Differential localisation of nPKC delta during cell cycle progression

nPKC delta is a phospholipid-dependent and calcium-independent PKC isoform, whose over expression in BL6T murine melanoma cells, modifies their proliferative and metastatic potential in vivo. We focus here on the possible relationship between the subcellular localisation of nPKC delta and distinct phase of the cell cycle. Our findings show a dynamic localisation of nPKC delta in dependence of the phase of the cell cycle. Actually, this isoform is preferentially localised to the cytoplasm in serum-starved cells, shifting to the nucleus during the S-phase and becoming peri-nuclear, associated to the Golgi apparatus, in G2-M phase. Therefore, taken together our findings demonstrate that the subcellular localisation of nPKC delta changes dynamically during the cell cycle in dependence of the requirement of the enzyme at a particular place of the cell.     

Nuclear localization signal of murine CMP-Neu5Ac synthetase includes residues required for both nuclear targeting and enzymatic activity

5-N-Acetylneuraminic acid (Neu5Ac) is the major sialic acid derivative found in animal cells. As a component of cell surface glycoconjugates, Neu5Ac is pivotal to numerous cellular recognition and communication processes including host-parasite interactions. A prerequisite for the synthesis of sialylated glycoconjugates is the activation of Neu5Ac to cytidine-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). The reaction is catalyzed by CMP-Neu5Ac-synthetase (syn), which, for unknown reasons, resides in the nucleus. Sequence analysis of the cloned murine CMP-Neu5Ac synthetase identified three clusters of basic amino acids (BC1-BC3) that might function as nuclear localization signals (NLS). In the present study chimeric protein and mutagenesis strategies were used to show that BC1 and BC2 are active NLS sequences when attached to the green fluorescent protein (enhanced GFP), but only BC2 is necessary and sufficient to mediate the nuclear import of CMP-Neu5Ac synthetase. Site-directed mutations identified the residues K(198)RXR to be essential for nuclear transport and Arg(202) to be necessary to complete the transport process. Cytoplasmic forms of CMP-Neu5Ac synthetase generated by single site mutations in BC2 demonstrated that (i) enzyme activity is independent of nuclear localization, and (ii) Arg(199) and Arg(202) are involved in both nuclear transport and synthetase activity. Comparison of all known and predicted CMP-sialic acid synthetases reveals Arg(202) and Gln(203) as highly conserved in evolution and critically important for optimal synthetase activity but not for nuclear localization. Combined, the data demonstrate that nuclear transport and enzyme activity are independent functions that share some common amino acid requirements in CMP-Neu5Ac synthetase.     

Identification and biochemical characterization of two functional CMP-sialic acid synthetases in Danio rerio

Sialic acids (Sia) form the nonreducing end of the bulk of cell surface-expressed glycoconjugates. They are, therefore, major elements in intercellular communication processes. The addition of Sia to glycoconjugates requires metabolic activation to CMP-Sia, catalyzed by CMP-Sia synthetase (CMAS). This highly conserved enzyme is located in the cell nucleus in all vertebrates investigated to date, but its nuclear function remains elusive. Here, we describe the identification and characterization of two Cmas enzymes in Danio rerio (dreCmas), one of which is exclusively localized in the cytosol. We show that the two cmas genes most likely originated from the third whole genome duplication, which occurred at the base of teleost radiation. cmas paralogues were maintained in fishes of the Otocephala clade, whereas one copy got subsequently lost in Euteleostei (e.g. rainbow trout). In zebrafish, the two genes exhibited a distinct spatial expression pattern. The products of these genes (dreCmas1 and dreCmas2) diverged not only with respect to subcellular localization but also in substrate specificity. Nuclear dreCmas1 favored N-acetylneuraminic acid, whereas the cytosolic dreCmas2 showed highest affinity for 5-deamino-neuraminic acid. The subcellular localization was confirmed for the endogenous enzymes in fractionated zebrafish lysates. Nuclear entry of dreCmas1 was mediated by a bipartite nuclear localization signal, which seemed irrelevant for other enzymatic functions. With the current demonstration that in zebrafish two subfunctionalized cmas paralogues co-exist, we introduce a novel and unique model to detail the roles that CMAS has in the nucleus and in the sialylation pathways of animal cells.     

CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives.

The gene encoding CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) in Escherichia coli serotype O7 K1 was isolated and overexpressed in E.coli W3110. Maximum expression of 8-10% of the soluble E.coli protein was achieved by placing the gene with an engineered 5'-terminus and Shine-Dalgarno sequence into a pKK223 vector derivative behind the tac promoter. The overexpressed synthetase was purified to greater than 95% homogeneity in a single step by chromatography on high titre Orange A Matrex dye resin. Enzyme purified by this method was used directly for the synthesis of CMP-NeuAc and derivatives. The enzymatic synthesis of CMP-NeuAc was carried out on a multigram scale using equimolar CTP and N-acetylneuraminic acid as substrates. The resultant CMP-NeuAc, isolated as its disodium salt by ethanol precipitation, was prepared in an overall yield of 94% and was judged to be greater than 95% pure by 1H NMR analysis. N-Carbomethoxyneuraminic acid and N-carbobenzyloxyneuraminic acid were also found to be substrates of the enzyme; 5-azidoneuraminic acid was not a substrate of the enzyme. N-Carbomethoxyneuraminic acid was coupled to CMP at a rate similar to that observed with NeuAc, whereas N-carbobenzyloxyneuraminic acid was coupled greater than 100-fold more slowly. The high level of expression achieved with the E.coli synthetase, together with the high degree of purity readily obtainable from crude cell extracts, make the recombinant bacterial enzyme the preferred catalyst for the enzymatic synthesis of CMP-N-acetylneuraminic acid.     

Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene

The Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase gene is located on a 3.3-kilobase (kb) HindIII fragment of the plasmid pSR23 which contains the genes for K1 capsule production (Vann, W. F., Silver, R. P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C. W., Lindner, W., and Kotsatos, M. (1987) J. Biol. Chem. 262, 17556-17562). The CMP-NeuAc synthetase gene expression was increased 10-30-fold by cloning of a 2.7-kb EcoRI-HindIII fragment onto the vector pKK223-3 containing the tac promoter. The complete nucleotide sequence of the gene encoding CMP-NeuAc synthetase was determined from progressive deletions generated by selective digestion of M13 clones containing the 2.7-kb fragment. CMP-NeuAc synthetase is located near the EcoRI site on this fragment as indicated by the detection of an open reading frame encoding a 49,000-dalton polypeptide. The amino- and carboxyl-terminal sequences of the encoded protein were confirmed by sequencing of peptides cleaved from both ends of the purified enzyme. The nucleotide deduced amino acid sequence was confirmed by sequencing several tryptic peptides of purified enzyme. The molecular weight is consistent with that determined from sodium dodecyl sulfate-gel electrophoresis. Gel filtration and ultracentrifugation experiments under nondenaturing conditions suggest that the enzyme is active as a 49,000-dalton monomer but may form aggregates.     

Novel method for enzymatic synthesis of CMP-NeuAc

A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67% yield.     

Cloning and Overexpression of a Tagged CMP-N-Acetylneuraminic Acid Synthetase from E. coli Using a Lambda Phage System and Application of the Enzyme to the Synthesis of CMP-N-Acetylneuraminic Acid

The gene coding from CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (Ec 2.7.7.43) was amplified from total DNA of E. coli strain K-235 through a primer-directed polymerase chain reaction. The gene was fused with a modified ribosome binding site of the original CMP-NeuAc synthetase gene and a decapeptide tag sequence which served as a marker for screening of expressed proteins. The gene was cloned into lambda ZAP vector at EcoRI and XbaI sites and overexpressed in E. coli Sure at a level approximately 1000 times that of the wild type. The decapeptide-containing enzyme retained almost the same specificity as indicated by the V_max and K_m values using CTP and NeuAc as substrates. A preparative synthesis of CMP-NeuAc based on the recombinant enzyme was demonstrated.     

大肠杆菌CMP-唾液酸合成酶最小活性域的研究

比较大肠杆菌与脑膜炎奈瑟氏球菌的CMP－唾液酸合成酶的氨基酸序列,发现大肠杆菌CMP－唾液酸合成酶的保守区域主要位于N－端,其C－末端似乎对其催化活性没有作用。通过PCR方法,对大肠杆菌CMP－唾液酸合成酶的C－末端进行了一系列截短,将得到的产物连接至表达载体pET-15b中,在大肠杆菌BL21（DE3）pLysS中表达。经IPTG诱导,发现从C－末端截去189个氨基酸酶仍有催化活性,说明大肠杆菌CMP－唾液酸合成酶的最小活性域主要集中在N－不端的229个氨基酸。在催化活性的C－端缺失突变合成酶的比活,最适pH及热稳定性发生变化,提示被截去的C－端氨基酸残基虽不直接参与构成酶的催化活性中心,但可影响催化活性域的构象,从而对酶的催化活性与稳定性产生影响。     

CMP-N-acetylneuraminic acid synthetase from Escherichia coli K1 is a bifunctional enzyme: identification of minimal catalytic domain for synthetase activity and novel functional domain for platelet-activating factor acetylhydrolase activity

Escherichia coli CMP-NeuAc synthetase (EC 2.7.7.43) catalyzes the synthesis of CMP-NeuAc from CTP and NeuAc, which is essential for the formation of capsule polysialylate for strain K1. Alignment of the amino acid sequence of E. coli CMP-NeuAc synthetase with those from other bacterial species revealed that the conserved motifs were located in its N termini, whereas the C terminus appeared to be redundant. Based on this information, a series of deletions from the 3'-end of the CMPNeuAc synthetase coding region was constructed and expressed in E. coli. As a result, the catalytic domain required for CMP-NeuAc synthetase was found to be in the N-terminal half consisting of amino acids 1-229. Using the strategy of tertiary structure prediction based on the homologous search of the secondary structure, the C-terminal half was recognized as an alpha1-subunit of bovine brain platelet-activating factor acetylhydrolase isoform I. The biochemical analyses showed that the C-terminal half consisting of amino acids 228-418 exhibited platelet-activating factor acetylhydrolase activity. The enzyme properties and substrate specificity were similar to that of bovine brain alpha1-subunit. Although its physiological function is still unclear, it has been proposed that the alpha1-subunit-like domain of E. coli may be involved in the traversal of the blood-brain barrier.     

Structure of a sialic acid-activating synthetase, CMP-acylneuraminate synthetase in the presence and absence of CDP

The x-ray crystallographic structure of selenomethionyl cytosine-5'-monophosphate-acylneuraminate synthetase (CMP-NeuAc synthetase) from Neisseria meningitidis has been determined at 2.0-A resolution using multiple-wavelength anomalous dispersion phasing, and a second structure, in the presence of the substrate analogue CDP, has been determined at 2.2-A resolution by molecular replacement. This work identifies the active site residues for this class of enzyme for the first time. The detailed interactions between the enzyme and CDP within the mononucleotide-binding pocket are directly observed, and the acylneuraminate-binding pocket has also been identified. A model of acylneuraminate bound to CMP-NeuAc synthetase has been constructed and provides a structural basis for understanding the mechanism of production of "activated" sialic acids. Sialic acids are key saccharide components on the surface of mammalian cells and can be virulence factors in a variety of bacterial species (e.g. Neisseria, Haemophilus, group B streptococci, etc.). As such, the identification of the bacterial CMP-NeuAc synthetase active site can serve as a starting point for rational drug design strategies.     

Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid. We have purified CMP-NeuAc synthetase from an Escherichia coli O18:K1 cytoplasmic fraction to apparent homogeneity by ion exchange chromatography and affinity chromatography on CDP-ethanolamine linked to agarose. The enzyme has a specific activity of 2.1 mumol/mg/min and migrates as a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis. The enzyme has a requirement for Mg2+ or Mn2+ and exhibits optimal activity between pH 9.0 and 10. The apparent Michaelis constants for the CTP and NeuAc are 0.31 and 4 mM, respectively. The CTP analogues 5-mercuri-CTP and CTP-2',3'-dialdehyde are inhibitors. The purified CMP-N-acetylneuraminic acid synthetase has a molecular weight of approximately 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding CMP-N-acetylneuraminic acid synthetase is located on a 3.3-kilobase HindIII fragment. The purified enzyme appears to be identical to the 50,000 Mr polypeptide encoded by this gene based on insertion mutations that result in the loss of detectable enzymatic activity. The amino-terminal sequence of the purified protein was used to locate the start codon for the CMP-NeuAc synthetase gene. Both the enzyme and the 50,000 Mr polypeptide have the same NH2-terminal amino acid sequence. Antibodies prepared to a peptide derived from the NH2-terminal amino acid sequence bind to purified CMP-NeuAc synthetase.     

Investigation of the kinetic mechanism of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi with new insights on rate-limiting steps from product inhibition analysis.

The presence of sialic acid as a component of cell surface lipooligosaccharides or capsular polysaccharides has been shown to be correlated with the virulence of a number of Gram-negative mucosal pathogens, including several Haemophilus and Neisseria spp. As part of our efforts to evaluate the role of sialic acid in the pathobiology of these organisms, we have initiated studies of the enzymes from Haemophilus ducreyi (the infectious agent of chancroid) responsible for the activation and attachment of sialic acid to the lipooligosaccharide. In this report, we describe results of an investigation of the steady-state kinetic mechanism of the activating enzyme, cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase. Using a combination of initial velocity, product inhibition, and dead-end inhibition studies, the reaction is shown to be freely reversible and to proceed through an ordered bi-bi kinetic mechanism in which CTP binds first and CMP-NeuAc dissociates last. In addition, a detailed analysis of the kinetic expressions for the observable constants is presented showing how the variation in apparent product inhibition constants (Kii) can be used to predict the rate-limiting step in kcat, which appears to be dissociation of CMP-NeuAc in this enzyme. To our knowledge, this relationship has not been previously recognized.     

Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.

Sulfation is a major modification of many molecules in eukaryotes that is dependent on the enzymatic synthesis of an activated sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). While sulfate activation has long been assumed to occur in the cytosol, we show in this study that human PAPS synthetase 1 (PAPSS1), a bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase enzyme sufficient for PAPS synthesis, accumulates in the nucleus of mammalian cells. Nuclear targeting of the enzyme is mediated by its APS kinase domain and requires a catalytically dispensable 21 amino acid sequence at the amino terminus. Human PAPSS1 and Drosophila melanogaster PAPSS localize to the nucleus in yeast and relieve the methionine auxotrophy of ATP sulfurylase- or APS kinase-deficient strains, suggesting that PAPSS1 is fully functional in vivo when targeted to the nucleus. A second PAPS synthetase gene, designated PAPSS2, has recently been described, mutations of which are responsible for abnormal skeletal development in human spondyloepimetaphyseal dysplasia and murine brachymorphism. We found that PAPSS2, which localizes to the cytoplasm when ectopically expressed in mammalian cells, is relocated to the nucleus when coexpressed with PAPSS1. Taken together, these results indicate that a sulfation pathway might exist in the nucleus of eukaryotic cells. -Besset, S., Vincourt, J.-B., Amalric, F., Girard, J.-P. Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.     

Enzymatic synthesis of cytidine 5'-monophospho-N-acetylneuraminic acid

We have established an efficient method for enzymatic production of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) from inexpensive materials, N-acetylglucosamine (GlcNAc) and cytidine 5'-monophosphate (CMP). The Haemophilus influenzae nanE gene encoding GlcNAc 6-phosphate (GlcNAc 6-P) 2-epimerase and the Campylobacter jejuni neuB1 gene encoding N-acetylneuraminic acid (NeuAc) synthetase, both of whose products are involved in NeuAc biosynthesis, were cloned and co-expressed in Escherichia coli cells. We examined the synthesis of NeuAc from GlcNAc via GlcNAc 6-P, N-acetylmannosamine (ManNAc) 6-P, and ManNAc by the use of E. coli cells producing GlcNAc 6-P 2-epimerase and NeuAc synthetase, in expectation of biological functions of E. coli such as the supply of phosphoenolpyruvate (PEP), which is an essential substrate for NeuAc synthetase, GlcNAc phospholylation by the PEP-dependent phosphotransferase system, and dephospholylation of ManNAc 6-P. Eleven mM NeuAc was synthesized from 50 mM GlcNAc by recombinant E. coli cells with the addition of glucose as an energy source. Next we attempted to synthesize CMP-NeuAc from GlcNAc and CMP using yeast cells, recombinant E. coli cells, and H. influenzae CMP-NeuAc synthetase, and succeeded in efficient production of CMP-NeuAc due to a sufficient supply of PEP and efficient conversion of CMP to cytidine 5'-triphosphate by yeast cells.     

Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase.

Glucokinase (GK), a key enzyme in the glucose homeostatic responses of the liver, changes its intracellular localisation depending on the metabolic status of the cell. Rat liver GK and Xenopus laevis GK, fused to the green fluorescent protein (GFP), concentrated in the nucleus of cultured rat hepatocytes at low glucose and translocated to the cytoplasm at high glucose. Three mutant forms of Xenopus GK with reduced affinity for GK regulatory protein (GKRP) did not concentrate in the hepatocyte nuclei, even at low glucose. In COS-1 and HeLa cells, a blue fluorescent protein (BFP)-tagged version of rat liver GK was only able to accumulate in the nucleus when it was co-expressed with GKRP-GFP. At low glucose, both proteins concentrated in the nuclear compartment and at high glucose, BFP-GK translocated to the cytosol while GKRP-GFP remained in the nucleus. These findings indicate that the presence of and binding to GKRP are necessary and sufficient for the proper intracellular localisation of GK and directly involve GKRP in the control of the GK subcellular distribution.     

De novo sn-glycerol-3-phosphorylcholine synthetase activity in lung and muscle and its subcellular location

The activity of glycerophosphorylcholine synthetase, a newly discovered enzyme involved in the synthesis of acyl-specific phosphatidylcholines, is reported in rat lung and muscle. Its subcellular location appears to be mitochondrial. The implication of these findings in the synthesis of lung surfactant and the pathology of muscular dystrophy are discussed.     

Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.     

Molecular determinants of the yeast Arc1p-aminoacyl-tRNA synthetase complex assembly

Eukaryotic aminoacyl-tRNA synthetases are usually organized into high-molecular-weight complexes, the structure and function of which are poorly understood. We have previously described a yeast complex containing two aminoacyl-tRNA synthetases, methionyl-tRNA synthetase and glutamyl-tRNA synthetase, and one noncatalytic protein, Arc1p, which can stimulate the catalytic efficiency of the two synthetases. To understand the complex assembly mechanism and its relevance to the function of its components, we have generated specific mutations in residues predicted by a recent structural model to be located at the interaction interfaces of the N-terminal domains of all three proteins. Recombinant wild-type or mutant forms of the proteins, as well as the isolated N-terminal domains of the two synthetases, were overexpressed in bacteria, purified and used for complex formation in vitro and for determination of binding affinities using surface plasmon resonance. Moreover, mutant proteins were expressed as PtA or green fluorescent protein fusion polypeptides in yeast strains lacking the endogenous proteins in order to monitor in vivo complex assembly and their subcellular localization. Our results show that the assembly of the Arc1p-synthetase complex is mediated exclusively by the N-terminal domains of the synthetases and that the two enzymes bind to largely independent sites on Arc1p. Analysis of single-amino-acid substitutions identified residues that are directly involved in the formation of the complex in yeast cells and suggested that complex assembly is mediated predominantly by van der Waals and hydrophobic interactions, rather than by electrostatic forces. Furthermore, mutations that abolish the interaction of methionyl-tRNA synthetase with Arc1p cause entry of the enzyme into the nucleus, proving that complex association regulates its subcellular distribution. The relevance of these findings to the evolution and function of the multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases is discussed.