bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational aspects of HIV‐1 integrase inhibition by a peptide derived from the enzyme central domain and by antibodies raised against this peptide
Authors: Maroun Richard G. , Krebs Daniel , Roshani Mahmoud , Porumb Horea , Auclair Christian , Troalen Frédéric , Fermandjian Serge
Published: March 2003

Monospecific antibodies were raised against a synthetic peptide K159 (SQGVVESMNKELKKIIGQVRDQAEHLKTA) reproducing the segment 147–175 of HIV‐1 integrase (IN). Synthesis of substituted and truncated analogs of K159 led us to identify the functional epitope reacting with antibodies within the C‐terminal portion 163–175 of K159. Conformational studies combining secondary structure predictions, CD and NMR spectroscopy together with ELISA assays, showed that the greater is the propensity of the epitope for helix formation the higher is the recognition by anti‐K159. Both the antibodies and the antigenic peptide K159 exhibited inhibitory activities against IN. In contrast, neither P159, a Pro‐containing analog of K159 that presents a kink around proline but with intact epitope conformation, nor the truncated analogs encompassing the epitope, were inhibitors of IN. While the activity of antibodies is restricted to recognition of the sole epitope portion, that of the antigenic K159 likely requires interactions of the peptide with the whole 147–175 segment in the protein [Sourgen F., Maroun, R.G., Frère, V., Bouziane, A., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765–773
]. Actually, of all tested peptides only K159 was found to fulfill conditions of minimal number of helical heptads to achieve the formation of a stable coiled‐coil structure with the IN 147–175 segment. The binding of antibodies and of the antigenic peptide to this segment of IN hampers the binding of IN to its DNA substrates in filter‐binding assays. This appears to be the main effect leading to inhibition of integration. Quantitative analysis of filter‐binding assay curves indicates that two antibody molecules react with IN implying that the enzyme is dimeric within these experimental conditions. Together, present data provide an insight into the structure–function relationship for the 147–175 peptide domain of the enzyme. They also strongly suggest that the functional enzyme is dimeric. Results could help to assess models for binding of peptide fragments to IN and to develop stronger inhibitors. Moreover, K159 antibodies when expressed in vivo might exhibit useful inhibitory properties.
Δ9‐Fatty acid desaturase from arachidonic acid‐producing fungus
Authors: Sakuradani Eiji , Kobayashi Michihiko , Shimizu Sakayu
Published: March 2003

Based on the sequence information for Δ9‐desaturase genes (from rat, mouse and yeast), which are involved in the desaturation of palmitic acid and stearic acid to palmitoleic acid and oleic acid, respectively, the corresponding cDNA and genomic gene were cloned from the fungal strain, Mortierella alpina 1S‐4, which industrially produces arachidonic acid. There was a cytochrome b5‐like domain linked to the carboxyl terminus of this Mortierella desaturase, as also seen in the yeast Δ9‐desaturase. The MortierellaΔ9‐desaturase genomic gene had only one intron, in which a novel phenomenon was observed: there was a GC‐end at the 5′‐terminus instead of a GT‐end that is, in general, found in introns of eukaryotic genes. The full‐length cDNA clone was expressed under the control of an amyB promoter in a filamentous fungus, Aspergillus oryzae, resulting in drastic changes in the fatty acid composition in the transformant cells; the contents of palmitoleic acid (16 : 1) and oleic acid (18 : 1) increased significantly, with accompanying decreases in palmitic acid (16 : 0) and stearic acid (18 : 0). These changes were controlled by the addition of maltose as a carbon source to the medium. Also, the expression of the gene caused a significant change in the lipid composition in the Aspergillus transformant. Genomic Southern blot analysis of the transformant with the MortierellaΔ9‐desaturase gene as a probe confirmed the integration of this gene into the genome of A. oryzae.
Isolation and structural analysis of phosphorylated oligosaccharides obtained from Escherichia coli J‐5 lipopolysaccharide
Authors: Müller‐Loennies Sven , Holst Otto , Lindner Buko , Brade Helmut
Published: March 2003

The chemical structure of the phosphorylated lipopolysaccharide (LPS) of Escherichia coli J‐5 was investigated because it is of biomedical interest in the context of septic shock, a syndrome often encountered in nosocomial infections with gram‐negative pathogens. The successive de‐O‐acylation and de‐N‐acylation of J‐5 LPS yielded phosphorylated oligosaccharides which represent the complete carbohydrate backbone. Five compounds were separated by high‐performance anion‐exchange chromatography and analysed by one‐dimensional and two‐dimensional homonuclear and heteronuclear 1H‐NMR, 13C‐NMR and 31P‐NMR spectroscopy. The main product was a nonasaccharide of the structure α‐
d‐Glcp‐(1→3)‐[α‐
d‐GlcpN‐(1→7)‐α‐
l,
d‐Hepp‐(1→7)]‐α‐
l,
d‐Hepp‐(1→3)‐α‐
l,
d‐Hepp‐4P‐(1→5)‐[α‐Kdop‐(2→4)]‐α‐Kdop‐(2→6)‐β‐
d‐GlcpN‐4P‐(1→6)‐α‐
d‐GlcpN‐1P wherein all sugars are present as
d‐pyranoses. Hep and Kdo represent
lglycerodmanno‐heptose and 3‐deoxy‐
dmanno‐oct‐2‐ulosonic acid, respectively. In addition, two octasaccharides and two heptasaccharides were isolated that were partial structures of the nonasaccharide. In one octasaccharide the terminal α‐
d‐GlcpN was missing and an additional phosphate group linked to O4 of the branched heptose was present, whereas in the other octasaccharide the side‐chain Kdo was missing. In both heptasaccharides the side‐chain α‐
d‐GlcpN‐(1→7)‐
l‐α‐
d‐Hepp‐disaccharide was absent; they differed in their phosphate substitution. Whereas both heptasaccharides contained two phosphates in the lipid‐A backbone (β‐1,6‐linked GlcpN‐disaccharide at the reducing end) and one phosphate group at O4 of the first heptose, only one of them was additionally substituted with phosphate at O4 of the second heptose.
Stimulation of premature retinoic acid synthesis in Xenopus embryos following premature expression of aldehyde dehydrogenase ALDH1
Authors: Ang Hwee Luan , Duester Gregg
Published: March 2003

In order for nuclear retinoic acid receptors to mediate retinoid signaling, the ligand retinoic acid must first be produced from its vitamin A precursor retinal. Biochemical studies have shown that retinal can be metabolized in vitro to retinoic acid by members of the aldehyde dehydrogenase enzyme family, including ALDH1. Here we describe the first direct evidence that ALDH1 plays a physiological role in retinoic acid synthesis by analysis of retinoid signaling in Xenopus embryos, which have plentiful stores of maternally derived retinal. The Xenopus ALDH1 gene was cloned and shown to be highly conserved with chick and mammalian homologs. Xenopus ALDH1 was not expressed at blastula and gastrula stages, but was expressed at the neurula stage. We used a retinoic acid bioassay to demonstrate that retinoic acid is normally undetectable in embryos from fertilization to the initial gastrula stage, but that a tremendous increase in retinoic acid occurs during neurulation when ALDH1 is first expressed. Overexpression of ALDH1 by injection of Xenopus embryos with mRNAs encoding the mouse, chick or Xenopus ALDH1 homologs induced high levels of retinoic acid detection during the blastula stage. Thus, premature expression of ALDH1 stimulates premature synthesis of retinoic acid. These findings reveal an important conserved role for ALDH1 in retinoic acid synthesis in vivo, and demonstrate that conversion of retinoids from the aldehyde form to the carboxylic acid form is a crucial regulatory step in retinoid signaling.
Lipopolysaccharide stimulates HepG2 human hepatoma cells in the presence of lipopolysaccharide‐binding protein via CD14
Authors: Nanbo Asuka , Nishimura Hitoshi , Muta Tatsushi , Nagasawa Shigeharu
Published: March 2003

Lipopolysaccharide (LPS)‐binding protein (LBP), an opsonin for activation of macrophages by bacterial LPS, is synthesized in hepatocytes and is known to be an acute phase protein. Recently, cytokine‐induced production of LBP was reported to increase 10‐fold in hepatocytes isolated from LPS‐treated rats, compared with those from normal rats. However, the mechanism by which the LPS treatment enhances the effect of cytokines remains to be clarified. In the present study, we examined whether LPS alone or an LPS/LBP complex directly stimulates the hepatocytes, leading to acceleration of the cytokine‐induced LBP production. HepG2 cells (a human hepatoma cell line) were shown to express CD14, a glycosylphosphatidylinositol‐anchored LPS receptor, by both RT/PCR and flow cytometric analyses. An LPS/LBP complex was an effective stimulator for LBP and CD14 production in HepG2 cells, but stimulation of the cells with either LPS or LBP alone did not significantly accelerate the production of these proteins. The findings were confirmed by semiquantitative RT/PCR analysis of mRNA levels of LBP and CD14 in HepG2 cells after stimulation with LPS alone and an LPS/LBP complex. In addition, two monoclonal antibodies (mAbs) to CD14 (3C10 and MEM‐18) inhibited LPS/LBP‐induced cellular responses of HepG2 cells. Furthermore, prestimulation of HepG2 cells with LPS/LBP augmented cytokine‐induced production and gene expression of LBP and CD14. All these findings suggest that an LPS/LBP complex, but not free LPS, stimulates HepG2 cells via CD14 leading to increased basal and cytokine‐induced LBP and CD14 production.
The role of water structure in conformational changes of nucleic acids in ambient and high‐pressure conditions
Authors: Barciszewski Jan , Jurczak Janusz , Porowski Sylwester , Specht Thomas , Erdmann Volker A.
Published: March 2003

This review describes and summarizes data on the structure and properties of water under normal conditions, at high salt concentration and under high pressure. We correlate the observed conformational changes in nucleic acids with changes in water structure and activity, and suggest a mechanism of conformational transitions of nucleic acids which accounts for changes in the water structure. From the biophysical, biochemical and crystallographic data we conclude that the Z‐DNA form can be induced only at low water activity produced by high salt concentrations or high pressure, and accompanied by the stabilizing conjugative effect of the cytidine O4′ electrons of the CG base pairs.
The strict molybdate‐dependence of glucose‐degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme – an aldehyde oxidoreductase
Authors: Kardinahl Simone , Schmidt Christian L. , Hansen Thomas , Anemüller Stefan , Petersen Arnd , Schäfer Günter
Published: March 2003

In order to investigate the effects of trace elements on different metabolic pathways, the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius (DSM 639) has been cultivated on various carbon substrates in the presence and absence of molybdate. When grown on glucose (but neither on glutamate nor casein hydrolysate) as sole carbon source, the lack of molybdate results in serious growth inhibition. By analysing cytosolic fractions of glucose adapted cells for molybdenum containing compounds, an aldehyde oxidoreductase was detected that is present in the cytosol to at least 0.4% of the soluble protein. With Cl2Ind (2,6‐dichlorophenolindophenol) as artificial electron acceptor, the enzyme exhibits oxidizing activity towards glyceraldehyde, glyceraldehyde‐3‐phosphate, isobutyraldehyde, formaldehyde, acetaldehyde and propionaldehyde. At its pH‐optimum (6.7), close to the intracellular pH of Sulfolobus, the glyceraldehyde‐oxidizing activity is predominant. The protein has an apparent molecular mass of 177 kDa and consists of three subunits of 80.5 kDa (α), 32 kDa (β) and 19.5 kDa (γ). It contains close to one Mo, four Fe, four acid‐labile sulphides and four phosphates per protein molecule. Methanol extraction revealed the existence of 1 FAD per molecule and 1 molybdopterin per molecule, which was identified as molybdopterin guanine dinucleotide on the basis of perchloric acid cleavage and thin layer chromatography. EPR‐spectra of the aerobically prepared enzyme exhibit the so‐called ‘desulpho‐inhibited’‐signal, known from chemically modified forms of molybdenum containing proteins. Anaerobically prepared samples show both, the signals arising from the active molybdenum‐cofactor as well as from the two [2Fe‐2S]‐clusters. According to metal‐, cofactor‐, and subunit‐composition, the enzyme resembles the members of the xanthine oxidase family. Nevertheless, the melting point and long‐term thermostability of the protein are outstanding and perfectly in tune with the growth temperature of S. acidocaldarius (80 °C).

The findings suggest the enzyme to function as a glyceraldehyde oxidoreductase in the course of the nonphosphorylated Entner‐Doudoroff pathway and thereby may attribute a new physiological role to this class of enzyme.
Purification and characterization of the assembly factor P17 of the lipid‐containing bacteriophage PRD1
Authors: Caldentey Javier , Hänninen Anna‐Liisa , Holopainen Juha M. , Bamford Jaana K. H. , Kinnunen Paavo K. J. , Bamford Dennis H.
Published: March 2003

Assembly factors, proteins assisting the formation of viral structures, have been found in many viral systems. The gene encoding the assembly factor P17 of bacteriophage PRD1 has been cloned and expressed in Escherichia coli. P17 acts late in phage assembly, after capsid protein folding and multimerization, and sorting of membrane proteins has occurred. P17 has been purified to near homogeneity. It is a tetrameric protein displaying a rather high heat stability. The protein is largely in an α‐helical conformation and possesses a putative leucine zipper which is not essential for protein function, as judged by in vitro mutagenesis and complementation analysis. Although heating does not cause structural changes in the conformation of the protein, the dissociation of the tetramer into smaller units is evident as diminished self‐quenching of the fluorescently labeled P17. Similarly, dissociation of the tetramer is also obtained by dialysis of the protein against 6‐m guanidine hydrochloride (GdnHCl) or 1% SDS. The reassembly of these smaller units upon cooling is evident from resonance energy transfer.
A 42‐kDa glycoprotein from chicken egg‐envelope, an avian homolog of the ZPC family glycoproteins in mammalian zona pellucida
Authors: Takeuchi Yukinari , Nishimura Keiji , Aoki Naohito , Adachi Takahiro , Sato Chihiro , Kitajima Ken , Matsuda Tsukasa
Published: March 2003

A glycoprotein with molecular mass of 42 kDa was identified as the major component of the chicken egg‐envelope, the filamentous, extracellular matrix known as the perivitelline layer. By using a DNA probe amplified with degenerative primers derived from the protein’s partial amino acid sequences, a cDNA clone encoding the egg‐envelope 42‐kDa glycoprotein (gp42) was isolated from a hen’s ovary cDNA library. The gp42 open reading frame encoded 435 amino acid residues, including a putative signal peptide of 20 amino acids. The deduced amino acid sequence of gp42 showed significant similarity to egg‐envelope glycoproteins of the ZPC family of several other vertebrate species, including human ZP3, mouse ZP3, Xenopus laevis gp43 and medaka (Oryzias latipes) ZI3 (LS‐F), which play important roles for sperm–egg interaction. A single N‐glycosylation site present in chicken gp42 is conserved among all five of these proteins: carbohydrate analysis of gp42 revealed the presence of a complex type glycan chain at this site. N‐terminal sequence analysis of the mature polypeptide suggests that C‐terminal processing of the pro‐protein occurs during synthesis and secretion. The 1.4‐kb gp42 transcript was detected only in follicles, and was found to be accumulated in granulosa cells in a manner dependent on ovarian follicular development. Furthermore, a metabolically radio‐labeled gp42 was immunopreciptated from both cell lysate and culture supernatant of the granulosa cells with specific anti‐gp42 antibody, suggesting granulosa cell‐specific synthesis and secretion of the glycoprotein.
The carboxy‐terminal sequence Asp427–Glu432 of β‐tubulin plays an important function in axonemal motility
Authors: Audebert Stéphane , White Daniel , Cosson Jacky , Huitorel Philippe , Eddé Bernard , Gagnon Claude
Published: March 2003

Flagellar motility is the result of specific interactions between axonemal microtubular proteins and the dynein motors. Tubulin, the main component of microtubule, is a very polymorphic protein resulting from the expression of several isogenes and from the existence of various post‐translational modifications. In order to characterize tubulin isoforms and tubulin domains that are important for flagellar movement, we prepared monoclonal antibodies against axonemal proteins from whole sea‐urchin sperm tails. The monoclonal antibodies obtained were screened for their potency to inhibit demembranated‐reactivated sperm models and for their monospecific immunoreactivity on immunoblot. Among the different antibodies we obtained, D66 reacted specifically with a subset of β‐tubulin isoforms. Limited proteolysis, HPLC, peptide sequencing, mass spectroscopy and immunoblotting experiments indicated that D66 recognized an epitope localized in the primary sequence Gln423–Glu435 of the C‐terminal domain of Lytechinus pictusβ2‐tubulin, and that this sequence belongs to class IVb. The use of synthetic peptides and immunoblotting analysis further narrowed the amino acids important for antibody recognition to Asp427–Glu432. Because the primary effect of this antibody on sperm motility is to decrease the flagellar beat frequency, we suggest that this sequence is involved in the tubulin–dynein head interaction.
Phosphorylation of components of the ER translocation site
Authors: Gruss Oliver J. , Feick Peter , Frank Rainer , Dobberstein Bernhard
Published: March 2003

In many eukaryotic cells, protein secretion is regulated by extracellular signalling molecules giving rise to increased intracellular Ca2+and activation of kinases and phosphatases. To test whether components involved in the first step of secretion, the translocation of proteins across the endoplasmic reticulum (ER) membrane, are regulated by Ca2+‐dependent phosphorylation and dephosphorylation, we have investigated the effect of Ca2+on kinases associated with the rough ER. Using purified rough microsomes from dog pancreas we found that Ca2+‐dependent isoforms of protein kinase C (PKC) are associated with the rough ER and phosphorylate essential components of the protein translocation machinery. Phosphorylation of microsomal proteins by PKCs increased protein translocation efficiencyin vitro. We also found that proteins of the translocation machinery became phosphorylated in intact cells. This suggests a further level of regulation of protein translocation across the ER membrane.
A molecular mechanism for the cleavage of a disulfide bond as the primary function of agonist binding to G‐protein‐coupled receptors based on theoretical calculations supported by experiments
Authors: Brandt Wolfgang , Golbraikh Alexander , Täger Michael , Lendeckel Uwe
Published: March 2003

A model of the binding site of δ‐opioids in the extracellular region of the G‐protein‐coupled opioid receptor based on modelling studies is presented. The distance between Asp288 and the disulfide bridge (Cys121–Cys198) formed between the first and second extracellular loops was found to be short. This model is consistent with site‐directed mutagenesis studies. The arrangement of the ligands found in the receptor led to the development of a reaction mechanism for the cleavage of the disulfide bond catalysed by the ligands. Semi‐empirical quantum chemical PM3 and AM1 calculations as well as ab initio studies showed that the interaction between the carboxylic acid side chain of aspartic acid and the disulfide bond leads to the polarization of, and withdrawal of a proton from, the protonated nitrogen of the ligand to one of the sulfur atoms. A mixed sulfenic acid and carboxylic acid anhydrate is formed as an intermediate as well as a thiol. The accompanying cleavage of the disulfide bond may produce a conformational change in the extracellular loops such that the pore formed by the seven‐helix bundle opens allowing entrance of the ligand, water and ions into the cell. Cleavage of the disulfide bond after opioid administration was demonstrated experimentally by flow‐cytometric measurements employing CMTMR and monobromobimane‐based analyses of membrane‐located thiols. The suggested mechanism may explain, in a consistent way, the action of agonists and antagonists and is assumed to be common for many G‐protein coupled receptors.
Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic isozyme
Authors: Janssen Marcel J. W. , Verheij Hubertus M. , Slotboom Arend J. , Egmond Maarten R.
Published: March 2003

Secretory phospholipases A2 (PLA2s) are small homologous proteins rich in disulphide bridges. These PLA2s have been classified into several groups based on the disulphide bond patterns found [Dennis, E. A. (1997)Trends Biochem. Sci.22, 1–2]. To probe the effect of the various disulphide bond patterns on folding, stability and enzymatic properties, analogues of the secretory PLA2s were produced by protein engineering of porcine pancreatic PLA2. Refolding experiments indicate that small structural variations play an important role in the folding of newly made PLA2 analogues. Introduction of a C‐terminal extension together with disulphide bridge 50–131 gives rise to an enzyme that displays full enzymatic activity having increased conformational stability. In contrast, introduction of a small insertion between positions 88 and 89 together with disulphide bridge 86–89 decreases the catalytic activity significantly, but does not change the stability. Both disulphide bridges 11–77 and 61–91 are important for the kinetic properties and stability of the enzyme. Disulphide bridge 11–77, but not 61–91, was found to be essential to resist tryptic breakdown of native porcine pancreatic PLA2.
Motor proteins of the kinesin family
Authors: Sack Stefan , Kull F. Jon , Mandelkow Eckhard
Published: March 2003

Microtubule‐dependent motors of the kinesin family convert the energy from ATP hydrolysis into mechanical work in order to transport vesicles and organelles along microtubules. The motor domains of several kinesins have been solved by X‐ray diffraction, but the conformational changes associated with force development remain unknown. Here we describe conformational properties of kinesin that might be related to the mechanism of action. First, we have evaluated the conformational variability among all known kinesin structures and find they are concentrated in six areas, most of which are functionally important either in microtubule binding or in linking the core motor to the stalk. Secondly, we show that there is an important difference between kinesins when compared with myosins or GTPases (with which kinesin motor domains bear structural and catalytic similarities); in the diphosphate‐state (with bound ADP), all kinesins show a ‘tight’ nucleotide‐binding pocket, comparable with myosin or GTPases in the triphosphate state, whose nucleotide‐binding pockets become open, or ‘loose’, following nucleotide hydrolysis. Thus, kinesin‐ADP appears to be in a tense state, resembling that observed in myosin‐ATP or p21ras‐GTP.
Substrate specificity studies of Flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan–protein linkage region
Authors: Tsuda Hiromi , Yamada Shuhei , Miyazono Hirofumi , Morikawa Kiyoshi , Yoshida Keiichi , Goto Fumitaka , Tamura Jun‐ichi , Neumann Klaus W. , Ogawa Tomoya , Sugahara Kazuyuki
Published: March 2003

Bacterial chondroitinases and heparitinases are potentially useful tools for structural studies of chondroitin sulfate and heparin/heparan sulfate. Substrate specificities of Flavobacterium chondroitinase C, as well as heparitinases I and II, towards the glycosaminoglycan–protein linkage region ‐HexA‐HexNAc‐GlcA‐Gal‐Gal‐Xyl‐Ser (where HexA represents glucuronic acid or iduronic acid and HexNAc represents N‐acetylgalactosamine or N‐acetylglucosamine) were investigated using various structurally defined oligosaccharides or oligosaccharide‐serines derived from the linkage region. In the case of oligosaccharide‐serines, they were labeled with a chromophore dimethylaminoazobenzenesulfonyl chloride (DABS‐Cl), which stably reacted with the amino group of the serine residue and rendered high absorbance for microanalysis. Chondroitinase C cleaved the GalNAc bond of the pentasaccharides or hexasaccharides derived from the linkage region of chondroitin sulfate chains and tolerated sulfation of the C‐4 or C‐6 of the GalNAc residue and C‐6 of the Gal residues, as well as 2‐O‐phosphorylation of the Xyl residue. In contrast, it did not act on the GalNAc–GlcA linkage when attached to a 4‐O‐sulfated Gal residue. Heparitinase I cleaved the innermost glucosaminidic bond of the linkage region oligosaccharide‐serines of heparin/heparan sulfate irrespective of substitution by uronic acid, whereas heparitinase II acted only on the glucosaminidic linkages of the repeating disaccharide region, but not on the innermost glucosaminidic linkage. These defined specificities of chondroitinase C, as well as heparitinases I and II, will be useful for preparation and structural analysis of the linkage oligosaccharides.
The expression of both domains of the 69/71 kDa 2′,5′ oligoadenylate synthetase generates a catalytically active enzyme and mediates an anti‐viral response
Authors: Marié Isabelle , Rebouillat Dominique , Hovanessian Ara G.
Published: March 2003

The 2′,5′ oligoadenylate synthetase (OAS) represents a family of interferon‐induced proteins which, when activated by double‐stranded (ds) RNA, polymerizes ATP into 2′,5′‐linked oligomers with the general formula pppA(2′p5′A)n, where n ≥ 1. The 69‐kDa form of human OAS has two isoforms (p69 and p71) that are identical for their first 683 amino acids and consist of two homologous and adjacent domains, each homologous to the small 40‐kDa OAS. Here, we demonstrate that mRNA species specific for the isoforms p69 and p71 are enhanced in interferon‐treated cells, with the p69 mRNA being more abundant than that of p71. In transfected cells, both isoforms could be expressed independently to generate enzymes with similar catalytic activity, typical of the natural 69‐kDa OAS from interferon‐treated cells. On the other hand, deletion mutants expressing either the N‐ or C‐terminal domain common in p69 and p71 were greatly unstable and were found to be devoid of catalytic activity, in spite of the capacity of the C‐terminal domain to bind dsRNA. Finally, we show that murine cell lines stably expressing either p69 or p71 isoforms partially resist infection by the encephalomyocarditis virus. These results indicate that both isoforms of the 69‐kDa form of 2′,5′ OAS are expressed in interferon‐treated cells, and that each isoform could be implicated in the mechanism of the anti‐viral action of interferon.
Expression in vitro of alternatively spliced variants of the messenger RNA for human apolipoprotein E receptor‐2 identified in human tissues by ribonuclease protection assays
Authors: Sun Xi‐Ming , Soutar Anne K.
Published: March 2003

The apolipoprotein E receptor‐2 (apoER2), also called LR7/8B, is a member of the low‐density lipoprotein (LDL)‐receptor family that is expressed in brain. We have identified mRNA splicing variants in human tissues by ribonuclease protection assays and found that some variants are preferentially amplified by reverse transcription‐polymerase chain reaction (RT‐PCR). Transcripts were found that lacked sequences encoding three repeats in the putative ligand‐binding domain, the O‐linked sugar domain or a novel region in the cytoplasmic domain. When mammalian expression vectors for eight potential protein isoforms were transfected into LDL‐receptor‐deficient Chinese hamster ovary cells, the proteins were all expressed on the cell surface, as detected by immunoblotting of cell extracts with a specific antipeptide antiserum to apoER2 before and after treatment of intact cells with pronase. Although cells expressing all the variants bound very low‐density lipoprotein of β mobility (β‐VLDL), it was with lower affinity and capacity than binding by the LDL‐receptor and none was able to degrade β‐VLDL. Ligand blotting of cell extracts showed that all variants bound recombinant histidine6‐tagged receptor‐associated protein (His6‐RAP) with high affinity, although variants lacking exon 5 bound less strongly. The presence of vestiges of the novel insert in the cytoplasmic domain of apoER2 in the LDL‐ or VLDL‐receptor genes was investigated, but nucleotide sequencing showed that no sequences homologous to it could be detected in the final intron of these genes.
Changing the efficiency and specificity of the esterase activity of human carbonic anhydrase II by site‐specific mutagenesis
Authors: Elleby Björn , Sjöblom Björn , Lindskog Sven
Published: March 2003

Rates of hydrolysis of 4‐, 3‐, and 2‐nitrophenyl acetate and 4‐nitrophenyl propionate catalyzed by wild‐type and mutant forms of human carbonic anhydrase II have been measured. The results show that the mutations Tyr7→Phe and Ala65→Leu lead to activity enhancements with all the investigated substrates, but there is no significant effect on the specificity. In contrast, some mutations at sequence position 200 have large effects on specificity. For example, while the mutation Thr200→Gly results in a threefold increase of the rate of hydrolysis of 4‐nitrophenyl acetate, the activity is enhanced 10 times with the meta‐substituted substrate and 380 times with the ortho‐substituted substrate. These results are interpreted in terms of the removal in the mutant of a steric interference between the 2‐NO2 group, in particular, and the side chain of Thr200. Mutants involving residues lining a hydrophobic pocket near the catalytically essential zinc ion have also been investigated. The most pronounced effect on specificity was found for the Val143→Gly mutant. This mutation leads to a sixfold decrease of the rate of hydrolysis of 4‐nitrophenyl acetate but a 20‐fold increase of the activity with the propionyl ester as substrate. These results suggest that the side chain of Val143 interferes sterically with the acyl moiety of 4‐nitrophenyl propionate. Based on these results, we have constructed a hypothetical model of the location of these ester substrates in the enzymic active site.
Enzymatic action of human glandular kallikrein 2 (hK2)
Authors: Lövgren Janita , Airas Kalervo , Lilja Hans
Published: March 2003

Human glandular kallikrein 2 (hK2) is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate‐specific antigen (PSA). Recently, hK2 was shown to activate the zymogen form of PSA (proPSA) in vitro and is likely to be the physiological activator of PSA in the prostate. hK2 is also able to activate urokinase and effectively cleave fibronectin. We studied the substrate specificity of hK2 and regulation of its activity by zinc and extracellular protease inhibitors present in the prostate and seminal plasma. The enzymatic activity and substrate specificity was studied by determining hK2 cleavage sites in the major gel proteins in semen, semenogelin I and II, and by measuring hydrolysis of various tripeptide aminomethylcoumarin substrates. HK2 cleaves substrates C‐terminal of single or double arginines. Basic amino acids were also occasionally found at several other positions N‐terminal of the cleavage site. Therefore, the substrate specificity of hK2 fits in well with that of a processor of protein precursors. Possible regulation mechanisms were studied by testing the ability of Zn2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn2+. The high affinity of PCI for hK2 (kass = 2.0 × 105 m−1·s−1) and the high concentrations of PCI (4 µm) and hK2 (0.2 µm) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn2+ at micromolar concentrations well below the 9 mm zinc concentration found in the prostate. The enzymatic activity of hK2 is likely to be reversibly regulated by Zn2+ in prostatic fluid. This regulation may be impaired in CAP and advanced metastatic cancer resulting in lack of control of the hK2 activity and a need for other means of control.
The structural basis for the regulation of tissue transglutaminase by calcium ions
Authors: Casadio Rita , Polverini Eugenia , Mariani Paolo , Spinozzi Francesco , Carsughi Flavio , Fontana Angelo , de Laureto Patrizia Polverino , Matteucci Gabriella , Bergamini Carlo M.
Published: March 2003

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three‐dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 × 4.2 × 11 nm) and comprises four domains, assembled pairwise into N‐terminal and C‐terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium‐free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis.

 The protein dimensions are consistent with results obtained with small‐angle neutron and X‐ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein. Proteolysis of an exposed loop connecting the N‐terminal and C‐terminal regions is linearly correlated with enzyme inactivation and prevents the calcium‐induced conformational changes.
Cysteine control over glutathione homeostasis in Chinese hamster fibroblasts overexpressing a γ‐glutamylcysteine synthetase activity
Authors: Vincent Bruno Robert de Saint , Mousset Simone , Jacquemin‐Sablon Alain
Published: March 2003

γ‐Glutamylcysteine synthetase (GCS) catalyses the first step of glutathione (GSH) biosynthesis and is considered to be the rate‐limiting step of this pathway. In several experimental systems, GCS overexpression has been associated with GSH pool expansion and drug resistance. In this report, we describe a mutant line of Chinese hamster fibroblasts that overexpress this activity by 4–5 times, due to the amplification of the gene encoding the catalytic subunit of GCS. These mutant cells contained a wild‐type steady‐state level of GSH and, after depletion, synthesized GSH at the same rate as wild‐type cells because their rate of endogenous production of cysteine was limiting. An exogenous supply of cysteine expanded the pool of GSH in mutant cells by 80% but did not increase that of wild‐type cells, and, in GSH‐depleted cells, increased the rate of GSH biosynthesis by eight and 35‐times in wild‐type and mutant cells, respectively. These experiments indicated that GCS overexpression had no consequence on the metabolism of GSH, unless a supply of cysteine was provided. Mutant cells were not resistant to cisplatin or nitrogen mustard.
Fluorescent phosphoinositide derivatives reveal specific binding of gelsolin and other actin regulatory proteins to mixed lipid bilayers
Authors: Tuominen Esa K. J. , Holopainen Juha M. , Chen Jian , Prestwich Glenn D. , Bachiller Patricia R. , Kinnunen Paavo K. J. , Janmey Paul A.
Published: March 2003

Fluorescent derivatives of phosphatidyl inositol (PtdIns)‐(4,5)‐P2 were synthesized and used to test the effects of the PtdIns‐(4,5)‐P2‐regulated proteins gelsolin, tau, cofilin, and profilin on labeled PtdIns‐(4,5)‐P2 that was either in micellar form or mixed with phosphatidylcholine (PtdCho) in bilayer vesicles. Gelsolin increased the fluorescence of 7‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD)‐ or pyrene‐labeled PtdIns‐(4,5)‐P2 and NBD‐PtdIns‐(3,4,5)‐P3. Cofilin and profilin produced no detectable change at equimolar ratios to PtdIns‐(4,5)‐P2, while tau decreased NBD‐PtdIns‐(4,5)‐P2 fluorescence. Fluorescence enhancement by gelsolin of NBD‐PtdIns‐(4,5)‐P2 in mixed lipid vesicles depended on the mole fraction of PtdIns‐(4,5)‐P2 in the bilayer. Specific enhancement of 3% NBD‐PtdIns‐(4,5)‐P2 : 97% PtdCho was much lower than that of 10% PtdIns‐(4,5)‐P2 : 90% PtdCho, but the enhancement of 3% NBD‐PtdIns‐(4,5)‐P2 could be increased by addition of 7% unlabeled PtdIns‐(4,5)‐P2. The gelsolin‐dependent increase in NBD‐PtdIns‐(4,5)‐P2 fluorescence was reversed by addition of Ca2+ or G‐actin. Significant, but weaker, fluorescence enhancement was observed with the gelsolin N‐terminal domain (residues 1–160) and a peptide comprised of gelsolin residues 150–169. Fluorescence energy transfer from gelsolin to pyrene‐PtdIns‐(4,5)‐P2 was much stronger with intact gelsolin than the N‐terminal region of gelsolin containing the PtdIns‐(4,5)‐P2 binding sites, suggesting that PtdIns‐(4,5)‐P2 may bind near a site formed by the juxtaposition of the N‐ and C‐terminal domains of gelsolin.
Cysteine‐scanning mutagenesis of an eukaryotic pore‐forming toxin from sea anemone
Authors: Anderluh Gregor , Barlič Ariana , Podlesek Zdravko , Maček Peter , Pungerčar Jože , Gubenšek Franc , Zecchini Maria Lucia , Serra Mauro Dalla , Menestrina Gianfranco
Published: March 2003

Equinatoxin II is a cysteineless pore‐forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine‐scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in Escherichia coli and purified. The accessibility of the thiol groups in lipid‐embedded cysteine mutants was studied by reaction with biotin maleimide. Most of the mutants were modified, except those with cysteines at positions 105 and 114. Mutants R144C and S160C were modified only at high concentrations of the probe. Similar results were obtained if membrane‐bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding. These results were further confirmed by labeling the mutants with acrylodan, a polarity‐sensitive fluorescent probe. When labeled mutants were combined with vesicles, the following mutants exhibited blue‐shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: the N‐terminal 13–20 region, probably forming an amphiphilic helix, and the tryptophan‐rich 105–120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with the membrane appears to be unique and different from that of bacterial pore‐forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel‐forming toxins.
Binding of AR‐1‐144, a tri‐imidazole DNA minor groove binder, to CCGG sequence analyzed by NMR spectroscopy
Authors: Yang Xiang‐Lei , Kaenzig Crystal , Lee Moses , Wang Andrew H.‐J.
Published: March 2003

The interactions of N‐[2‐(dimethylamino)ethyl]‐1‐methyl‐4‐[1‐methyl‐4‐[4‐formamido‐1‐methylimidazole‐2‐carboxamido]imidazole‐2‐carboxamido]imidazole‐2‐carboxamide (AR‐1‐144), a tri‐imidazole polyamide minor groove binder, with DNA have been investigated by NMR and CD spectroscopy. A series of DNA oligonucleotides with a C/G‐containing four‐bp core, i.e. CCGG, CGCG, GGCC, and GCGC, have been titrated with AR‐1‐144 at different ratios. AR‐1‐144 favors the CCGG sequence. The flanking sequence of the CCGG core also influences the binding preference, with a C or T being favored on the 3′‐side of the CCGG core. The three‐dimensional structure of the symmetric 2 : 1 side‐by‐side complex of AR‐1‐144 and GAACCGGTTC, determined by NOE‐constrained NMR refinement, reveals that each AR‐1‐144 binds to four base pairs, i.e. at C5‐G6‐G7‐T8, with every amide‐imidazole unit forming two potential hydrogen bonds with DNA. The same DNA binding preference of AR‐1‐144 was also confirmed by circular dichroism spectroscopy, indicating that the DNA binding preference of AR‐1‐144 is independent of concentration. The cooperative binding of an AR‐1‐144 homodimer to the (purine)CCGG(pyrimidine) core sequence appears to be weaker than that of the distamycin A homodimer to A/T sequences, most likely due to the diminished hydrophobic interactions between AR‐1‐144 and DNA. Our results are consistent with previous footprinting data and explain the binding pattern found in the crystal structure of a di‐imidazole drug bound to CATGGCCATG.
Purification and characterization of a highly enantioselective epoxide hydrolase from Aspergillus niger
Authors: Morisseau Christophe , Archelas Alain , Guitton Carole , Faucher Didier , Furstoss Roland , Baratti Jacques C.
Published: March 2003

The epoxide hydrolase from Aspergillus niger was purified to homogeneity using a four‐step procedure and p‐nitrostyrene oxide (pNSO) as substrate. The enzyme was purified 246‐fold with 4% activity yield. The protein is a tetramer composed of four identical subunits of molecular mass 45 kDa. Maximum activity was observed at 40 °C, pH 7.0, and with dimethylformamide as cosolvent to dissolve pNSO. Hydrolysis of pNSO was highly enantioselective, with an E value (i.e. enantiomeric ratio) of 40 and a high regioselectivity (97%) for the less hindered carbon atom of the epoxide. This enzyme may be a good biocatalyst for the preparation of enantiopure epoxides or diols.
Mouse fibulin‐2 gene
Authors: Grässel Susanne , Sicot François‐Xavier , Gotta Susan , Chu Mon‐Li
Published: March 2003

Fibulin‐2, an extracellular matrix protein containing tandem arrays of calcium‐binding epidermal growth factor‐like motifs, is present in the basement membrane and stroma of many tissues. Its expression pattern suggested an essential role in organogenesis, particularly in embryonic heart development. In this study, we cloned the extreme 5′ end of the mouse fibulin‐2 cDNA, isolated phage and cosmid clones encoding the entire gene, and functionally characterized the promoter. The gene was found to consist of 18 exons spanning 55 kb of DNA. The exon–intron organization reflected the modular structure of the protein. Exon 9 was subjected to alternative splicing. All splice junctions conformed to the GT/AG rule, except that GC instead of GT was found in the splice donor site of exon 4. The gene lacked TATA and CAAT boxes but contained an initiator element (Inr) and several consensus Sp1 binding sites surrounding the transcription start sites. By transient transfection of promoter deletion constructs, a 0.46‐kb region containing the clustered Sp1 sites was found to confer a high promoter activity.
Cloning, expression and characterization of an A6‐related protein
Authors: Rohwer Andreas , Kittstein Walter , Marks Friedrich , Gschwendt Michael
Published: March 2003

By interaction cloning (yeast two‐hybrid system) using the catalytic domain of protein kinase Cζ (PKCζ) as bait, we cloned a human full‐length cDNA with 62% nucleotide homology to the A6 protein recently cloned and characterized by Beeler et al. [Beeler, J.F., LaRochelle, W.J., Chedid, M., Tronick, S.R. & Aaronson, S. A. (1994) Mol. Cell. Biol.14, 982–988]. The deduced amino acid sequence (349 amino acids) of the A6‐related protein (A6rp) contained potential actin‐binding sites and ATP‐binding sites. We also cloned the murine homolog of A6rp. Human A6rp was expressed in an in‐vitro transcriptional/translational system with an apparent molecular mass of 40 kDa and as a glutathione S‐transferase (GST) fusion protein in bacteria. A polyclonal anti‐(A6rp) was raised in rabbits and used for the identification of A6rp by immunoblotting. A6rp was found to be expressed at the mRNA and the protein levels in all cells and tissues investigated. GST–A6rp was phosphorylated by PKCζ but not significantly by other PKC isoenzymes. Moreover, it was phosphorylated by casein kinase 2 and most effectively by the tyrosine kinase Src. In contrast to GST–A6rp, GST–A6 was also phosphorylated by PKC isoforms other than PKCζ and strongly by CK2, but just weakly by Src. In contrast to the results of Beeler et al. on β‐galactosidase–A6, we were unable to demonstrate autokinase activity or tyrosine phosphorylation of either GST–A6 or GST–A6rp. In accordance with the potential ATP‐binding sites, both proteins were able to bind ATP.
Chemical and antigenic structure of the O‐polysaccharide of the lipopolysaccharides from two Acinetobacter haemolyticus strains differing only in the anomeric configuration of one glycosyl residue in their O‐antigens
Authors: Pantophlet Ralph , Haseley Simon R. , Vinogradov Evgeny V. , Brade Lore , Holst Otto , Brade Helmut
Published: March 2003

In a previous study [Pantophlet, R., Brade, L., Dijkshoorn, L., and Brade, H. (1998) J. Clin. Microbiol.36, 1245–1250] the O‐polysaccharide of the lipopolysaccharides (LPS) from Acinetobacter haemolyticus strains 57 and 61 exhibited indistinguishable banding‐patterns following Western blot and immunostaining with homologous or heterologous rabbit antiserum. In this report, the molecular basis for the observed cross‐reactivity was elucidated, by determining the chemical structure of the polysaccharides by compositional analysis and NMR spectroscopy. The structures are:

for strain 57, and

for strain 61 [GulpNAcA, 2‐acetamido‐2‐deoxy‐gulopyranosyluronic acid; ManpNAcA, 2‐acetamido‐2‐deoxy‐mannopyranosyluronic acid; QuipN4N, 2,4‐diamino‐2,4,6‐trideoxy‐glucopyranose; acyl (S)‐3‐hydroxybutyryl], thus, differing only in the anomeric configuration of the QuipN4N residue.

 The antigenic structures were determined by generating murine monoclonal antibodies, which were characterized by Western blot using LPS as antigen, by ELISA using LPS and de‐O‐acylated LPS as solid‐phase antigens, and by ELISA inhibition studies using LPS, polysaccharide, and de‐O‐acylated LPS as inhibitors. Of the four antibodies selected, two were specific for the respective LPS moieties and two were cross‐reactive. All antibodies were found to require the presence of the O‐acetyl group for reactivity.