Published: March 2003

The gene encoding threonyl‐tRNA synthetase (Thr‐tRNA synthetase) from the extreme thermophilic eubacterium Thermus thermophilus HB8 has been cloned and sequenced. The ORF encodes a polypeptide chain of 659 amino acids (Mr 75 550) that shares strong similarities with other Thr‐tRNA synthetases. Comparative analysis with the three‐dimensional structure of other subclass IIa synthetases shows it to be organized into four structural modules: two N‐terminal modules specific to Thr‐tRNA synthetases, a catalytic core and a C‐terminal anticodon‐binding module. Comparison with the three‐dimensional structure of Escherichia coli Thr‐tRNA synthetase in complex with tRNAThr enabled identification of the residues involved in substrate binding and catalytic activity. Analysis by atomic absorption spectrometry of the enzyme overexpressed in E. coli revealed the presence in each monomer of one tightly bound zinc atom, which is essential for activity.

Despite strong similarites in modular organization, Thr‐tRNA synthetases diverge from other subclass IIa synthetases on the basis of their N‐terminal extensions. The eubacterial and eukaryotic enzymes possess a large extension folded into two structural domains, N1 and N2, that are not significantly similar to the shorter extension of the archaebacterial enzymes. Investigation of a truncated Thr‐tRNA synthetase demonstrated that domain N1 is not essential for tRNA charging. Thr‐tRNA synthetase from T. thermophilus is of the eubacterial type, in contrast to other synthetases from this organism, which exhibit archaebacterial characteristics. Alignments show conservation of part of domain N2 in the C‐terminal moiety of Ala‐tRNA synthetases. Analysis of the nucleotide sequence upstream from the ORF showed the absence of both any anticodon‐like stem‐loop structure and a loop containing sequences complementary to the anticodon and the CCA end of tRNAThr. This means that the expression of Thr‐tRNA synthetase in T. thermophilus is not regulated by the translational and trancriptional mechanisms described for E. coli thrS and Bacillus subtilis thrS and thrZ. Here we discuss our results in the context of evolution of the threonylation systems and of the position of T. thermophilus in the phylogenic tree.
Biochemical characterization of the murine S100A9 (MRP14) protein suggests that it is functionally equivalent to its human counterpart despite its low degree of sequence homology
Authors: Nacken Wolfgang , Sopalla Claudia , Pröpper Christian , Sorg Clemens , Kerkhoff Claus
Published: March 2003

Due to the low degree of sequence similarity it has been speculated that murine and human S100A9 (MRP14), an inflammatory marker protein belonging to the S100 protein family, may have different cellular functions in mouse and man. The present study was undertaken to investigate the murine S100A9 protein (mS100A9) biochemically. We demonstrate that in murine peripheral CD11b+ cells up to 20% of the protein of the cytosolic fraction consists of mS100A9 and that several minor mS100A9 isoforms are present. Cell fractionation experiments with CD11b+ murine leukocytes showed that mS100A9 is found in the cytosol as well as in the insoluble fraction. Transient expression of a green fluorescence protein–mS100A9 fusion in mammalian cells revealed that mS100A9 is localized in neither the nucleus nor the vesicles. Recombinantly expressed murine S100A9 interacts in vitro with murine and human S100A8 in an in vitro glutathione S‐transferase pull‐down assay. Homodimerization was not observed. For further biochemical analysis the myeloid 32D cell line is presented as a suitable model, to study murine myeloid expressed S100 proteins. Both murine S100A9 and its dimerization partner mS100A8 are expressed at the onset of granulocyte‐colony stimulating factor induced myeloid differentiation. Substantial amounts of this complex are constitutively secreted by granulocytic 32D cells into the medium. In summary, these data suggest, that the human and murine S100A9 may share a higher degree of functional homology than of sequence similarity.
Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cells
Authors: Drori Stavit , Sprecher Hannah , Shemer Gidi , Jansen Gerrit , Goldman I. David , Assaraf Yehuda G.
Published: March 2003

Human CEM‐7A cells established by gradual deprivation of leucovorin from the growth medium, display 100‐fold overexpression of methotrexate transport activity. We found that this was associated with 10‐fold reduced folate carrier gene amplification and 50‐fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6. This deletion consisted of the 3′‐most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3′‐UTR. These resulted in a truncated reduced folate carrier protein, which lacks the C‐terminal 160 amino acids, but instead contains 58 new C‐terminal amino acids obtained from reading through the 3′‐UTR. Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C‐terminus predicted to reside at the extracellular face. Western analysis with plasma‐membrane fraction from CEM‐7A cells revealed marked overexpression of both a broadly migrating ≈ 65–90 kDa native reduced folate carrier and a ≈ 40–45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS‐[3H]MTX and NHS‐[3H]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement. The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed.
Spectroscopic characterization of the conformational adaptability of Bombyx mori apolipophorin III
Authors: Narayanaswami Vasanthy , Yamauchi Yoshio , Weers Paul M. M. , Maekawa Hideaki , Sato Ryoichi , Tsuchida Kozo , Oikawa Kim , Kay Cyril M. , Ryan Robert O.
Published: March 2003

Apolipophorin III (apoLp‐III) from the silkmoth, Bombyx mori, has been over‐expressed in Escherichia coli, purified and characterized. Far‐UV CD spectroscopic analysis revealed 65% α‐helix secondary structure. Near‐UV CD spectra obtained in buffer or complexed with dimyristoylglycerophosphocholine (DMPC), provided evidence that apoLp‐III α‐helices reorient upon interaction with lipid, indicative of a protein conformational change. In guanidine hydrochloride (GdnHCl) denaturation studies, a transition midpoint of 0.33 m was observed, corresponding to a ΔGDH2O = 2.46 kcal·mol−1. Fluorescence studies of the sole tryptophan residue (Trp40) in apoLp‐III revealed an emission λmax = 327 nm. Compared to free tryptophan, Stern–Volmer constants (KSV) for acrylamide and KI quenching of Trp40 fluorescence were decreased by 20‐fold and sevenfold, respectively. In studies of apoLp‐III–DMPC disc complexes, far‐UV CD spectroscopy revealed an increase in α‐helix content to ≈ 85% and a ninefold increase in the GdnHCl‐induced denaturation transition midpoint to 3 m. In studies of lipid interaction, apoLp‐III was shown to disrupt both negatively charged and zwitterionic phospholipid bilayer vesicles, transforming them into discoidal complexes. Characterization of apoLp‐III–DMPC discs, using 5‐doxyl or 12‐doxyl stearic acid as lipid‐based quenching agents, revealed that Trp40 localizes near the phospholipid polar head groups. KSV values for acrylamide and KI quenching of intrinsic fluorescence of apoLp‐III–DMPC discs indicate that Trp40 is embedded in the lipid milieu, with little or no accessibility to the aqueous quenchers. Given the large amount of α‐helix in apoLp‐III, the data presented support a model in which amphipathic α‐helical segments are stabilized by helix–helix interactions and lipid association induces a protein conformational change which results in substitution of helix–helix interactions for helix–lipid contacts.