Monophosphate N-acetylneuraminic Acid Synthetase Research Articles

Unraveling the impact of sialic acids on the immune landscape and immunotherapy efficacy in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Despite the successful application of immune checkpoint blockade in a range of human cancers, immunotherapy in PDAC remains unsuccessful. PDAC...

Cytidine monophosphate N-acetylneuraminic acid synthetase enhances invasion of human triple-negative breast cancer cells.

BackgroundCancer cells have altered bioenergetics, which contributes to their ability to proliferate, survive in unusual microenvironments, and invade other tissues. Changes in glucose metabolism can have pleomorphic effects on tumor cells.MethodsTo investigate potential mechanisms responsible for the increased malignancy associated with altered glucose metabolism, we used an unbiased nuclear magnetic resonance spectroscopy screening method to identify glucose metabolites differentially produced in a highly malignant human triple-negative breast cancer (TNBC) cell line (BPLER) and a less malignant isogenic TNBC cell line (HMLER).ResultsN-acetylneuraminic acid (Neu5Ac), the predominant sialic acid derivative in mammalian cells, which forms the terminal sugar on mucinous cell surface glycoproteins, was the major glucose metabolite that differed. Neu5Ac was ~7-fold more abundant in BPLER than HMLER. Loss of Neu5Ac by enzymatic removal or siRNA knockdown of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS), which activates cellular sialic acids for glycoprotein conjugation, had no significant effect on cell proliferation, but decreased the ability of BPLER to invade through a basement membrane. Conversely, overexpressing CMAS in HMLER increased invasivity. TNBCs in The Cancer Genome Atlas also had significantly more CMAS copy number variations and higher mRNA expression than non-TNBC, which have a better prognosis. CMAS knockdown in BPLER ex vivo blocked xenograft formation in mice.ConclusionNeu5Ac is selectively highly enriched in aggressive TNBC, and CMAS, the enzyme required for sialylation, may play an important role in TNBC tumor formation and invasivity.

Molecular cloning and bioinformatic analysis of the Streptococcus agalactiae neuA gene isolated from tilapia.

Cytidine monophosphate (CMP) N-acetylneuraminic acid (NeuNAc) synthetase, which is encoded by the neuA gene, can catalyze the activation of sialic acid with CMP, and plays an important role in Streptococcus agalactiae infection pathogenesis. To study the structure and function of the S. agalactiae neuA gene, we isolated it from diseased tilapia, amplified it using polymerase chain reaction (PCR) with specific primers, and cloned it into a pMD19-T vector. The recombinant plasmid was confirmed by PCR and restriction enzyme digestion, and identified by sequencing. Molecular characterization analyses of the neuA nucleotide amino acid sequence were performed using bioinformatic tools and an online server. The results showed that the neuA nucleotide sequence contained a complete coding region, which comprised 1242 bp, encoding 413 amino acids (aa). The aa sequence was highly conserved and contained a Glyco_tranf_GTA_type superfamily and an SGNH_hydrolase superfamily conserved domain, which are related to sialic acid activation catalysis. The NeuA protein possessed many important sites related to post-translational modification, including 28 potential phosphorylation sites and 2 potential N-glycosylation sites, had no signal peptides or transmembrane regions, and was predicted to reside in the cytoplasm. Moreover, the protein had some B-cell epitopes, which suggests its potential in development of a vaccine against S. agalactiae infection. The codon usage frequency of neuA differed greatly in Escherichia coli and Homo sapiens genes, and neuA may be more efficiently expressed in eukaryotes (yeast). S. agalactiae neuA from tilapia maintains high structural homology and sequence identity with CMP-NeuNAc synthetases from other bacteria.

Characterization of a Bifunctional Cytidine 5′-Monophosphate N-acetylneuraminic acid Synthetase Cloned from Streptococcus Agalactiae

Recombinant CMP-sialic acid synthetase, cloned from Streptococcus agalactiae serotype V strain 2603 V/R, is bifunctional having both CMP-sialic acid synthetase and acetylhydrolase (acylesterase) activities. The enzyme is active over a wide pH range with an optimal CMP-sialic acid synthetase activity at pH 9.0 and an optimal acetylhydrolase activity at pH 8.0. A metal cofactor (either Mg(2+) or Mn(2+)) is required for the CMP-sialic acid synthetase activity but is not for acetylhydrolase activity. Both catalytic functions, however, are impaired by high concentrations of Mn(2+).

Cloning and overexpression of a cytidine 5′-monophosphate N-acetylneuraminic acid synthetase from Escherichia coli

The gene for cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC2.7.7.43) was amplified from the total DNA of Escherichia coli 44277 through a primer-directed polymerase chain reaction and cloned into pUC19 between the sites EcoRI and SalI. The sequence analysis showed the cloned DNA was the same as the published neuA except for a codon missing for Asp230 and four base substitutions at the sites 153, 438, 618, and 687. The four base substitutions did not change the amino acid sequence. Based on the preliminary expression experiment and computer-aided analysis of the gene structure, six bases were mutated in order to achieve high-level expression. The gene mutated by PCR was cloned into the expression vector pET15b. Upon the induction of IPTG, the recombinant enzyme was shown to be 26% of the total bacterial proteins, which was 850-fold higher than that of the wild strain. Under the optimal inducing condition, the enzyme yield reached 100 U/l cell culture. Using the partially purified recombinant enzyme, the synthesis of CMP-NeuAc was carried out in 90% yield.

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.

Covalent modification of Lys19 in the CTP binding site of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.

Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min(-1) and a K(I) of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with beta-elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The beta-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP. This compound also formed a stable reduced morpholino adduct with CMP-NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its beta-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the beta-elimination product of oCTP.

Identification, expression and tissue distribution of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase activity in the rat.

We report the postnatal developmental profiles of N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase) in different rat tissues. This enzyme, which catalyses the activation of NeuAc to CMP-Neu5Ac, was detected in brain, kidney, heart, spleen, liver, stomach, intestine, lung, thymus, prostate and urinary bladder but not in skeletal muscle. Comparative analysis of the different specific activity profiles obtained shows that the expression of CMP Neu5Ac synthetase is tissue-dependent and does not seem to be embryologically determined. Changes in the level of sialylation during development were also found to be intimately related to variations in the expression of this enzyme, at least in brain, heart, kidney, stomach, intestine and lung.

Purification, Cloning, and Expression of a Cytidine 5′-Monophosphate N-Acetylneuraminic Acid Synthetase from Haemophilus ducreyi

An N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) was isolated from a Haemophilus ducreyi strain 35000 cell lysate and partially characterized. The enzyme catalyzes the reaction of CTP and NeuAc to form CMP-NeuAc, which is the nucleotide sugar donor used by sialyltransferases. Previous studies have shown that the outer membrane lipooligosaccharides of H. ducreyi contain terminal sialic acid attached to N-acetyllactosamine and that this modification is likely important to its pathogenesis. Therefore, to investigate the role of sialic acid in H. ducreyi pathogenesis, the gene encoding the CMP-NeuAc synthetase was cloned using degenerate oligonucleotide probes derived from NH2-terminal sequence data, and the nucleotide sequence was determined. The derived amino acid sequence of the CMP-NeuAc synthetase gene has homology to other CMP-NeuAc synthetases and to a lesser extent to CMP-2-keto-3-deoxy-D-manno-octulosonic acid synthetases. The gene was cloned into a T7 expression vector, the protein expressed in Escherichia coli, and purified to apparent homogeneity by anion exchange, Green 19 dye, and hydrophobic interaction chromatography. The final step yielded 20 mg of pure protein/liter of culture. The protein has a predicted molecular mass of 25440.6 Da, which was confirmed by electrospray mass spectrometry (Mexpt = 25439.9 +/- 1.4 Da). The enzyme appears to exist as a dimer by size exclusion chromatography. In contrast to other bacterial CMP-NeuAc synthetases, the H. ducreyi enzyme exhibited a different substrate specificity, being capable of also using N-glycolylneuraminic acid as a substrate.

Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver.

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.

Regulation of colominic acid biosynthesis by temperature: Role of cytidine 5′-monophosphate N-acetylneuraminic acid synthetase

Synthesis of colominic acid in Escherichia coli K-235 is strictly regulated by temperature. Evidence for the role of cytidine 5′-monophospho- N-acetylneuraminic acid (CMP-Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37°C. No activity was found in E. coli grown at 20°C. CMP-Neu5Ac started to be quickly synthesized when bacteria grown at 20°C were transferred to 37°C and was halted when cells grown at 37°C were transferred to 20°C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP-Neu5Ac necessary for the biosynthesis of colominic acid.

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.

Effects of essential fatty acid deficiency during late gestation on brain N-acetylneuraminic acid metabolism and behaviour in the progeny.

1. Rat dams given a diet containing 100 g maize oil/kg for approximately two weeks before mating and during the first 14 d of gestation, were given the same diet or one containing 100 g hydrogenated coconut oil/kg (essential fatty acid (EFA)-deficient) in place of maize oil until parturition. After parturition the dams were given the same diets and all progeny were weaned to the maize oil diet at 21 d of age. Brain N-acetylneuraminic acid (NeuNAc) content as well as neuraminidase (sialidase; (EC 3.2.1.18), and cytidine monophosphate N-acetylneuraminic acid synthetase (CMP-NeuNAc synthetase) activities were measured at days, 7, 14, 21 and 168 in the progeny. Y-maze learning was measured at 168 d. 2. Brain weight was independent of dietary fat at all ages. 3. Lack of EFA in the maternal diet during gestation and lactation depressed ganglioside and glycoprotein NeuNAc levels and the activities of sialidase and CMP-NeuNAc synthetase. 4. Maternal dietary deprivation of EFA irreversibly impaired learning behaviour of the progeny. A relationship exists between early exposure to EFA deficiency and learning potential of the progeny.