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PubMed1997

Multiple forms of alkaline phosphatase in horse bone, liver, kidney, duodenum, caecum and serum separated by agarose gel electrophoresis with and without neuraminidase pretreatment.

Horse bone, liver, duodenum, caecum and kidney alkaline phosphatases were separated by a commercial agarose gel electrophoresis method with and without neuraminidase pretreatment, following the manufacturer's directions. Tissue extracts were obtained in saline solution and ALP extracted from cell membranes by the butanol method. Electrophoresis was performed using a TRIS/barbital/sodium barbital buffer with detergent, pH 8.6 to 9.0, at 250 V for 30 minutes. Bone, liver and kidney untreated extracts showed two ALP bands each, but with different relative migration (compared to albumin migration). When they were preincubated with neuraminidase, the two bone bands showed a marked decrease in their migration, followed by the kidney ALP bands and the most anodic band of liver Both intestinal untreated extracts showed three bands but with different mobilities. After preincubation with neuraminidase, the three bands of caecum mucosa decreased in their migration, and the most anodic duodenum band disappeared, overlapping the second one. When tissue extracts were incubated with wheat germ-lectin (WGL), 74.5% of bone extract ALP and 67.2% of caecum extract ALP precipitated, which demonstrated that the ALP band of both tissues have similar groups in the carbohydrate side chains. Horse serum showed two electrophoretic bands, which increased to three bands when treated with neuraminidase. ALP from hepatocytes was the dominant isoform, followed by a caecum band. Because the electrophoretic mobilities of some of the tissue bands studied were identical, the neuraminidase agarose electrophoretic method appeared to be a satisfactory alternative to separate them.

Journal Article↗
PubMed2003

Mutations in human parainfluenza virus type 3 hemagglutinin-neuraminidase causing increased receptor binding activity and resistance to the transition state sialic acid analog 4-GU-DANA (Zanamivir).

Entry and fusion of human parainfluenza virus type 3 (HPF3) require the interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-GU-DANA, a potent inhibitor of influenza virus neuraminidase, inhibits not only HPF3 neuraminidase but also the receptor binding activity of HPF3 HN and thus its ability to promote attachment and fusion. We previously generated a 4-GU-DANA-resistant HPF3 virus variant (ZM1) with a markedly fusogenic plaque morphology that harbored two HN gene mutations resulting in amino acid alterations. The present study using cells that express the individual mutations of ZM1 HN shows that one of these mutations is responsible for the increases in receptor binding and neuraminidase activities as well as the diminished sensitivity of both activities to the inhibitory effect of 4-GU-DANA. To examine the hypothesis that increased receptor binding avidity underlies 4-GU-DANA resistance, parallel studies were carried out on the high-affinity HN variant virus C22 and cells expressing the C22 variant HN. This variant also exhibited reduced sensitivity to 4-GU-DANA in terms of receptor binding and infectivity but without concomitant changes in the neuraminidase activity of HN. Another high-affinity HN variant, C0, was not resistant in terms of infectivity; however, a small increase in the receptor binding activity of C0 HN and a partial resistance of this activity to 4-GU-DANA were revealed by sensitive methods that we developed. In each virus variant, one mutation in HN accounted for both increased receptor binding avidity and 4-GU-DANA resistance; the higher affinity for the receptor overcomes the inhibitory effect of 4-GU-DANA. Thus, in contrast to influenza viruses for which 4-GU-DANA escape variants include hemagglutinin mutants with decreased receptor binding avidity that promotes virion release, for HPF3, HN mutants with increased receptor binding avidity are those that can escape the growth inhibitory effect of 4-GU-DANA.

Animals↗
PubMed1992

The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid.

Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R-factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co-ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.

Amino Acid Sequence↗
PubMed1992

Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities.

Trypomastigotes, the blood stage form of the human parasite Trypanosoma cruzi, contain an enzyme on their surface, trans-sialidase, which catalyses the transfer of sialic acid from host glycoconjugates to acceptors on its own cell surface. At least a subset of the sialic acid-bearing acceptor molecules are involved in parasite invasion of host cells, an essential step in the life cycle of the parasite. Another trypomastigote surface enzyme that affects host cell invasion is neuraminidase and recent evidence suggests that both trans-sialidase and neuraminidase activities may be expressed by the same proteins on the parasite surface. We describe here the isolation and expression of several members of a trans-sialidase--neuraminidase gene family from T.cruzi. One of the isolated genes does indeed encode a protein with both trans-sialidase and neuraminidase activities, while other members of the gene family encode closely related proteins that express neither enzymatic activity. Chimeric protein constructs combining different portions of active and inactive genes identified a region of the gene necessary for enzymatic activity. Sequence analysis of this portion of the gene revealed a limited number of amino acid differences between the predicted active and inactive gene products.

Amino Acid Sequence↗
PubMed1995

Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase.

The three-dimensional X-ray structure of a complex of the potent neuraminidase inhibitor 4-guanidino-Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 A resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the inhibitor, and the active site residues show no significant conformational changes on binding. Sialic acid, the natural substrate, binds in a half-chair conformation that is isosteric to the inhibitor. The conformation of the inhibitor in the active site of the X-ray structure concurs with that obtained by theoretical calculations and validates the structure-based design of the inhibitor. Comparison of known high-resolution structures of neuraminidase subtypes N2, N9, and B shows good structural conservation of the active site protein atoms, but the location of the water molecules in the respective active sites is less conserved. In particular, the environment of the 4-guanidino group of the inhibitor is strongly conserved and is the basis for the antiviral action of the inhibitor across all presently known influenza strains. Differences in the solvent structure in the active site may be related to variation in the affinities of inhibitors to different subtypes of neuraminidase.

Antiviral Agents↗
PubMed1987

Three-dimensional structure of neuraminidase of subtype N9 from an avian influenza virus.

Neuraminidases from different subtypes of influenza virus are characterized by the absence of serological cross-reactivity and an amino acid sequence homology of approximately 50%. The three-dimensional structure of the neuraminidase antigen of subtype N9 from an avian influenza virus (A/tern/Australia/G70c/75) has been determined by X-ray crystallography and shown to be folded similarly to neuraminidase of subtype N2 isolated from a human influenza virus. This result demonstrates that absence of immunological cross-reactivity is no measure of dissimilarity of polypeptide chain folding. Small differences in the way in which the subunits are organized around the molecular fourfold axis are observed. Insertions and deletions with respect to subtype N2 neuraminidase occur in four regions, only one of which is located within the major antigenic determinants around the enzyme active site.

Amino Acid Sequence↗
PubMed1998

GS4071 is a slow-binding inhibitor of influenza neuraminidase from both A and B strains.

The kinetics of inhibition of purified influenza neuraminidases from A/Tokyo/3/67 and B/Memphis/3/89 influenza viruses by (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene- 1-carboxylic acid (GS4071) were investigated. Progress curve experiments established that GS4071 is a time dependent inhibitor of both A and B strains of influenza neuraminidase. The apparent association and dissociation rate constants, as well as the overall Ki values, were only modestly different for the two neuraminidase strains. The time dependent inhibition phenomenon, often referred to as slow-binding inhibition, appears to be a consequence of the very slow rate of dissociation of the compound from influenza neuraminidase.

Acetamides↗
PubMed1993

Three-dimensional structure of influenza A N9 neuraminidase and its complex with the inhibitor 2-deoxy 2,3-dehydro-N-acetyl neuraminic acid.

We present here the three-dimensional structure of neuraminidase (E.C. 3.2.1.18) from influenza virus A/Tern/Australia/G70c/75 (N9), determined by the method of multiple isomorphous replacement, and the structure of the neuraminidase complexed with an inhibitor, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid (DANA). Native and inhibitor complex crystals are isomorphous and belong to space group I432 with unit cell dimensions of 183.78 A. The native enzyme structure and the inhibitor complex structure have been refined at 2.5 A and 2.8 A resolution, respectively, with crystallographic R-factor values of 0.193 for the native enzyme, and 0.179 for the inhibitor complex. The current enzyme model includes 387 amino acid residues which comprise the asymmetric unit. The root-mean-square deviation from ideal values is 0.013 A for bond lengths and 1.6 degree for bond angles. The neuraminidase (NA), as proteolytically cleaved from the virus, retains full enzymatic and antigenic activity, and is a box-shaped tetramer with edge lengths of 90 A and a maximal depth of 60 A. The NA tetramers are composed of crystallographically equivalent monomers related by circular 4-fold symmetry. Each monomer folds into six antiparallel beta-sheets of four strands. The secondary structure composition is 50% beta-sheet. The remaining 50% of the residues form 24 strand-connecting loops or turns. One of the loops contains a small alpha-helix. The structure of the complex of NA with DANA, a transition state analog, has enabled us to identify and characterize the site of enzyme catalysis. The center of mass of bound inhibitor is 32 A from the 4-fold axis of the tetramer, lodged at the end of a shallow crater of diameter 16 A with a depth of 8 to 10 A. There are 12 amino acid residues that directly bind DANA, with a further six conserved amino acids lining the active site pocket. The neuraminidase inhibitor complex provides a three-dimensional model which will be used to further the understanding of enzymatic hydrolysis and aid the design of specific, antineuraminidase antiviral compounds.

Binding Sites↗
PubMed1999

Novel aromatic inhibitors of influenza virus neuraminidase make selective interactions with conserved residues and water molecules in the active site.

The active site of type A or B influenza virus neuraminidase is composed of 11 conserved residues that directly interact with the substrate, sialic acid. An aromatic benzene ring has been used to replace the pyranose of sialic acid in our design of novel neuraminidase inhibitors. A bis(hydroxymethyl)pyrrolidinone ring was constructed in place of the N-acetyl group on the sialic acid. The hydroxymethyl groups replace two active site water molecules, which resulted in the high affinity of the nanomolar inhibitors. However, these inhibitors have greater potency for type A influenza virus than for type B influenza virus. To resolve the differences, we determined the X-ray crystal structure of three benzoic acid substituted inhibitors bound to the active site of B/Lee/40 neuraminidase. The investigation of a hydrophobic aliphatic group and a hydrophilic guanidino group on the aromatic inhibitors shows changes in the interaction with the active site residue Glu275. The results provide an explanation for the difference in efficacy of these inhibitors against types A and B viruses, even though the 11 active site residues of the neuraminidase are conserved.

Benzoic Acid↗
PubMed1995

Extracellular neuraminidase production by Pasteurella species isolated from infected animals.

A total of 721 field isolates of various Pasteurella species (haemolytica, multocida, and testudinis) from various regions of the United States were examined for extracellular neuraminidase production. All strains were grown and tested in the same way. Included were 372 P. haemolytica serotype 1 isolates, 181 P. haemolytica serotype 2 isolates, 63 P. haemolytica serotype 6 isolates, 101 Pasteurella multocida isolates, and 4 Pasteurella testudinis isolates. All Pasteurella species examined produced the enzyme. The data revealed the following: (1) Several transfers of P. haemolytica strains on blood agar medium did not cause a decrease in enzyme activity. (2) P. haemolytica serotypes 2 and 6 produce more neuraminidase than P. haemolytica serotype 1, P. multocida, and P. testudinis isolates. (3) There was no apparent change in neuraminidase production by P. haemolytica serotypes 1 and 2 obtained from the same animal taken on different days in the feedyard. (4) There was no significant change in neuraminidase production by P. haemolytica serotype 2 isolates taken from the same animal at the auction market and later at the feedyard.

Animals↗
PubMed1983

Functional defects of fowl plague virus temperature-sensitive mutant having mutation in the neuraminidase.

A fowl plague virus (FPV) temperature-sensitive mutant ts 5 having mutation lesions in the gene coding for the neuraminidase has been obtained. The mutant induced synthesis of cRNA, vRNA and proteins in cells under non-permissive conditions, but formation of virions including non-infectious ones was defective. The neuraminidase and haemagglutinin synthesized under non-permissive conditions possessed functional activity and could migrate from the rough endoplasmic reticulum into plasma membranes; however, cleavage of the haemagglutinin was reduced. In ts 5-infected cells under non-permissive conditions the synthesis of segments 5 and 8 of cRNA and vRNA was predominant both early and late in the reproduction cycle, and the synthesis of P1, P2, P3, HA and M proteins was reduced after approximately 3 hours. The data obtained suggest that involvement of the neuraminidase in the formation of infectious virions may have no direct association with the enzymatic activity of this protein, and that the mutation in the neuraminidase may affect regulation of replication and transcription processes.

Cell Membrane↗
PubMed1979

Selective inactivation of hemagglutinin and neuraminidase on mumps virus.

The thermal stability and the effect of guanidine on the hemagglutinin and neuraminidase of three strains of mumps virus were compared. The heat inactivation of hemagglutinin resulted in the concomitant loss of neuraminidase. The effect of guanidine at various molarities showed that the neuraminidase was more sensitive than the hemagglutinin and a selective inactivation was obtained after exposure to 1.5 M guanidine. However differences in sensitivity of both activities (hemagglutinin and neuraminidase) to heat and guanidine inactivations were observed among strains and correlated with differential susceptibility to non-specific inhibitors of the strains.

Allantois↗
PubMed1995

Changes in the neuraminidase of neurovirulent influenza virus strains.

The influenza virus A/WS/33 has been adapted to mouse brain to produce two neurovirulent derivatives, A/NWS/33 (NWS) and A/WSN/33 (WSN), with the viral neuraminidase gene shown to be the major determinant of neurovirulence. The complete nucleotide sequence of the NA genes from each strain has been determined, which has allowed the identification of changes that have occurred during adaptation to mouse brain. Five changes are shared by the neurovirulent strains. Comparison to the known neuraminidase structure has identified four of these that may affect the active site of the enzyme. In addition, significant differences in the properties of the neuraminidase from the neurovirulent strains were observed relative to the parent strain. While no correlation was observed between neurovirulence and overall neuraminidase activity or preference for a particular N-substitution, the enzymes from both neurovirulent strains showed an increased preference for small substrates and those with 2-->3 linkages, and their activity was potentiated by Ca2+ ions.

Adaptation, Physiological↗
PubMed2001

Clinical presentation of congenital sialidosis in a patient with a neuraminidase gene frameshift mutation.

UNLABELLED: Congenital sialidosis is a rare lysosomal storage disease caused by a primary neuraminidase deficiency which results from defects in the neuraminidase gene on chromosome 6p. The inheritance is autosomal recessive. Patients exhibit excessive urinary excretion of bound sialic acid and decreased or undetectable amounts of neuraminidase activity in various tissues. The clinical expression is variable, but ascites and hepatosplenomegaly are hallmarks of the disease. Skeletal abnormalities, facial dysmorphism and inguinal herniae have been described in most of the few reported cases. We describe a baby girl with biochemically proven sialidosis, who in addition to the above clinical features, had severely dilated coronary arteries, excessive retinal vascular tortuosity and an erythematous, macular rash. Homozygosity for a frameshift mutation at residue 623 of the neuraminidase cDNA was found. We speculate that the additional features found in our patient might be associated with the here described genotype of congenital sialidosis. CONCLUSION: Severely dilated coronary arteries, excessive retinal vascular tortuosity and an erythematous macular rash might be associated features of congenital sialidosis.

Abnormalities, Multiple↗
PubMedDate not supplied

Evaluation of a neuraminidase detection assay for the rapid detection of influenza A and B virus in children.

A prototype version of a new diagnostic assay for influenza A and B (Zstat Flutrade mark) based on detection of viral neuraminidase was evaluated and compared to culture in 196 clinical samples. Children with respiratory illnesses were prospectively evaluated at a pediatrician's office and at a large children's hospital using the neuraminidase assay and viral culture performed on respiratory secretions. Influenza virus was isolated from 51 samples and 83 were positive by the neuraminidase assay. When compared to culture the sensitivity of the assay was 96%, specificity was 77%, positive predictive value was 59%, and negative predictive value was 98%. Testing in the laboratory of pure cultures of bacteria and non-influenza viruses frequently found in the respiratory tract showed 0% cross-reactivity with the neuraminidase assay and 100% specificity for influenza virus in vitro. This new assay provided useful information for the preliminary diagnosis of influenza A and B infections and appears to be suitable for both point-of-care use in the physician's office and rapid diagnosis in a virology laboratory. The high sensitivity makes it particularly useful as a screening test for exclusion of influenza A and B infections. To confirm the diagnosis and exclude a false-positive result, as well as to determine the influenza virus type, a viral culture may be considered.

Animals↗
PubMed1983

The interaction of substrate-related ketals with bacterial and viral neuraminidases.

Arthrobacter sialophilus neuraminidase catalyzes the hydration of 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic acid (2,3-dehydro-AcNeu) with Km and kcat values of 8.9 X 10(-4) M and 6.40 X 10(-4) s-1, respectively. The methyl ester of 2,3-dehydro-AcNeu as well as 2,3-dehydro-4-epi-AcNeu are also hydrated by the enzyme. The product resulting from the enzymatic hydration of 2,3-dehydro-AcNeu is N-acetylneuraminic acid. A series of derivatives of 2,3-dehydro-AcNeu (K1, 1.60 X 10(-6) M) including 2,3-dehydro-4-epi-AcNeu (2.10 X 10(-4) M) and 2,3-dehydro-4-keto-AcNeu (K1 = 6.10 X 10(-5) M) were each competitive inhibitors of the enzyme. The methyl esters of these ketal derivatives were also competitive enzyme inhibitors. Dissociation constants for these ketals were determined independently by fluorescence enzyme titrations which gave values similar to those found kinetically. These six relatives of 2,3-dehydro-AcNeu were also competitive inhibitors for the influenza viral neuraminidases. For the viral neuraminidases, the dissociation constant for 2,3-dehydro-AcNeu and its methyl ester were 2.40 X 10(-6) and 1.17 X 10(-3) M, respectively. The interpretation placed upon the K1 values determined for these ketals against the Arthrobacter versus influenza neuraminidases is that the bacterial enzyme has a more flexible glycone binding site.

Arthrobacter↗
PubMed1983

Two genetically different MU-NANA neuraminidases in human leucocytes.

Human leucocytes contain two different MU-NANA neuraminidases, which can be distinguished by Concanavalin A binding. The Con A binding form is predominant in lymphocytes (more than 80%) and the non-binding form predominates in granulocytes. The pH optima of both these neuraminidases as well as their subcellular localization as determined by Percoll gradient centrifugation suggest that they are both lysosomal. Immunological studies indicate that the Con A binding form is present in a complex with beta-galactosidase whereas the non-binding form is not. Leucocytes from patients with sialidosis or galactosialidosis are deficient in the Con A binding neuraminidase, whereas the non-binding form is normal. In sialolipidosis both forms are normal. These results demonstrate that leucocytes contain at least two genetically different MU-NANA neuraminidases. Thus, the use of leucocytes should be avoided for the diagnosis of sialidosis and galactosialidosis, and isolated lymphocytes should be used to obtain reliable results.

Chromatography, Affinity↗
PubMed1979

Neuraminidase activity in liver and brain from patients with I-cell disease.

The activity of neuraminidase in liver and brain from I-cell disease (Mucolipidosis II) was investigated. Neuraminidase activities using two substrates [alpha-L-N-acetylneuraminosyl(2 leads to 3)lactose and alpha-L-N-acetylneuraminosyl(2 leads to 6)lactose] were reduced in the supernatant and sedimentable fractions obtained in isotonic KCl. The activity of beta-D-galactosidase was also reduced in the liver; on the other hand, both neuraminidase fractions were normal, although beta-galactosidase activities were markedly reduced. In view of these results, it is suggested that the defect of neuraminidase is not directly responsible for the primary etiology of I-cell disease.

Brain↗