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PubMed1975

Experimental pneumococcal meningitis. Effects of neuraminidase and other pneumococcal constituents on cerebrospinal fluid in the intact dog.

The intracisternal administration of a Type I pneumococcus to mongrel dogs resulted in spinal fluid abnormalities and clinical course and outcome similar to those of meningitis in humans. In order to define the pathogenic role of pneumococcal neuraminidase and other pneumococcal extracts in experimental meningitis, the following preparations were derived from the same Type I pneumococcus: crude neuraminidase, partially purified neuraminidase, heat-inactivated crude neuraminidase, and heat-killed pneumococci. These preparations were administered intracisternally daily for 5 days to a series of mongrel dogs. These substances did not produce clinical morbity similar to that observed in infected animals, even though there was significant decrease in the NANA content of cortical brain subcellular structures in the neuraminidase-treated dogs. It was concluded that the substances investigated were not responsible for morbidity and mortality in experimental pneumococcal meningitis. Both heat-killed pneumococci and the crude neuraminidase preparation elicited significant alterations in spinal fluid glucose and protein concentrations which were similar to those recorded in infected animals; however these abnormalities were not associated with significant morbidity. It is proposed that spinal fluid glucose and protein abnormalities may not be directly linked to brain damage or dysfunction in this experimental model, or in man.

Animals↗

PubMed1986

Botulinum A neurotoxin unlike tetanus toxin acts via a neuraminidase sensitive structure.

The binding and effects of tetanus and botulinum A neurotoxins were studied on mouse spinal cord cultures treated with neuraminidase. In untreated cultures both neurotoxins blocked synaptic transmission. Treatment of the cell cultures with neuraminidase, 25 mU/ml for 24 hr, decreased the potency of botulinum A neurotoxin. At 7 X 10(-11) M no toxin effect on inhibitory or excitatory synapses was observed, whereas at higher concentrations of the toxin the concentration-response curve was shifted to the right by a factor of about 30. Surprisingly, the action of tetanus toxin over a large concentration range was unaffected by pretreatment of the neurones with the enzyme. Accordingly, neurones treated with neuraminidase failed to bind 125I-botulinum A neurotoxin, whereas labelled tetanus toxin was still fixed by cell bodies, as well as by neurites, as shown by histoautoradiography. Chromatographic extraction of gangliosides from cultures prelabelled with 14C-glucosamine showed a dramatic loss in the contents of polysialogangliosides following treatment with neuraminidase. Our results indicate that neuraminidase-sensitive structures might be important for the action of botulinum A neurotoxin. The effect of tetanus toxin appears to be mediated by a different site which is insensitive to neuraminidase.

Action Potentials↗

PubMed2004

Neuraminidase activity assays for monitoring MDCK cell culture derived influenza virus.

Three assay methods were investigated for monitoring the time-course of neuraminidase (NA) activity of tissue culture derived equine influenza A virus from large-scale microcarrier cultivation and several steps of downstream processing required for the production of inactivated vaccines. Measurements of neuraminidase activity by a thiobarbituric acid (TBA) and a fluorometric method using Amplex Red as a fluorogen (FL-AR) did not correlate with the increase of hemagglutinin (HA) during virus replication. Samples analysed by the TBA method showed unspecific interference from low molecular weight compounds (< 3 kDa) of cell growth medium and virus maintenance medium. Further investigations showed that this was probably caused by interfering reactions between reducing sugars and amino acids that can be overcome by dialysis of samples. On the other hand, the sensitivity of the FL-AR method was not sufficient for the required measuring range. However, a reliable and sensitive fluorometric assay method (FL-MU-NANA) was obtained using 4-methylumbelliferyl-alpha-d-N-acetylneuraminic acid (4-MU-NANA) as a substrate, which allowed the detection of neuraminidase activities as low as 0.09 mU/mL. In this assay, time-course of neuraminidase activities correlated well with increasing hemagglutinin activities during virus replication in a bioreactor. Analysis of samples from various downstream processing steps comprising of clarification, inactivation, ultrafiltration (UF) and size-exclusion chromatography for the purification of influenza virus showed that neuraminidase activity was preserved at comparatively high levels. Based on the hemagglutinin and neuraminidase activity of the clarified and inactivated virus harvest, the overall recovery after gel filtration was about 34.4% and 119.5%, respectively.

Amino Acids↗

PubMed2003

Neuraminidase treatment of respiratory syncytial virus-infected cells or virions, but not target cells, enhances cell-cell fusion and infection.

Respiratory syncytial virus (RSV) infection of HeLa cells induces fusion, but transient expression of the three viral glycoproteins induces fusion poorly, if at all. We found that neuraminidase treatment of RSV-infected cells to remove sialic acid (SA) increases fusion dramatically and that the same treatment of transiently transfected cells expressing the three viral glycoproteins, or even cells expressing the fusion (F) protein alone, results in easily detectable fusion. Neuraminidase treatment of the effector cells, expressing the viral glycoproteins, enhanced fusion while treatment of the target cells did not. Likewise, infectivity was increased by treating virions with neuraminidase, but not by treating target cells. Reduction of charge repulsion by removal of the negatively charged SA is unlikely to explain this effect, since removal of negative charges from either membrane would reduce charge repulsion. Infection with neuraminidase-treated virus remained heparan-sulfate-dependent, indicating that a novel attachment mechanism is not revealed by SA removal. Interestingly, neuraminidase enhancement of RSV infectivity was less pronounced in a virus expressing both the G and the F glycoproteins, compared to virus expressing only the F glycoprotein, possibly suggesting that the G protein sterically hinders access of the neuraminidase to its fusion-enhancing target.

Animals↗

PubMed1996

Design and expression of a stable bispecific scFv dimer with affinity for both glycophorin and N9 neuraminidase.

We have designed and produced a stable bispecific scFv dimer (bisFv) by non-covalent association of two hybrid VH-VL pairs derived from an anti-neuraminidase antibody (NC10) and an anti-glycophorin antibody (1C3). The bisFv dimer was demonstrated to have binding activity to the two respective target antigens and was evaluated as a reagent for rapid whole blood agglutination assays. The bisFv was expressed in the periplasm of Escherichia coli, from a secretion vector which comprised two cistrons in tandem under the control of a single lac promoter, inducible with IPTG. Each cistron encoded one of the hybrid VH-VL pairs, with V domains separated by a linker region encoding the five amino acids, Gly4Ser. The short linker region was designed to prevent association of VH and VL regions of the same molecule and favour the formation of dimers. The protein synthesized from each hybrid scFv cistron was directed to the E. coli periplasm by the inclusion of distinctive signal secretion sequences preceding each hybrid gene; from pel B of Erwinia cartovora and from gene III of fd phage. The bisFv was affinity-purified from culture supernatants via the C-terminal tag epitope FLAG and was shown, by FPLC on a Superose 6 column, to be consistent in size with that of a scFv dimer. The bisFv was stable for more than 4 months at 4 degrees C and was shown by BIAcore analysis to bind to either target antigen, human glycophorin, or tern N9 neuraminidase. Simultaneous binding to both target antigens was demonstrated when a pre-formed bisFv-neuraminidase complex was shown to bind to immobilized glycophorin. In whole blood agglutination assays, the bisFv dimer was able to agglutinate red blood cells when crosslinked with an anti-idiotype antibody (3-2G12) binding to the NC10 combining site, but no agglutination occurred on binding the antigen neuraminidase. These results are a function of the topology of the epitopes on neuraminidase and have implications for the use of relatively rigid bifunctional molecules (as bisFv dimers) to cross link two large membrane-anchored moieties, in this case, red blood cell glycophorin and neuraminidase, an M(r) 190,000 tetramer.

Antibodies, Bispecific↗

PubMed1985

Enzymological heterogeneity of influenza B virus neuraminidase demonstrated by the fluorometric assay method.

The neuraminidase activity of 26 strains of influenza B virus isolated from all over the world was investigated colorimetrically, using fetuin as a substrate, and fluorometrically, using 4-methylumbelliferyl(4-MU)-N-Ac-alpha-D-neuraminide as a substrate, with special reference to enzymological heterogeneity. The activity of influenza A viral neuraminidases and of a commercially available pure viral one was strongly inactivated by either ethylenediaminetetraacetic acid or glycoletherdiaminetetraacetic acid, when measured by the fluorometric assay method, whereas that of influenza B ones was not at all. However, the viral neuraminidases of both influenza A and B viruses were found to be calcium ion-dependent by the colorimetric assay method. A difference in the catalytic rate between the two assay methods was observed with influenza B viral neuraminidase to a much greater extent as compared with that of influenza A. A difference in substrate specificity of these enzymes was demonstrated to be due to a difference in the degree of competitive inhibition by N-acetylneuraminic acid. These findings strongly suggest that enzymological heterogeneity in influenza B viral neuraminidase may be attributed to delicate structural differences, between the enzymes of influenza A and B viruses demonstratable only by the fluorometric neuraminidase assay method using 4-MU-N-Ac-alpha-D-neuraminide as a substrate.

Clostridium perfringens↗

PubMed1994

Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain.

BACKGROUND: Vibrio cholerae neuraminidase is part of a mucinase complex which may function in pathogenesis by degrading the mucin layer of the gastrointestinal tract. The neuraminidase, which has been the target of extensive inhibitor studies, plays a subtle role in the pathology of the bacterium, by processing higher order gangliosides to GM1, the receptor for cholera toxin. RESULTS: We report here the X-ray crystal structure of V. cholerae neuraminidase at 2.3 A resolution. The 83 kDa enzyme folds into three distinct domains. The central catalytic domain has the canonical neuraminidase beta-propeller fold, and is flanked by two domains which possess identical legume lectin-like topologies but without the usual metal-binding loops. The active site has many features in common with other viral and bacterial neuraminidases but, uniquely, has an essential Ca2+ ion which plays a crucial structural role. CONCLUSIONS: The environment of the small intestine requires V. cholerae to secrete several adhesins, and it is known that its neuraminidase can bind to cell surfaces, and remain active. The unexpected lectin-like domains possibly mediate this attachment. These bacterial lectin folds represent additional members of a growing lectin superfamily.

Bacterial Adhesion↗

PubMed1994

N9 neuraminidase complexes with antibodies NC41 and NC10: empirical free energy calculations capture specificity trends observed with mutant binding data.

X-ray crystallographic coordinates of influenza virus N9 neuraminidase complexed with monoclonal antibodies NC41 and NC10 [Tulip et al. (1992) J. Mol. Biol. 227, 122-148] served as a starting point for calculations aimed at estimating free energy changes (delta G) of complex formation between the two antibodies and the neuraminidase. Using an empirical function incorporating hydrophobic, electrostatic, and conformational entropy effects, we estimated contributions individual neuraminidase residues make to complex formation (delta G(residue)) and compared the calculated values to experimentally measured differences in antibody binding between the wild-type and mutated neuraminidases [Nuss et al. (1993) Proteins 15, 121-132; calculations done without prior knowledge of the experimental data]. A good correspondence was found between the calculated delta G(residue) values and the mutant binding data in that side chains with large calculated delta G contributions (delta G(residue) < -1 kcal/mol) lie at sites of mutation which cause a marked reduction in antibody binding, and side chains for which delta G(residue) > -1 kcal/mol are sites at which a mutation does not have a marked effect on binding. Because most of the delta G(residue) < -1 kcal/mol side chains also make hydrogen bonds/salt bridges with the antibody, the correspondence of the effect of antibody binding with these electrostatic interactions (18 out of 27 for NC41 and, tentatively, 5 out of 7 for NC10) is about as good as that with predicted energetic residues. All the delta G(residue) < -1 kcal/mol neuraminidase side chains cluster around the most protruding surface regions and are thus spread over different epitope segments. Surprisingly, different residues were found to make the most critical contributions to the NC41 and NC10 complex stabilities despite the fact that the NC41 and NC10 antigenic epitopes overlap, having approximately 70% of surface residues in common. It is thus possible, for two different antibodies, to recognize the same protein surface in strikingly different ways. As only a fraction of the neuraminidase residues appear to make large contributions to antibody binding, the results also support the hypothesis of a "functional" epitope in antigen-antibody interactions. Positive trends between both backbone rigidity and residue accessibility in the complexed state, and contributions of these residues to binding, were also observed for the NC41 complex.

Antibodies, Monoclonal↗

PubMed1987

Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism.

Different isolates of influenza virus show a high degree of amino acid sequence variation in their surface glycoproteins. Conserved residues located in the substrate-binding pocket of the influenza virus neuraminidase are therefore likely to be involved in substrate binding or enzyme catalysis. In order to study the structure and function of the active site of this protein, a full-length cDNA clone of the neuraminidase gene from influenza A/Tokyo/3/67 was subcloned into aN M13 vector and amino acid substitutions were made in selected residues by using the oligonucleotide mismatch technique. The mutant neuraminidase genes were expressed from a recombinant SV40 vector, and the proteins were analyzed for synthesis, transport to the cell surface, and proper three-dimensional folding by internal and surface immunofluorescence. The mutant neuraminidase proteins were then assayed to determine the effect of the amino acid substitution on enzyme activity. Twelve of the 14 mutant proteins were correctly folded and were transported to the cell surface in a manner identical with that of the wild type. Two of these have full enzyme activity, but seven mutants, despite correct three-dimensional structure, have completely lost neuraminidase activity. Two mutants were active at low pH. The properties of the mutant enzymes suggest a possible mechanism of neuraminidase action.

Binding Sites↗

PubMed2000

Characterization of the 1918 "Spanish" influenza virus neuraminidase gene.

The "Spanish" influenza pandemic of 1918 was characterized by exceptionally high mortality, especially among young adults. The surface proteins of influenza viruses, hemagglutinin and neuraminidase, play important roles in virulence, host specificity, and the human immune response. The complete coding sequence of hemagglutinin was reported last year. This laboratory has now determined the complete coding sequence of the neuraminidase gene of the 1918 virus. Influenza RNA fragments were isolated from lung tissue of three victims of the 1918 flu; complete sequence was generated from A/Brevig Mission/1/18, with confirmatory sequencing carried out on A/South Carolina/1/18 and A/New York/1/18. The 1918 neuraminidase gene sequence was compared with other N1 subtype neuraminidase genes, including 9 N1 strains newly sequenced for this study. The 1918 neuraminidase shares many sequence and structural characteristics with avian strains, including the conserved active site, wild-type stalk length, glycosylation sites, and antigenic sites. Phylogenetically, the 1918 neuraminidase gene appears to be intermediate between mammals and birds, suggesting that it was introduced into mammals just before the 1918 pandemic.

Adult↗

PubMed1969

Independent variation in nature of hemagglutinin and neuraminidase antigens of influenza virus: distinctiveness of hemagglutinin antigen of Hong Kong-68 virus.

Antigenic variations of the two virus-coded surface proteins of influenza virus-hemagglutinin and neuraminidase-were examined in seven strains of influenza A(2) virus (including the Hong Kong/68 strain) isolated from 1957 to 1968. Changes in the two antigens were found to occur independently in nature, resulting in new viruses which differ from older strains more with respect to one antigen than the other. The Hong Kong/68 strain is markedly different from previous A(2) strains in its hemagglutinin antigen but possesses neuraminidase indistinguishable antigenically from that of recent strains. Immunization experiments in mice provided evidence that only the neuraminidase component of an earlier A(2) strain provided protection against Hong Kong virus challenge. Segregation of hemagglutinin and neuraminidase antigens through recombination of each of the seven strains of influenza A(2) virus with A(0)/NWS virus made it possible to investigate antigenic variation of the two dissociated surface proteins independently in a common plaque assay system. Comparison of these hybrid viruses with the parent A(2) strains provided evidence that all the cross-reactivity of the Hong Kong strain with previous A(2) viruses is explicable on the basis of its similar neuraminidase component. It is proposed that the taxonomy of influenza A viruses must take into account differences in neuraminidase as well as hemagglutinin antigens.

Animals↗

PubMed2005

Lysosomal sialidase (neuraminidase-1) is targeted to the cell surface in a multiprotein complex that facilitates elastic fiber assembly.

We have established previously that the 67-kDa elastin-binding protein (EBP), identical to the spliced variant of beta-galactosidase, acts as a recyclable chaperone that facilitates secretion of tropoelastin. (Hinek, A., Keeley, F. W., and Callahan, J. W. (1995) Exp. Cell Res. 220, 312-324). We now demonstrate that EBP also forms a cell surface-targeted molecular complex with protective protein/cathepsin A and sialidase (neuraminidase-1), and provide evidence that this sialidase activity is a prerequisite for the subsequent release of tropoelastin. We found that treatment with sialidase inhibitors repressed assembly of elastic fibers in cultures of human skin fibroblasts, aortic smooth muscle cells, and ear cartilage chondrocytes and caused impaired elastogenesis in developing chick embryos. Fibroblasts derived from patients with congenital sialidosis (primary deficiency of neuraminidase-1) and galactosialidosis (secondary deficiency of neuraminidase-1) demonstrated impaired elastogenesis, which could be reversed after their transduction with neuraminidase-1 cDNA or after treatment with bacterial sialidase, which has a similar substrate specificity to human neuraminidase-1. We postulate that neuraminidase-1 catalyzes removal of the terminal sialic acids from carbohydrate chains of microfibrillar glycoproteins and other adjacent matrix glycoconjugates, unmasking their penultimate galactosugars. In turn, the exposed galactosugars interact with the galectin domain of EBP, thereby inducing the release of transported tropoelastin molecules and facilitating their subsequent assembly into elastic fibers.

Aorta↗

PubMed2005

Human parainfluenza virus 3 neuraminidase activity contributes to dendritic cell maturation.

Mechanisms of dendritic cells (DCs) immunomodulation by parainfluenza viruses have not been characterized. We analyzed whether the human parainfluenza 3 (HPF3) virus hemagglutinin-neuraminidase glycoprotein (HN) might influence DC maturation. HN possesses a receptor binding function and a neuraminidase or desialidating activity. To assess whether the neuraminidase activity of HN affects DC maturation, human myeloid DCs were exposed to either live or UV-inactivated HPF3 viruses containing wild type or a mutated form of HN with decreased neuraminidase activity. Exposure of human DCs to either UV-inactivated or live virus induced up-regulation of CD83 and CD86 surface markers, morphological changes, and a cytokine expression pattern consistent with maturation. However, the level of maturation was found to be lower in DCs infected with the neuraminidase deficient variant as compared to the wild type. These results suggest that during the course of viral infection, HN's neuraminidase activity may play an important role contributing to maturation and activation of DCs.

Cell Differentiation↗

PubMed1980

Fluorometric assay for measurement of viral neuraminidase--application to the rapid detection of influenza virus in nasal wash specimens.

A sensitive assay was developed for the measurement of neuraminidase with use of the 4-methylumbelliferkyl-alph-ketoside of N-acetyl-neuraminic acid as a fluorescent substrate. Neuraminidase activity was detected in preparation containing small quantities of cultivated influenza virus as well as in some nasal wash specimens from human volunteers experimentally infected with influenza A/Alaska/78 virus. Activity due to influenza viral neuraminidase could be distinguished from activity due to bacterial or mammalian (fibroblast) neuraminidase by means of enzyme inhibition with ethylenediaminetetraacetate, a chelating agent. The same substrate could be used in a solid-phase assay to determine the antigenic type of the influenza viral neuraminidase. The fluorescent neuraminidase assay takes 2-4 hr to perform and uses equipment available in many laboratories. Thus, it offers promise for the rapid detection of influenza viruses in cell cultures and clinical specimens.

Animals↗

PubMed1985

Neuraminidase production by streptococci from patients with glomerulonephritis.

Autoantigenic events in acute poststreptococcal glomerulonephritis (APSGN) may result from neuraminidase-induced alterations in immunoglobulin levels. Whether streptococci from patients with APSGN produced neuraminidase and, if so, what substrates could be used for screening purposes and what were of potential clinical relevance were determined. Group A streptococci cultured from 20 patients with APSGN and four patients with acute rheumatic fever and group B, C, D, and G streptococci cultured from other individuals were reacted with the substrates to determine neuraminidase activity by the thiobarbituric acid assay. Neuraminidase production was detected in 16 of 20 streptococci from patients with APSGN. Partial purification by Sephadex G-150 chromatography gave two peaks, and polyacrylamide gel electrophoresis gave two bands with neuraminidase activity. Bovine mucin was the most useful substrate to detect neuraminidase production by nephritogenic streptococci, and with respect to human substrates, IgM was the most sensitive and renal basement membrane the least sensitive to enzyme action.

Glomerulonephritis↗

PubMed1989

Differential exposure of the major gangliosides in rabbit thymocytes to Vibrio cholerae neuraminidase.

Neuraminidase treatment of lymphocytes is known to cause changes of cellular responses in several biological phenomena, but the molecules modified on the cell surface by neuraminidase are not known in detail. Rabbit thymocytes, which contain tissue-characteristic gangliosides, were treated with Vibrio cholerae neuraminidase, and the susceptibility of the cell surface sialic acid residues was examined. The amount of sialic acid released from the thymocytes at the highest level was 42.4 nmol per 1 X 10(9) cells, among which 26.5% was from gangliosides. Ninety-three percent of the VI3NeuGc-nLc6Cer, 84% of the IV3NeuGc-nLc4Cer, and 50% of the II3NA2-LacCer in the thymocytes was hydrolyzed to nLc6Cer, nLc4Cer, and LacCer, respectively, but II3NA-LacCer was completely cryptic. Also, among the molecular species of II3NA2-LacCer, C20:0- to C24:0-containing, but not C16:0- to C18:0-containing molecules, were susceptible to neuraminidase. After neuraminidase treatment, nLc4Cer and nLc6Cer became the major glycosphingolipids, and a 15-fold increase of radioactivity incorporated into the glycosphingolipids was observed by the galactose oxidase-sodium borotritide procedure, suggesting that the beta-galactose of the glycosphingolipids produced by neuraminidase treatment is accessibly to the several ligands which are functionally associated with lymphocytes.

Animals↗

PubMed1982

Catalytically defective ganglioside neuraminidase in mucolipidosis IV.

Cultured skin fibroblasts from patients with mucolipidosis IV were found to be deficient in neuraminidase activity toward GD1a and GD1b gangliosides radiolabelled in C8 and C7 analogs of their sialic acid residues. Neuraminidase activities toward 4-methylumbelliferyl-N-acetyl-neuraminic acid, neuraminlactose, and radiolabelled neuraminlactitol, fetuin and alpha 1-acid glycoprotein were within the range of normal controls. Fibroblasts from parents of patients with mucolipidosis IV demonstrated intermediate levels of ganglioside neuraminidase activity and normal levels of glycoprotein neuraminidase activity. The residual acidic neuraminidase activity toward GD1a ganglioside in the patients' fibroblasts did not differ from that of controls in its pH optimum and thermostability, but had an abnormal apparent Km which was about 18 times higher than that of the normal enzyme. These findings suggest that mucolipidosis IV is a ganglioside sialidosis due to a catalytically defective ganglioside neuraminidase.

Fibroblasts↗

PubMed1985

Evidence for sialylated type 1 blood group chains on human erythrocyte membranes revealed by agglutination of neuraminidase-treated erythrocytes with Waldenström's macroglobulin IgMWOO and hybridoma antibody FC 10.2.

Haemagglutination studies have been performed with untreated and neuraminidase-treated human erythrocytes of the three Lewis antigen types Le(a-b-), Le(a+b-) and Le(a-b+) using two monoclonal antibodies, IgMWOO and FC 10.2, which were previously shown to recognize the type 1 based blood group chains: Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc/GlcNAc (for explanation of abbreviations see table IV legend). Both antibodies behaved as cold agglutinins with neuraminidase-treated but not with untreated erythrocytes of the three Lewis antigen types. Neuraminidase-treated erythrocytes of i antigen type were similarly agglutinated. This haemagglutination was specifically inhibited by the type 1 based milk oligosaccharide lacto-N-tetraose. Thus, there is strong evidence for the occurrence of sialylated type 1 chains on human erythrocyte membranes of I and i antigen types. In addition, evidence for the presence of type 1 chains which are both sialylated and fucosylated was obtained by (1) haemagglutination of Le(a+b-) erythrocytes with the monoclonal antibody 19.9; (2) increased haemagglutination of neuraminidase-treated erythrocytes with anti-H antibodies of Bombay serum; (3) increased haemagglutination of neuraminidase-treated Le(a+b-) cells with anti-Lea antibodies, and (4) the appearance of Lea antigen activity on neuraminidase-treated erythrocytes of Le(a-b+) type.

ABO Blood-Group System↗