bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels
Authors: CHITLARU Theodor , KRONMAN Chanoch , ZEEVI Menachem , KAM Michal , HAREL Adrian , ORDENTLICH Arie , VELAN Baruch , SHAFFERMAN Avigdor 
Published: August 2015

Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver β-galactoside α-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t½β, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.
Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI–BchD complex
Authors: GIBSON Lucien C. D. , JENSEN Poul Erik , HUNTER C. Neil 
Published: August 2015

The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses the insertion of Mg into protoporphyrin IX, the first committed step in (bacterio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchDand bchH genes. These genes have been expressed in Escherichia coli so that the BchI, BchD and BchH proteins are produced with N-terminal His6 affinity tags, which has led to the production of large amounts of highly purified, highly active Mg chelatase subunits from a single chromatography step. Furthermore, BchD has been purifed free of contamination with the chaperone GroEL, which had proven to be a problem in the past. BchD, present largely as an insoluble protein in E. coli, was purified in 6 M urea and refolded by addition of BchI, MgCl2 and ATP, yielding highly active protein. BchI/BchD mixtures prepared in this way were used in conjunction with BchH to determine the kinetic parameters of R. sphaeroides Mg chelatase for its natural substrates. We have been able to demonstrate for the first time that BchI and BchD form a complex, and that Mg2+ and ATP are required to establish and maintain this complex. Gel filtration data suggest that BchI and BchD form a complex of molecular mass 200 kDa in the presence of Mg2+ and ATP. Our data suggest that, in vivo, BchD is only folded correctly and maintained in its correct conformation in the presence of BchI, Mg2+ and ATP.
Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 Å resolution
Authors: VARROT Annabelle , HASTRUP Sven , SCHÜLEIN Martin , DAVIES Gideon J. 
Published: August 2015

The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 Å. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reeseiequivalent. The structure features both O- (α-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme.
Co-ordinates for the structure described in this paper have been deposited with the Brookhaven Protein Data Bank with accession code 1BVW.PDB.
Isolation of pig mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene promoter: characterization of a peroxisome proliferator-responsive element
Authors: ORTIZ José A. , MALLOLAS Judith , NICOT Carine , BOFARULL Josep , RODRÍGUEZ Joan C. , HEGARDT Fausto G. , HARO Diego , MARRERO Pedro F. 
Published: August 2015

Low expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene during development correlates with an unusually low hepatic ketogenic capacity and lack of hyperketonaemia in piglets. Here we report the isolation and characterization of the 5´ end of the pig mitochondrial HMG-CoA synthase gene. The 581 bp region proximal to the transcription start site permits transcription of a reporter gene, confirming the function of the promoter. The pig mitochondrial HMG-CoA synthase promoter is trans-activated by the peroxisomal proliferator-activated receptor (PPAR), and a functional response element for PPAR (PPRE) has been localized in the promoter region. Pig PPRE is constituted by an imperfect direct repeat (DR-1) and a downstream sequence, both of which are needed to confer PPAR-sensitivity to a thymidine kinase promoter and to form complexes with PPAR·retinoid X receptor heterodimers. A role of PPAR trans-activation in starvation-associated induction of gene expression is suggested.
Epidermal growth factor increases the level of the cyclin-dependent kinase (CDK) inhibitor p21/CIP1 (CDK-interacting protein 1) in A431 cells by increasing the half-lives of the p21/CIP1 transcript and the p21/CIP1 protein
Authors: JOHANNESSEN Lene E. , KNARDAL Sigrun L. , MADSHUS Inger Helene 
Published: August 2015

DNA synthesis was inhibited in A431 cells by epidermal growth factor (EGF) in a p21/CIP1-dependent manner [where CIP1 is cyclin-dependent kinase (CDK)-interacting protein 1]. When 1 or 10 nM EGF was added, the level of p21/CIP1 was increased to the same extent, and the protein level peaked after approx. 5 h of incubation. The increase in p21/CIP1 mRNA upon addition of EGF was rapid, and was enhanced in the presence of cycloheximide. The half-life of p21/CIP1 mRNA in EGF-treated A431 cells was increased approx. 2-fold; this is in contrast with the case in MCF-7 cells with normal p53, in which the half-life of p21/CIP1 mRNA was not increased upon addition of EGF. This increased stability accounts for most of the increase in mRNA levels observed in A431 cells during short incubation periods. Additionally, upon prolonged incubation of A431 cells with EGF, the half-life of the protein was also increased compared with that in untreated cells and in cells treated with EGF for short time periods. Nuclear run-on assays demonstrated only marginal stimulation of transcription by 10 or 1 nM EGF, or by 10 ng/ml tumour necrosis factor α. Our results indicate that the most important mechanisms by which EGF increases p21/CIP1 protein levels in A431 cells are post-transcriptional and post-translational stabilization.
6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme
Authors: TETAUD Emmanuel , HANAU Stefania , WELLS Jeremy M. , Le PAGE Richard W. F. , ADAMS Margaret J. , ARKISON Scott , BARRETT Michael P. 
Published: August 2015

A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1.44) was identified from the homofermentative lactic acid bacterium Lactococcus lactis, by complementation of Escherichia coli mutants. The cloned gene was then expressed to high levels in E. coli and the protein purified for kinetic analysis. The enzyme had a Km for 6-phosphogluconate of 15.4±1.4 µM and for NADP of 1.9±0.2 µM at pH 7.5. Sequence comparison of the L. lactis 6-PGDH with the corresponding enzyme derived from the pathogenic protozoan Trypanosoma brucei and sheep liver revealed the substrate-binding residues to be identical in all three species, although the three coenzyme-binding pockets differed slightly. A totally conserved arginine residue (Arg-447), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan. In each case enzyme activity was lost, confirming an essential role for this residue on activity. A second arginine (Arg-34), believed to be critical in binding the 2´-phosphate of cofactor NADP+, was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis. This alteration led to decrease in affinity for NADP+ of nearly three orders of magnitude. A second 6-PGDH gene has been identified from the genome of B. subtilis. This second isoform contains an arginine (Arg-34) in this position, suggesting that B. subtilis has two 6-PGDHs with different coenzyme specificities.
A transforming mutation enhances the activity of the c-Kit soluble tyrosine kinase domain
Authors: LAM Lily P. Y. , CHOW Rowena Y. K. , BERGER Stuart A. 
Published: August 2015

An activating mutation (DY814) located in the catalytic domain of the c-Kit receptor has been found in mastocytomas from human, mouse and rat. We evaluated the enzymic properties of purified wild-type (WT) and DY814 tyrosine kinase domains expressed in Pichia pastoris. A linker encoding the Flag epitope was fused to c-Kit cDNA species, enabling affinity purification of the proteins with anti-Flag antibodies. Yeast lysates expressing DY814 contained multiple tyrosine-phosphorylated proteins, whereas WT lysates had no detectable tyrosine phosphorylation. Purification of the WT and mutant kinases in the presence of vanadate demonstrated that both enzymes undergo autophosphorylation. Kinetic analyses of WT and DY814 kinases indicated that at 20 nM enzyme concentration the mutation increases the specific activity 10-fold and decreases the apparent Km for ATP 9-fold. WT activity displayed a hyperbolic dependence on enzyme concentration, consistent with a requirement for dimerization or aggregation for activity. This activity was also enhanced by anti-Flag antibodies. In contrast, the dependence of DY814 activity on enzyme concentration was primarily linear and only marginally enhanced by anti-Flag antibodies. Gel-filtration analysis showed that the WT kinase migrated as a monomer, whereas the DY814 mutant migrated as a dimer. These results indicate that this point mutation promotes dimerization of the c-Kit kinase, potentially contributing to its transforming potential in mast cells.
Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin
Authors: MONIAUX Nicolas , NOLLET Séverine , PORCHET Nicole , DEGAND Pierre , LAINE Anne , AUBERT Jean-Pierre 
Published: August 2015

The MUC4 gene, which encodes a human epithelial mucin, is expressed in various epithelial tissues, just as well in adult as in poorly differentiated cells in the embryo and fetus. Its N-terminus and central sequences have previously been reported as comprising a 27-residue peptide signal, followed by a large domain varying in length from 3285 to 7285 amino acid residues. The present study establishes the whole coding sequence of MUC4 in which the C-terminus is 1156 amino acid residues long and shares a high degree of similarity with the rat sialomucin complex (SMC). SMC is a heterodimeric glycoprotein complex composed of mucin (ascites sialoglycoprotein 1, ASGP-1) and transmembrane (ASGP-2) subunits. The same organization is found in MUC4, where the presence of a GlyAspProHis proteolytic site may cleave the large precursor into two subunits, MUC4α and MUC4β. Like ASGP-2, which binds the receptor tyrosine kinase p185neu, MUC4β possesses two epidermal growth factor-like domains, a transmembrane sequence and a potential phosphorylated site. MUC4, the human homologue of rat SMC, may be a heterodimeric bifunctional cell-surface glycoprotein of 2.12 µm. These results confer a new biological role for MUC4 as a ligand for ErbB2 in cell signalling.
Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin
Authors: ROSTAGNO Agueda A. , SCHWARZBAUER Jean E. , GOLD Leslie I. 
Published: August 2015

Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1–COOH), expressed in COS-1 cells, and 10F1–12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction.
A novel class of protein from wheat which inhibits xylanases
Authors: McLAUCHLAN W. Russell , GARCIA-CONESA Maria T. , WILLIAMSON Gary , ROZA Martinus , RAVESTEIN Peter , MAAT Jan 
Published: August 2015

We have purified a novel class of protein that can inhibit the activity of endo-β-1,4-xylanases. The inhibitor from wheat (Triticum aestivum, var. Soisson) is a glycosylated, monomeric, basic protein with a pI of 8.7–8.9, a molecular mass of 29 kDa and a unique N-terminal sequence of AGGKTGQVTVFWGRN. We have shown that the protein can inhibit the activity of two family-11 endo-β-1,4-xylanases, a recombinant enzyme from Aspergillus niger and an enzyme from Trichoderma viride.The inhibitory activity is heat and protease sensitive. The kinetics of the inhibition have been characterized with the A. niger enzyme using soluble wheat arabinoxylan as a substrate. The Km for soluble arabinoxylan in the absence of inhibitor is 20±2 mg/ml with a kcat of 103±6 s-1. The kinetics of the inhibition of this reaction are competitive, with a Ki value of 0.35 µM, showing that the inhibitor binds at or close to the active site of free xylanase. This report describes the first isolation of a xylanase inhibitor from any organism.
Position-independent and copy-number-related expression of a goat bacterial artificial chromosome α-lactalbumin gene in transgenic mice
Authors: STINNAKRE Marie-Georges , SOULIER Solange , SCHIBLER Laurent , LEPOURRY Laurence , MERCIER Jean-Claude , VILOTTE Jean-Luc 
Published: August 2015

A bacterial artificial chromosome goat insert comprising the α-lactalbumin-encoding transcription unit with approximately 150 and 10 kb of 5´- and 3´-flanking sequences, respectively, was micro-injected into mouse eggs. In six out of seven transgenic lines, the level of mammary tissue- and stage-specific expression was position-independent and copy-number-dependent. The exogenous α-lactalbumin yield, about 0.8 mg/ml of milk per copy, compared favourably with the α-lactalbumin content of mouse and goat milks, about 0.8 and &gt; 1 mg/ml, respectively. This suggests that the insert contains most if not all of the cis-acting elements involved in the full and specific expression of the goat α-lactalbumin gene and opens up opportunities to use this vector to target expression of foreign genes in the lactating mammary gland of transgenic animals. The transgene was silent in the seventh line for an unknown reason.
Protein structure and gene cloning of Syncephalastrum racemosum nuclease
Authors: HO Heng-Chien , LIAO Ta-Hsiu 
Published: August 2015

The complete amino acid sequence of the fungus Syncephalastrum racemosum (Sr-) nuclease has been delineated on the basis of protein sequencing of the intact protein and its protease-digested peptides. The resulting 250-residue sequence shows a carbohydrate side chain attached at Asn134 and two half-cystine residues (Cys242 and Cys247) cross-linked to form a small disulphide loop. On the basis of the sequence of Sr-nuclease, a computer search in the sequence database yielded 60% and 48% positional identities with the sequences of Cunninghamella echinulata nuclease C1 and yeast mitochondria nuclease respectively, and very little similarity to those of several known mammalian DNases I. Sequence alignment of the three similar nucleases reveals that the single small disulphide loop is unchanged but the carbohydrate attachment in Sr-nuclease is absent from the other two nucleases. Alignment also shows a highly conserved region harbouring Sr-nuclease His85, which is assigned as one of the essential residues in the active site. The cDNA encoding Sr-nuclease was amplified by using reverse transcriptase-mediated PCR with degenerate primers based on its amino acid sequence. Subsequently, specific primers were synthesized for use in the 3´ and 5´ rapid amplification of cDNA ends (RACE). Direct sequencing of the RACE products led to the deduction of a 1.1 kb cDNA sequence for Sr-nuclease. The cDNA contains an open reading frame of 320 amino acid residues including a 70-residue putative signal peptide and the 250-residue mature protein. Finally, the recombinant Sr-nuclease was expressed in Escherichia coli strain BL21(DE3) in which the recombinant protein, after solubilization with detergent and renaturation, showed both DNase and RNase activities. The assignment of His85 to the active site was further supported by evidence that the mutant protein Sr-nuclease (H85A), in which His85 was replaced by Ala, was not able to degrade DNA or RNA.
Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine
Authors: HENNEBERRY Annette L. , McMASTER Christopher R. 
Published: August 2015

Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.