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PubMed1977

Distribution of neuraminidase in Arthrobacter and its purification by affinity chromatography.

Neuraminidase [sialidase, EC 3.2.1.18] was found to be widely distributed in bacteria belonging to Arthrobacter. Among these bacteria, Arthrobacter ureafaciens, A. oxydans, and A. aurescens produced relatively potent neuraminidase activities. For the production of this enzyme, not only colominic acid, a homopolymer of N-acetylneuraminic acid, but also N-acetylneuraminic acid, the reaction product of this enzyme, are effective as sources of carbon. An affinity adsorbent specific for neuraminidase was prepared by cross-linking colominic acid with soluble starch by means of epichlorohydrin. Neuraminidase from A. ureafaciens could be purified on this affinity column. The purified neuraminidase was shown to be free from protease, N-acetylneuraminic acid aldolase, phospholipase C, and glycosidases. Aminoff's assay procedure for sialic acid was modified to avoid the centrifugation step. The modified procedure gave a higher molecular extinction coefficient.

Arthrobacter↗
PubMed1976

The effect of neuraminidase on the sensitivity of tumor cells toward lysis by antibody and complement or by sensitized lymphocytes.

The effect of neuraminidase treatment on the antigenicity of sarcoma I (Sal) and mastocytoma P-815-X2 cells was compared, both in terms of their reaction with various antisera (C57BL/6 anti-SaI, C57BL/6 anti-mastocytoma, rabbit and horse ALS) and complements (guinea pig, rabbit) or with sensitized lymphocytes (C57BL/6 anti-SaI, C57BL/6 anti-mastocytoma, A anti-mastocytoma). With sensitized lymphocytes the reactivity of neuraminidase-treated tumor cells was similar to that of nontreated cells. In contrast, after neuraminidase treatment, the tumor cells showed increased specific lysis with the antisera and complement. Rabbit complement was highly cytotoxic to the neuraminidase-treated cells but this nonspecific activity could be removed by absorption with agar or agarose. The results of this study would strongly suggest that neuraminidase treatment does not change the antigenic determinants of tumor cells but increases their interaction with complement after removal of negatively charged sialic acids.

Animals↗
PubMed1985

Further studies on the role of neuraminidase and the mechanism of low pH dependence in influenza virus-induced membrane fusion.

The role of neuraminidase and the mechanism of low pH dependence in influenza virus-induced membrane fusion have been studied further using fowl plague virus (FPV, H7N1). Two specific anti-FPV neuraminidase antisera obtained from chickens immunized with recombinant virus strains inhibited viral neuraminidase activity without influencing its haemagglutinating activity. These sera totally inhibited the FPV-induced fusion of erythrocytes and partially reduced haemolysis. But both fusion and haemolysis activities could be restored by external addition of Vibrio cholerae neuraminidase, indicating participation of neuraminidase in FPV-induced membrane fusion. With regard to low pH-dependent fusion by influenza virus, it was found that erythrocytes of various species showed different pH optima for haemolysis by FPV and that erythrocytes could be sensitized for fusion and haemolysis by FPV at neutral pH if they had been pretreated with a low pH buffer. These results demonstrated that surface properties of erythrocytes rather than that of the virus are critical in the low pH-dependent fusion and haemolysis by influenza viruses.

Animals↗
PubMed1994

Calcium is needed for the thermostability of influenza B virus neuraminidase.

The activity and stability of influenza virus neuraminidase is known to depend on the presence of calcium ions. The atomic structure of the tetrameric neuraminidase head shows two distinct Ca2+ binding sites, one with low affinity on the molecular fourfold symmetry axis and one with high affinity close to the active site in each of the monomers. Here we show that Ca is essential for the thermostability of the isolated neuraminidase tetramer. Inactivation of Ca-free neuraminidase at high temperatures is accompanied by changes in protein structure leading to protease sensitivity. More than one Ca ion per tetramer is involved in stabilization, suggesting a role for the high affinity Ca binding site and the cooperative stabilization of the subunits. Sites which are located close to the fourfold axis of the neuraminidase tetramer and which are able to bind a variety of different metal ions are also described.

Calcium↗
PubMed1995

N1 neuraminidase of influenza virus A/FPV/Rostock/34 has haemadsorbing activity.

The neuraminidase (NA) gene of influenza virus A/FPV/Rostock/34 virus (H7N1) was cloned and expressed in Sf9 cells using a baculovirus vector. The expression product had the expected molecular mass and showed neuraminidase activity. NA expressed in Sf9 cells also showed haemagglutinating activity as indicated by its ability to induce haemadsorption of chicken red blood cells. Haemadsorption depended on the presence of neuraminic acid on the erythrocytes, but was not blocked by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, a specific inhibitor of the enzymatic activity. These observations suggest that the N1 neuraminidase has two distinct reactive sites: a catalytic site and a receptor binding site. This concept was confirmed by site-directed mutagenesis which showed that the haemadsorbing activity of FPV NA was abolished when amino acids located on two surface loops at the putative binding site were exchanged. Substitutions on either of the two loops were sufficient for this effect. The mutated NAs retained full neuraminidase activity. In contrast, a mutated NA lacking neuraminidase activity still had haemadsorbing activity.

Animals↗
PubMed1994

The role of neuraminidase in haemagglutination and adherence to colon WiDr cells by Bacteroides fragilis.

The role of neuraminidase in haemagglutination and adherence to colon WiDr cells by eight strains of Bacteroides fragilis and four strains of oral black-pigmented gram-negative anaerobes was studied. Neuraminidase treatment resulted in a very small increase of haemagglutination by some of the strains but had no effect on adherence to WiDr cells by all bacterial strains tested except one strain of Prevotella intermedia (HG 110). Inhibition of neuraminidase had no effect on haemagglutination or adherence, nor was any correlation found between haemagglutinating ability and neuraminidase activity in the B. fragilis strain. The results indicated that haemagglutination and adherence of B. fragilis to WiDr cells were not mediated by neuraminidase.

Bacterial Adhesion↗
PubMed2004

Molecular dynamics and free energy analysis of neuraminidase-ligand interactions.

We report molecular dynamics calculations of neuraminidase in complex with an inhibitor, 4-amino-2-deoxy-2,3-didehydro-N-acetylneuraminic acid (N-DANA), with subsequent free energy analysis of binding by using a combined molecular mechanics/continuum solvent model approach. A dynamical model of the complex containing an ionized Glu119 amino acid residue is found to be consistent with experimental data. Computational analysis indicates a major van der Waals component to the inhibitor-neuraminidase binding free energy. Based on the N-DANA/neuraminidase molecular dynamics trajectory, a perturbation methodology was used to predict the binding affinity of related neuraminidase inhibitors by using a force field/Poisson-Boltzmann potential. This approach, incorporating conformational search/local minimization schemes with distance-dependent dielectric or generalized Born solvent models, correctly identifies the most potent neuraminidase inhibitor. Mutation of the key ligand four-substituent to a hydrogen atom indicates no favorable binding free energy contribution of a hydroxyl group; conversely, cationic substituents form favorable electrostatic interactions with neuraminidase. Prospects for further development of the method as an analysis and rational design tool are discussed.

Antiviral Agents↗
PubMed1984

Expression of SSEA-I antigen (3-fucosyl-N-acetyl-lactosamine) on normal and leukaemic human haemopoietic cells: modulation by neuraminidase treatment.

Several mouse monoclonal antibodies (MoAbs) considered specific for the myeloid lineage recognize the same carbohydrate structure (3-fucosyl-N-acetyl-lactosamine) which is similar to the murine antigen SSEA-I. We have investigated the expression of this antigen with six different well-characterized murine IgM MoAbs on normal, leukaemic, and cultured cells by immunofluorescence and immunoelectron microscope cytochemistry. The cells were studied before and after neuraminidase treatment since epitopes recognized by these MoAbs may be masked by sialic acid. Among the recognizable normal marrow or blood cells, all these MoAbs specifically labelled the granulocytic lineage from the promyelocyte to the polymorph. After neuraminidase treatment, monocytes became labelled. All the other lineages remained unstained. Several cell lines were studied. Six of eight lymphoblastoid cell lines were stained by these MoABs; reactivity was increased by neuraminidase. One Burkitt cell line and two T cell lines were also found to be positive. These antibodies were tested on leukaemic cells. In acute non-lymphocytic leukaemia they usually labelled promyelocytes, more mature granulocytic and monocytic precursors but did not label myeloblasts; after neuraminidase treatment, these myeloblasts became stained. No labelling was observed on leukaemic proerythroblasts and promegakaryoblast before and after neuraminidase treatment except in one case of promegakaryoblastic leukaemia in which the SSEA-I antigen and platelet peroxidase were expressed in the same cell. In addition, six cases of common acute lymphoblastic leukaemia were studied; the blasts became positive after desialylation. Two examples of T cell acute leukaemia were essentially negative. We conclude, therefore, that the reactivity of haemopoietic cells with these MoAbs alone does not represent a criterion sufficient to sustain their myeloid origin since the SSEA-I antigen may be expressed at the surface of all cell lineages in the early phases of haemopoietic differentiation.

Acute Disease↗
PubMed1975

Effects of proteases and neuraminidase on RBC surface charge and agglutination. A kinetic study.

Electrophoretic mobility, membrane sialic acid content and agglutinability by "incomplete" antisera against Rh-o, hr' and k antigens were determined for red blood cells in the course of treatment with trypsin, ficin and neuraminidase. Neuraminidase gradually produces a slight to moderate agglutinability as it reduced surface charge density in proportion to the amount of sialic acid removed. Proteases acted in two distinct steps. The first stage is characterized by the cells rapidly becoming highly agglutinable and by the unmasking of new negative charge as the first half of the sialic acid is removed. In the second stage the cells show a slight gain in agglutinability as surface charge is removed in proportion to sialic acid removal as in the case of neuraminidase. Neuraminidase-treated cells are considerably less agglutinable than cells reduced to the same zeta-potential by protease treatment. The greater efficacy of proteases compared to neuraminidase in making cells agglutinable could be because they not only reduce surface charge density but also increase antigen-antibody bond strength, render antigens more mobile in the membrane to allow clustering in regions of cell to cell antibody bridging and remove glycopeptide chains which may be causing steric hindrance to antigen-antibody binding or to cell-cell contact.

Clostridium perfringens↗
PubMed1997

Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase.

The head domain of recombinant neuraminidase of A/Victoria/3/75 influenza virus was produced in a secreted form in the methylotrophic yeast Pichia pastoris using the P. pastoris alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-mating-factor signal sequence. Cultures in shake flasks provided expression levels of approximately 2.5-3 mg/l. Recombinant neuraminidase was purified from the culture medium to over 99% homogeneity. Although P. pastoris-secreted products are believed to carry shorter N-linked carbohydrate side chains than glycoproteins of S. cerevisiae, secreted neuraminidase was hyperglycosylated, with N-glycans of the high-mannose type containing up to 30-40 mannose residues. N-glycans were phosphorylated and only partially sensitive to alpha-mannosidase treatment. Balb/c mice immunized three times with 2 microg purified recombinant neuraminidase were 50% protected against a lethal challenge of mouse-adapted homologous virus; removal of glycosylation at the top of neuraminidase resulted in improved protection. The results provide a system for the production of an effective recombinant influenza vaccine that can easily be scaled up.

Animals↗
PubMed1986

Further characterization of a myelin-associated neuraminidase: properties and substrate specificity.

A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.

Animals↗
PubMed1997

Neuraminidase (sialidase) activity of Haemophilus parasuis.

Neuraminidase (sialidase), a potential virulence factor in bacteria, was demonstrated in Haemophilus parasuis, an invasive swine pathogen, but not in four other pathogens of the Pasteurellaceae family: H. influenzae, H. somnus, H. paragallinarum, or Actinobacillus pleuropneumoniae. H. parasuis neuraminidase had an acidic pH optimum and a specificity for several substrates also cleaved by other bacterial neuraminidases. Similar to the neuraminidase of Pasteurella multocida, H. parasuis neuraminidase was cell associated and did not require divalent cations for activity. Exogenous sialic acid added to growth medium of H. parasuis was cleared after a lag of about 10 h and these cultures grew to a greater final density than cultures without added sialic acid, indicating that exogenous sialic acid is metabolized. The role of sialidase in providing nutrients to H. parasuis may be an important factor in its obligate parasitism.

Animals↗
PubMed1989

Increased microvascular resistance to blood flow in the rat hindlimb after perfusion with neuraminidase.

1. The ability of blood to flow through capillaries with diameters smaller than that of erythrocytes and leucocytes has been explained hypothetically on the basis of electrostatic repulsion between vessel walls and circulating cells, but in the absence of in vivo evidence. Following our discovery of extraordinarily high densities of sialic acids on endothelial surfaces, we have now devised experiments to determine to what extent microcirculatory blood flow might depend on these negative charges. 2. Hindlimbs of rats anaesthetized with ether and pentobarbitone were perfused through the femoral artery at constant volume with continuous recording of inflow resistance: first with carotid arterial blood containing continually infused acetylcholine at concentrations which minimized peripheral resistance without affecting heart rate; then for 5 min with buffered saline containing acetylcholine at the same concentration plus neuraminidase (from Vibrio cholerae, at 0.1 mg/ml) to remove endothelial sialic acids (for control experiments without neuraminidase); and then again with acetylcholine-infused carotid blood. 3. As a consequence of the saline perfusion, there was a rapid initial rise and fall of inflow resistance. The subsequent changes in resistance were calculated as the difference between its lowest value in the first 10 min and the value after 60 min. Controls and neuraminidase-perfused groups were compared using the t test for unpaired measurements. 4. In four control experiments inflow resistance decreased by 10-40% (14 +/- 10%, mean +/- S.E.M.). In three experiments with neuraminidase, inflow resistance increased by 24-38% (29 +/- 4%, mean +/- S.E.M.). The difference between the saline and the neuraminidase-perfused groups was significant (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed2006

Detection of influenza viruses resistant to neuraminidase inhibitors in global surveillance during the first 3 years of their use.

Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) develops at a low level following drug treatment, and person-to-person transmission of resistant virus has not been recognized to date. The Neuraminidase Inhibitor Susceptibility Network (NISN) was established to follow susceptibility of isolates and occurrence of NAI resistance at a population level in various parts of the world. Isolates from the WHO influenza collaborating centers were screened for susceptibilities to oseltamivir and zanamivir by a chemiluminescent enzyme inhibition assay, and those considered potentially resistant were analyzed by sequence analysis of the neuraminidase genes. During the first 3 years of NAI use (1999 to 2002), 2,287 isolates were tested. Among them, eight (0.33%) viruses had a >10-fold decrease in susceptibility to oseltamivir, one (0.22%) in 1999 to 2000, three (0.36%) in 2000 to 2001, and four (0.41%) in 2001 to 2002. Six had unique changes in the neuraminidase gene compared to neuraminidases of the same subtype in the influenza sequence database. Although only one of the mutations had previously been recognized in persons receiving NAIs, none were from patients who were known to have received the drugs. During the 3 years preceding NAI use, no resistant variants were detected among 1,054 viruses. Drug use was relatively stable during the period, except for an approximate 10-fold increase in oseltamivir use in Japan during the third year. The frequency of variants with decreased sensitivity to the NAIs did not increase significantly during this period, but continued surveillance is required, especially in regions with higher NAI use.

Antiviral Agents↗
PubMed2001

Novel alpha- and beta-amino acid inhibitors of influenza virus neuraminidase.

In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 alpha- and beta-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited K(i) values in the 50 microM range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the alpha-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the beta-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the alpha- or beta-amino groups were replaced with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design.

Amino Acids↗
PubMed2002

In vitro characterization of A-315675, a highly potent inhibitor of A and B strain influenza virus neuraminidases and influenza virus replication.

A-315675 is a novel, pyrrolidine-based compound that was evaluated in this study for its ability to inhibit A and B strain influenza virus neuraminidases in enzyme assays and influenza virus replication in cell culture. A-315675 effectively inhibited influenza A N1, N2, and N9 and B strain neuraminidases with inhibitor constant (K(i)) values between 0.024 and 0.31 nM. These values were comparable to or lower than the K(i) values measured for oseltamivir carboxylate (GS4071), zanamivir, and BCX-1812, except for the N1 enzymes that were found to be the most sensitive to BCX-1812. The time-dependent inhibition of neuraminidase catalytic activity observed with A-315675 is likely due to its very low rate of dissociation from the active site of neuraminidase. The half times for dissociation of A-315675 from B/Memphis/3/89 and A/Tokyo/3/67 (H3N2) influenza virus neuraminidases of 10 to 12 h are significantly slower than the half times measured for oseltamivir carboxylate (33 to 60 min). A-315675 inhibited the replication of several laboratory strains of influenza virus in cell culture with potencies that were comparable or superior to those for oseltamivir carboxylate and BCX-1812, except for the A/H1N1 viruses that were found to be two- to fourfold more susceptible to BCX-1812. A-315675 and oseltamivir carboxylate exhibited comparable potencies against a panel of A/H1N1 and A/H3N2 influenza virus clinical isolates, but A-315675 was found to be significantly more potent than oseltamivir carboxylate against the B strain isolates. The favorable in vitro results relative to other clinically effective agents provide strong support for the further investigation of A-315675 as a potential therapy for influenza virus infections.

Acetamides↗
PubMed1983

Neuraminidase in Bacteroides fragilis.

A neuraminidase from Bacteroides fragilis was purified 542-fold by isoelectric focusing, adsorption chromatography on Affi-Gel 202, and gel filtration chromatography on Sephadex G-200. On isoelectric focusing the neuraminidase was resolved into three differently charged fractions with pI values of 6.8, 7.1, and 7.4. The major component of pI 7.1 was used for further purification. The purified enzyme had optimal activity at pH 6.4 with N-acetylneuraminlactose as the substrate. Its molecular weight, determined by Sephadex G-200 gel filtration chromatography, was 92,000. The neuraminidase hydrolyzed terminal neuraminic acid residues from N-acetylneuraminlactose, fetuin, bovine submaxillary mucin, and porcine stomach lining mucin. A new method for the detection of neuraminidase activity is described which is based on rocket affinoelectrophoresis. It utilizes the differences in the interaction of sialylated and desialylated mucin with Helix pomatia lectin, enzymatic activity being detected by formation of affinorockets after incubation of the neuraminidase with bovine submaxillary mucin.

Bacteroides fragilis↗
PubMed1991

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.

Actinomyces viscosus↗