CMAS and ST3GAL4 Play an Important Role in the Adsorption of Influenza Virus by Affecting the Synthesis of Sialic Acid Receptors.

Influenza A viruses (IAVs) initiate infection by attaching Hemagglutinin (HA) on the viral envelope to sialic acid (SA) receptors on the cell surface. Importantly, HA of human IAVs has a higher affinity for α-2,6-linked SA receptors, and avian strains prefer α-2,3-linked SA receptors, whereas swine strains have a strong affinity for both SA receptors. Host gene CMAS and ST3GAL4 were found to be essential for IAV attachment and entry. Loss of CMAS and ST3GAL4 hindered the synthesis of sialic acid receptors, which in turn prevented the adsorption of IAV. Further, the knockout of CMAS had an effect on the adsorption of swine, avian and human IAVs. However, ST3GAL4 knockout prevented the adsorption of swine and avian IAV and the impact on avian IAV was more distinct, whereas it had no effect on the adsorption of human IAV. Collectively, our findings demonstrate that knocking out CMAS and ST3GAL4 negatively regulated IAV replication by inhibiting the synthesis of SA receptors, which also provides new insights into the production of gene-edited animals in the future.

Synthesis of sulfone-based nucleotide isosteres: identification of CMP-sialic acid synthetase inhibitors.

A modular replacement approach to the synthesis of sulfo-nucleotide analogs prepared from condensation of nucleoside aldehydes with bis phosphonate Horner-Wadsworth-Emmons reagents is disclosed. These analogs were shown to be inhibitors of Neisseria meningitidis CSS (NmCSS), which is a key enzyme in the biosynthesis of the capsular polysaccharides required for bacterial infection.

Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase.

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12-->Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in k(cat), K(m) and K(s) for CTP, which implicates Arg-12 in catalysis and substrate binding.

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.

Partial purification and characterization of cytidine-5'-monophosphosialate synthase from rainbow trout liver.

Trout liver is a rich source of sialate cytidylyltransferase activity. Three procedures are described by which the enzyme was enriched between 67- and 647-fold with high specific activities varying between 0.67 and 1.88 U/mg protein. In the simplest procedure studied, 100,000 x g supernatant of liver homogenate was chromatographed on Q-Sepharose and beta-[3-(2-aminoethylthio)propyl]-N- acetylneuraminic acid as affinity matrix, leading to an enzyme preparation (0.67 U/mg protein) well suited for the synthesis of CMP-N-acetylneuraminic acid. The synthase has a molecular mass of 160 kDa, a temperature optimum of 28 degrees C, a pH-optimum of 9.3 and exhibits Km-values for CTP, N-acetylneuraminic acid and N-glycoloylneuraminic acid of 1.7 mM, 2.1 mM and 2.9 mM, respectively. It is inactive with N-acetyl-9-O-acetylneuraminic acid. The enzyme is inhibited by CMP, CDP and 2'-deoxy-CTP. The sialic acid fraction of trout liver after hydrolysis is composed by N-acetylneuraminic acid (86%), N-acetyl-9-O-acetylneuraminic acid (12%) and N-acetyl-9-O-lactoylneuraminic acid (2%).

Synthesis of sialic acid-containing nucleotide sugars: CMP-sialic acid analogs.

Syntheses of some sialic acid-containing nucleotide sugars are reported. The reaction of methyl [(2-hydroxy)ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto -2-nonulopyranosid]onate (4) with various fully protected hydrogen phosphonates of nucleosides (5a-c) in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl), gave, after oxidation and deprotection, the corresponding sialic acid-containing nucleotide sugar analogs (8a-c).

The phospholipid analogue hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis in Madin-Darby canine kidney cells.

The influence of the phospholipid analogue hexadecylphosphocholine on phosphatidylcholine biosynthesis was investigated in Madin-Darby canine kidney (MDCK) cells. It inhibits the incorporation rate of [methyl-3H]choline into phosphatidylcholine at a concentration of 50 microM by about 50%. The radiolabelled precursor accumulates in the phosphocholine pool indicating that hexadecylphosphocholine inhibits the formation of phosphatidylcholine via the CDP-choline pathway at the level of the rate-limiting enzyme. CTP:phosphocholine cytidylyl transferase (EC 2.7.7.15). This was verified by the determination of the activity of the enzyme in vitro. In consequence of its inhibitory effect it could be shown that the treatment of MDGK cells for 24 h with 50 microM hexadecylphosphocholine induces alterations of the phospholipid composition. Whereas in treated cells the relative phosphatidylcholine content was decreased from the control level of 36.0 +/- 0.9% to 29.9 +/- 0.2%; in contrast, the relative content of phosphatidylethanolamine was increased from 19.3 +/- 0.9% to 24.3 +/- 0.9%.

Interaction of N-acetyl-4-epi-D-neuraminic acid with key enzymes of sialic acid metabolism.

In spite of the axially orientated hydroxy group at C-4, the benzyl alpha-glycoside of N-acetyl-4-epi-D-neuraminic acid (4-epi-NeuAc) is a substrate for sialidases from Vibrio cholerae, Clostridium perfringens, and Arthrobacter ureafaciens, although to an extent which differs depending on the enzyme. Surprisingly, V. cholerae sialidase is by far the slowest acting enzyme; this is in contrast to its usual behavior. Fowl plague virus sialidase and bovine testis sialidase also cleave this glycoside slowly. 4-Epi-NeuAc is not a substrate for N-acetylneuraminic acid aldolase from C. perfringens but reversibly inhibits the enzyme with a Ki = 2.3 mM. The N-acetylneuraminic acid analogue is not converted to the corresponding CMP-glycoside by CMP-sialic acid synthase from bovine brain; however, it is an effective reversible inhibitor of the enzyme. The kinetic properties were analyzed with an assay system at pH 9 as well as an assay system at pH 7.5. The results from Dixon and Hanes plots did not agree. Therefore, no conclusions about the mechanism of the inhibition could be reached. This is the first reported sialic acid analogue which can act as an inhibitor of CMP-sialic acid synthase.