Studies on N-acetylneuraminic acid aldolase

N-Acetylneuraminic acid aldolase from Clostridium perfringens was irreversibly inactivated by 1mm-bromopyruvate with a half-life of 4.2min at pH7.2 and 37 degrees C. The rate of inactivation was diminished in the presence of pyruvate but not with N-acetyl-d-mannosamine, indicating that the inhibitor acted at, or close to, the pyruvate-binding site. The apparent K(i) for bromopyruvate, calculated from the variation of half-life with inhibitor concentration, was 0.46mm, compared with a competitive K(i) 3.0mm for pyruvate. Incubation of the enzyme with radioactive bromopyruvate gave a radioactive, enzymically inactive, protein in which the bromopyruvate had alkylated cysteine residues. Incubation of the enzyme with radioactive pyruvate, followed by reduction with sodium borohydride, led to inactivation of the enzyme and binding of the pyruvate to the protein by reduction of a Schiff's base initially formed with the in-amino group of a lysine residue; only one-twentieth as many pyruvyl residues were bound by this method, showing that bromopyruvate is not specific for the active site. After protection of the enzyme active site with pyruvate, treatment with unlabelled bromopyruvate and dialysis, the enzyme retained 72% activity. When this treated enzyme was separately incubated with radioactive bromopyruvate, or radioactive pyruvate followed by sodium borohydride, the ratio of radioactive pyruvyl residues bound by the two methods was 2.3:1. After reduction and hydrolysis of the bromopyruvate-treated enzyme, the only detectable radioactive amino acid derivative was chromatographically and electrophoretically identical with S-(3-lactic acid)-cysteine. The enzyme was fully active in the presence of EDTA and was not stimulated by bivalent metal ions. It was strongly inhibited by silver and mercuric ions. The apparent molecular weight, determined by Sephadex chromatography, was 250000. A mechanism of action is proposed for the enzyme. Bromopyruvate reacts rapidly at pH6.0 with thiol-containing amino acids. Cysteine appears to react anomalously.     

Lipase-catalyzed acetylation of N-acetylneuraminic acid derivative

A facile preparation of triacetylated derivative of 2-phenylthioglycoside of N-acetylneuraminic acid (4) was achieved by treatment with lipase PS in vinyl acetate. The major product 4 has a free hydroxyl group at C-7. Results of time-course HPLC analysis indicate that the reactivity of the hydroxyl groups under this condition is in the following order; C-9 > C-4 > C-8 > C-7.     

Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase

Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase. Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. 1H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme.     

Synthesis of N-acetylneuraminic acid and of CMP-N-acetylneuraminic acid in the rat liver cell.

Adult male rats, under starving and normal conditions, were injected intravenously with N-acetyl[3H]mannosamine and after various time intervals the specific radioactivities of free N-acetylneuraminic acid (NeuAc) and CMP-N-acetylneuraminic acid were determined in the liver. The specific radioactivity of free NeuAc was high even within 20s after injection; the maximum was reached between 7 and 10 min. The specific radioactivity of CMP-NeuAc showed a lag phase of approx. 1 min. Thereafter it increased quickly and rose above the specific radioactivity of free NeuAc, reaching a maximum about 20 min after injection. These results point to a channelling of the newly synthesized NeuAc molecules into a special compartment, from which they are preferentially used by the enzyme CMP-sialic acid synthetase. It is suggested that the cytosolic enzyme N-acetylneuraminic acid 9-phosphate phosphatase is working in concert with the nuclear localized enzyme CMP-N-acetylneuraminic acid synthetase. Incorporation of radioactive sialic acid into sialoglycoproteins in liver occurred 2 min after injection, and after 10 min bound radioactivity began to appear in the circulation, indicating a transport time of 8 min of sialoglycoproteins from the point of attachment of sialic acid to the point of excretion.     

A monoclonal antibody to free N-acetylneuraminic acid

A monoclonal antibody (70-A) to free N-acetylneuraminic acid was obtained by immunizing mice with its synthetic beta-glycoside, sodium O-[(5-acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-2- nonulopyranosyl)onate]-(2----3)-1,2-di-O-tetradecyl-sn-glyce rol, followed by fusing the isolated spleen cells with mouse myeloma cells and cloning positive fusions. 70-A reacted with various synthetic beta-glycosides of N-acetylneuraminic acid and also with cytidine-5'-monophosphate-N- acetylneuraminic acid, known as its sole naturally occurring beta-glycoside. The inhibition assay showed that N-glycolylneuraminic acid had slightly lower reactivity than N-acetylneuraminic acid, but other monosaccharides tested, such as N-acetylglucosamine, N-acetylgalactosamine or N-acetylmannosamine, had no reactivity toward 70-A. Reactivity of 70-A with free N-acetylneuraminic acid was confirmed by measuring the specific binding of N-[14C]acetylneuraminic acid to the antibody. The association constant of 70-A with N-acetylneuraminic acid was determined to be 5.96.10(4) M-1 by equilibrium dialysis.     

Thyroid-stimulating hormone binding to beef thyroid membranes. Role of N-acetylneuraminic acid.

The effect of sugars on 125I-thyroid-stimulating hormone binding to beef thyroid membranes was studied to determine their role in thyroid-stimulating hormone (TSH) binding. At 0.1 M concentration, N-acetylneuraminic acid produced a 3- to 7-fold increase in TSH binding, was the only sugar to enhance TSH binding, and did so whether binding was determined in the cyclase medium or under conditions of optimum binding. The enhanced TSH binding remained after the membranes were removed from the high NeuAc concentration and an effect was observed at concentrations of 10 mM NeuAc. NeuAc did not alter the kinetics of TSH binding but the pH optimum for TSH binding shifted from pH 5.5 to 7.5 in the presence of NeuAc. Incubation of the membranes with increasing concentrations of NeuAc resulted in increased sialic acid content of the membranes. The NeuAc concentration curve of membrane sialic acid and TSH binding were roughly parallel. The capacity of the low affinity site increased from 0.74 to 2.5 nmol/mg of protein in the presence of NeuAc. The apparent affinity (0.88 X 10(6) M-1) of this site was unaffected by NeuAc. With the high affinity site, NeuAc increased both the apparent affinity and capacity from 2.2 X 10(8)M-1 to 5.5 X 10(8) M-1 and 1.6 to 3.1 pmol/mg of protein, respectively. Neuraminidase or neuraminidase plus beta-galactosidase incubation of the membranes removed approximately 60% of the sialic acid from the membranes within 15 to 30 min but did not affect TSH binding. Large quantities of sialic acid were detected in the soluble fractions during isolation of the membranes, 4 to 5% of which was ultrafilterable and not associated with high molecular weight proteins. It is concluded that among the sugars tested, NeuAc exhibits an unique effect on TSH binding that may have physiological significance. The inability to alter TSH binding by enzymatic removal of endogenous sialic acid suggests that either NeuAc resistant to hydrolysis is sufficient to maintain TSH binding or that NeuAc important in TSH binding is removed during membrane preparation but is replaced by incubation with exogenous NeuAc.     

N-acetylneuraminic acid accumulation in a buoyant lysosomal fraction of cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured fibroblasts from control individuals and two patients affected with the infantile variant of generalized N-acetylneuraminic acid (NeuAc) storage disease were disrupted by nitrogen cavitation, and the post-nuclear supernatant fractions were subjected to subcellular fractionation on Percoll gradients. Accumulating NeuAc in affected fibroblasts (approx. 150 nmol/mg protein) co-localized with the lysosomal marker N-acetyl-beta-hexosaminidase (Hex), in a fraction with a mean density of 1.035 g/ml. In contrast, more than 70% of the Hex activity of control cells sedimented in comparable gradients with a density of more than 1.07 g/ml. The lysosomal localization of NeuAc accumulation in affected fibroblasts was confirmed by treatment of post-nuclear supernatant fractions with 0.5 mM Gly-Phe-beta-naphthylamide (20 min, 37 degrees C) prior to centrifugation, which resulted in the simultaneous loss of latency of Hex and free NeuAc, and their association with the soluble fraction on Percoll gradients. The results provide direct evidence for the accumulation of free NeuAc in a unique buoyant lysosomal fraction of affected fibroblasts.     

The influence of N-acetylneuraminic acid on the properties of human orosomucoid.

Little is known of the relationships that may exist among the three principal functionalities of glycoproteins. Orosomucoids of closely defined N-acetylneuraminic acid content were examined for evidence of influence of N-acetylneuraminic acid content on the physical properties of the glycoprotein. Fluorescence spectroscopy gave no indication of conformational change in the protein core upon desialylation. Small changes in the chromatographic partition coefficient, sigma, and thermal stability, Td, are interpreted to reflect loss of water of hydration and increased glycan stem-protein interaction without a major repositioning of the chains. Ligand-binding measurements indicate no alteration in the hydrophobic binding domain and a possible interaction between chlorpromazine and N-acetylneuraminic acid. All changes seen are progressive and occur through a region where changes in biological activity are not found. It is suggested that the dependence of biological activity on N-acetylneuraminic acid content in orosomucoid reflects, not coupled changes in protein conformation, but a charge-density-related interaction such that, below a contribution of four or five N-acetylneuraminic acid residues, activity is modified.     

Distribution of free N-acetylneuraminic acid in rat organs

The concentration of free N-acetylneuraminic acid in various rat organs was estimated by gas chromatography/mass spectrometry. Its concentration was in the range of 3.95 to 104.72 micrograms/g wet tissues, being particularly high in the endocrine glands. The ratio of free N-acetylneuraminic acid to total N-acetylneuraminic acid varied from 0.031 to 0.183, being especially high in the adrenal gland (0.181) and heart (0.183).     

Biosynthesis of sialylated lipooligosaccharides in Haemophilus ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using N-acylmannosamine analogues, N-glycolylneuraminic acid, and 13C-labeled N-acetylneuraminic acid

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.     

Cell surface changes accompanying viral transformation: N-acetylneuraminic acid ectotransferase system activity.

The activity of the sialyl ectotransferase system of normal chick embryo fibroblasts (CEF) and chick embryo fibroblasts transformed with the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) have been compared. Neuraminidase treatment of the intact cells increased the sialyl ectotransferase system activity of control and transformed cells two to three times. The ectotransferase system activity increased as the pH was decreased from 7.8 to 6.0. The temperature optimum for both systems was 40 degrees C. Approximately 60% of the 14C-sialic acid incorporated at pH 6.5 or above could be removed with neuraminidase. The activity of the transformed cell system with or without neuraminidase treatment was more stimulated by addition of Mn2+ ions, particularly above pH 7.0. This difference in ion sensitivity indicates that a different cell surface phenomenon is being studied after transformation.