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Results for “NEURAMINIDASE”

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PubMed1996

Association of N-acetylgalactosamine-6-sulfate sulfatase with the multienzyme lysosomal complex of beta-galactosidase, cathepsin A, and neuraminidase. Possible implication for intralysosomal catabolism of keratan sulfate.

N-Acetylgalactosamine-6-sulfate sulfatase (GALNS) catalyzes the first step of intralysosomal keratan sulfate (KS) catabolism. In Morquio type A syndrome GALNS deficiency causes the accumulation of KS in tissues and results in generalized skeletal dysplasia in affected patients. We show that in normal cells GALNS is in a 1.27-MDa complex with three other lysosomal hydrolases: beta-galactosidase, alpha-neuraminidase, and cathepsin A (protective protein). GALNS copurifies with the complex by different chromatography techniques: affinity chromatography on both cathepsin A-binding and beta-galactosidase-binding columns, gel filtration, and chromatofocusing. Anti-human cathepsin A rabbit antiserum coprecipitates GALNS together with cathepsin A, beta-galactosidase, and alpha-neuraminidase in both a purified preparation of the 1. 27-MDa complex and crude glycoprotein fraction from human placenta extract. Gel filtration analysis of fibroblast extracts of patients deficient in either beta-galactosidase (beta-galactosidosis) or cathepsin A (galactosialidosis), which accumulate KS, demonstrates that the 1.27-MDa complex is disrupted and that GALNS is present only in free homodimeric form. The GALNS activity and cross-reacting material are reduced in the fibroblasts of patients affected with galactosialidosis, indicating that the complex with cathepsin A may protect GALNS in the lysosome. We suggest that the 1.27-MDa complex of lysosomal hydrolases is essential for KS catabolism and that the disruption of this complex may be responsible for the KS accumulation in beta-galactosidosis and galactosialidosis patients.

Animals↗

PubMed1983

Effect of trypsin and neuraminidase on the infectivity of Plasmodium gallinaceum sporozoites.

An investigation was carried out to examine the effect of trypsin and neuraminidase on the infectivity of Plasmodium gallinaceum sporozoites. The sporozoites were obtained from freshly dissected salivary glands of Aedes aegypti: their infectivity was assayed by their ability to produce a patent parasitaemia following their inoculation into day-old chicks. Infectivity was abolished by treatment of the sporozoites with trypsin at a concentration of 100 micrograms ml-1 for 30 minutes at 30 degrees C. Incubation of sporozoites with neuraminidase (1 unit ml-1 for 30 minutes at 37 degrees C), however, had no effect on their infectivity. It is suggested that the proteolytic action of the trypsin removed a presumed receptor for the cells of the reticulo-endothelial system from the surface of the sporozoite.

Aedes↗

PubMed1988

Separation of hemagglutinin and neuraminidase from influenza virus membrane by column displacement electrophoresis (isotachophoresis) with preservation of their activities.

Triton X-100-solubilized membrane glycoproteins (neuraminidase and hemagglutinin) from purified equine influenza virus particles were separated by column displacement electrophoresis (isotachophoresis) in the presence of Pharmalyte spacers. Electrophoresis was performed in a 1.80 cm glass electrophoresis column with Sephadex G-25 Fine serving as supporting medium. Triton X-100 was present in the system to suppress protein aggregation. Neuraminidase and hemagglutinin activities were preserved and appeared in the electropherogram as separate peaks with some overlapping.

Cell Membrane↗

PubMed2001

Molecular modeling of sialyloligosaccharide fragments into the active site of influenza virus N9 neuraminidase.

Molecular modeling studies have been carried out to investigate the interactions between substrate sialyloligosaccharide (SOS) fragments bearing different glycosidic linkages and influenza virus N9 neuraminidase, a surface glycoprotein of influenza virus subtype N9. The studies revealed that the allowed orientation for sialic acid (SA) is less than 1% in the Eulerian space at the active site. The active site of this enzyme has enough space to accommodate various SOS fragments, NeuNAcalpha(2-3)Gal, NeuNAcalpha(2-6)Gal, NeuNAcalpha(2-8)NeuNAc and NeuNAcalpha(2-9)NeuNAc, but on specific conformations. In the bound conformation, among these substrates there exists a conformational similarity leading to a structural similarity, which may be an essential requirement for the cleavage activity of the neuraminidases irrespective of the type of glycosidic linkage.

Carbohydrate Conformation↗

PubMed2004

Protection of mice with recombinant influenza virus neuraminidase.

Contemporary influenza vaccines are standardized with respect to their content of hemagglutinin, the major virus antigen. Although the immunizing effect of viral neuraminidase--the less abundant of the 2 major surface glycoproteins--has been well documented in experimental animals, the importance of the purified recombinant protein has not yet been adequately assessed in animals or humans. We demonstrate that different lots of a baculovirus-derived recombinant N2 protein, in the absence of other influenza virus proteins, can induce neuraminidase-specific antibodies, reduce the replication of both homologous and heterovariant virus in mice, and suppress disease, as it is manifested by total body weight loss.

Animals↗

PubMed1997

Design of aromatic inhibitors of influenza virus neuraminidase.

Structure-based drug design, a terminology used to describe rational drug design by complementing the structure, spatially and chemically, of the target macromolecule, is rapidly developing as one of the innovative approaches to drug discovery. A growing volume of protein structure data and new techniques of protein structure determination make this all possible. The method of structure-based drug design and a specific example of the design of influenza virus neuraminidase is briefly presented. A whole new class of influenza virus neuraminidase inhibitors has been designed that can potentially be developed as antiinfluenza drugs.

Antiviral Agents↗

PubMed1997

Antigenic structure, function, and evolution of the hemagglutinin-neuraminidase protein of human parainfluenza virus type 1.

Twenty-two monoclonal antibodies directed to the hemagglutinin-neuraminidase protein of human parainfluenza virus type 1 (HPIV-1) were used in competition assays to create an antigenic map of neutralization sites. Eighty-seven clinical strains isolated over 35 years from multiple geographic regions were reacted in ELISA, hemagglutinin-inhibition, and microneutralization assays with these monoclonal antibodies. Together these assays revealed 21 epitopes on five nonoverlapping antigenic sites (I, III-VI) with a sixth (II) bridging site connecting sites I, III, and IV. Only 7 (33%) of these epitopes were conserved among all isolates. Previously described HPIV-1 genotypes were associated with the presence or absence of specific antigenic sites and evidence of probable immune selection within genotypes. Two sites were present on all isolates tested (III, V), and one (VI, genotype A) has not been found for 15 years. Forty hemagglutinin-neuraminidase nucleotide sequences were analyzed in terms of homology, structure, and evolution. These data may be useful in future epidemiologic, therapeutic, or vaccine-related work.

Antibodies, Monoclonal↗

PubMed1979

Evaluation of a neuraminidase-specific influenza A virus vaccine in children: antibody responses and effects on two successive outbreaks of natural infection.

Three groups of children were immunized with an inactivated Port Chalmers (H3ChN2Ch) influenza vaccine (group A), a neuraminidase-specific (Heq1N2Ch) influenza vaccine (group B), or a placebo. Immunization induced seroconversion for H3Ch and N2Ch-specific antibody in group A and for N2Ch antibody in group B. The protective efficacies observed against naturally acquired illness with the Port Chalmers strain of influneza A virus were 68.7% and 37.4% in groups A and B, respectively, in comparison to the placebo group, and those against illness produced by the subsequent outbreak of the Victoria strain were 80.0% and 72.7%. These data support the role of neuraminidase-specific immunization in protection against influenza. Although the degree of protection after vaccination with the Heq1N2Ch vaccine was less than that provided by the biphasic H3ChN2Ch vaccine against the Port Chalmers strain, it appeared to be similar in the two vaccine study groups against the Victoria strain.

Adolescent↗

PubMed2003

A new millennium conundrum: how to use a powerful class of influenza anti-neuraminidase drugs (NAIs) in the community.

Influenza A and B viruses cause serious medical problems and social disruption every year in particular countries of the world. The virus is notoriously fickle and may attack citizens in say two adjacent countries but not the third. More rarely a global pandemic virus emerges causing millions of deaths worldwide. The SARS outbreak has illuminated weaknesses in planning for sudden outbreaks of disease in a modern society and in particular how panic can grip and cause intense economic disruption. Many communities in the world are neither prepared for a global pandemic nor a very acute epidemic of influenza. The neuraminidase inhibitors (NAIs) are a new class of antiviral drug targeting a viral influenza enzyme, the neuraminidase, which acts both to facilitate virus infection of cells by clearing a passage through otherwise protective respiratory fluids and also by helping release of the virus by cutting the chemical umbilical cord which links up the virus to the infected cell. Extensive laboratory studies of the two molecules zanamivir and oseltamivir have shown that they block all influenza A and B viruses yet tested and would, in theory, even inhibit the 1918 pandemic virus. Both drugs can be used prophylactically to prevent spread of infection in families and communities where 80-90% protection has been documented. The therapeutic effects are also strong in adults and children abbreviating infection, reducing quantities of excreted virus and reducing antibiotic prescriptions. The drugs have to be taken within 48 h of the onset of symptoms. Drug resistance is not a problem at present because although such mutants occur the mutants are compromised and are less virulent than their drug-sensitive parents and they spread less easily. The two drugs could be stockpiled to prepare for an influenza pandemic but, importantly, clinical and scientific experience need to be gained by using these inhibitors in the yearly conflagrations of epidemic influenza, which unchecked do great harm to our communities.

Antiviral Agents↗

PubMed2003

Variation in the divalent cation requirements of influenza a virus N2 neuraminidases.

Influenza virus N2 neuraminidases were chromatographically purified from several vaccine candidate strains from 1957 to 1994. Enzymatic kinetic parameters and immunogenicity were tested for each strain. For each NA tested, with ionic strength held constant, Ca(2+) or Mg(2+) increased the initial rate of enzymatic activity. Earlier N2-NA strains had the highest initial velocity, V(max)/K(m) and V(max). There were significant differences among the influenza virus strains in enzymatic activity before and after addition of Ca(2+) or Mg(2+): V(max)/K(m) varied from 0.54 M(-1) s(-1) to 0.88 M(-1) s(-1) and V(max) varied from 2.45 s(-1) to 4.3 s(-1) before the addition of a divalent cation; and increased approximately 2-fold each of these kinetic parameters for each strain after the addition of exogenous Ca(2+) or Mg(2+). Exhaustive dialysis with EDTA reduced the initial velocity of each strain with significant differences found among strains, with a range of 0.1% to 8% of original activity. Activity was partially restored by the addition of exogenous Ca(2+) or Mg(2+), varying from 8% to 60% of pre-dialysis levels, but original rates were not achieved. This reduction in enzymatic activity for the tested strains (i.e., A/Japan/57 and A/Johannesburg/94) was accompanied by a parallel decrease in NA-immunogenicity, with antibody response decreasing by as much as 76% as measured by NI titer, and ELISA titer decreasing by as much as 68%. The addition of Ca(2+) or Mg(2+) to the post-dialysis sample restored immunogenicity to as much as 80% of pre-dialysis NI titers and as much as 78% of pre-dialysis ELISA titers. Dialysis had the least effect on early strains as measured by enzymatic kinetic parameters and immunogenicity studies. Zn(2+) had a slight inhibitory effect on the activity of all tested strains. Review of the nucleic acid sequence of each of these strains could not predict their enzymatic activity, immunogenicity or response to dialysis. If immunity against neuraminidase is desirable in vaccination against influenza, selection of vaccine candidate strains must include not only analysis of antigenic changes and sequence analysis but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production.

Amino Acid Sequence↗

PubMed1975

Augmented uptake of neuraminidase-treated sheep red blood cells: participation of opsonic factors.

Neuraminidase from Vibrio cholerae (VCN) was used to treat sheep red blood cells (SRBC) which were then incubated in vitro with murine peritoneal macrophages. The uptake of VCN-treated SRBC by macrophages was greater than the uptake of SRBC not treated with VCN. SRBC opsonized with normal mouse serum (NMS) were taken up to a greater extent than untreated SRBC. SRBC treated with VCN and opsonized with NMS were phagocytosed to a greater extent than untreated SRBC, VCN-treated SRBC, or opsonized SRBC. Evidence demonstrated that factors in serum from normal C3H/HeJ mice augmented the uptake of VCN-treated SRBC in greater amounts than of normal SRBC. These findings were discussed in relation to the increased immunogenicity of neuraminidase-treated cells.

Animals↗

PubMed1982

Sequence of the N2 neuraminidase from influenza virus A/NT/60/68.

The complete sequence of the neuraminidase gene of influenza virus A/NT/60/68 (N2 subtype) was determined following cloning of full length complementary DNA into pBR322. Comparison of the predicted amino acid sequence with a closely related neuraminidase from A/Udorn/72 suggests that point mutations over an extensive region of the primary sequence can contribute to antigenic drift, although the region between amino acid residues 308 and 371 may be particularly significant.

Amino Acid Sequence↗

PubMed1981

Beta-galactosidase-neuraminidase deficiency: restoration of beta-galactosidase activity by protease inhibitors.

Beta-Galactosidase was partially restored by protease inhibitors, leupeptin, chymostatin and E-64 in cultured fibroblasts from three patients with beta-galactosidase-neuraminidase deficiency. Pepstatin did not activate this enzyme. Neuraminidase was not affected by any of these compounds in the culture medium. It was concluded that the activating effect was produced by a specific inhibition of thiol proteases.

Cells, Cultured↗

PubMed1984

Production of all follicle-stimulating hormone isohormones from a purified preparation by neuraminidase digestion.

Graded removal of sialic acid residues from a purified preparation of rat follicle-stimulating hormone (FSH; NIADDK-FSH-I-5) by neuraminidase digestion resulted in the production of FSH isohormones with isoelectric points identical to those found within pituitary tissue. In addition, each neuraminidase-produced FSH form exhibited a radioreceptor assay:radioimmunoassay ratio similar to that of its endogenously produced counterpart. Thus, the molecular basis for FSH microheterogeneity appears to be due to the varying degree of sialylation into a common FSH protein core. We have demonstrated that the pituitary gland produces different amounts of these FSH isohormones depending upon the surrounding endocrine environment. The results of the present studies suggest the existence of biochemical mechanisms within the pituitary that influence sialic acid incorporation into FSH. These mechanisms appear to be sensitive to the surrounding hormonal milieu and serve to alter the intensity of the FSH stimulus delivered to the gonad.

Animals↗

PubMed1998

Analysis of human ocular mucus: effects of neuraminidase and chitinase enzymes.

PURPOSE: Our goal was to establish the characteristic migration pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of high molecular weight mucins from human ocular mucus and the effects of treatment with exo- and endoglycosidases. METHODS: Chromatography by gel filtration with Sepharose CL-4B was performed on samples collected from normal subjects. Human ocular mucins from the high molecular weight fraction were digested with exoglycosidases (neuraminidase, N-acetyl-beta-D-glucosaminidase, beta-D-glucosidase) and endoglycosidases (chitinase, lysozyme); and the resulting products were analyzed by electrophoresis. Carbohydrate identification was performed using lectin probes. RESULTS: The migration of the ocular mucins on SDS-PAGE stopped after treatment with neuraminidase, which removes the terminal negatively charged sialic acid residues from mucin. Chitinase (beta(1-4)N-acetylglucosaminidase) treatment increased the electrophoretic migration of mucins. Staining with wheat germ agglutinin and Maackia amurensis agglutinin lectins showed that these mucins contain beta(1-4)NAcGlc and SAa(2-3)Gal linkages. CONCLUSIONS: These studies demonstrate that the mobility of human ocular mucins on SDS-PAGE is determined by their intrinsic total negative charge and is not dependent on SDS treatment. It is interesting to note that human ocular mucus contains chitinous material resistant to lacrimal lysozyme, which is accessible to chitinase, an enzyme now found to degrade human ocular mucins. These chitinous linkages could be in part responsible for the mucus resistance.

Adolescent↗

PubMed1990

Resistance to human serum of gonococci in urethral exudates is reduced by neuraminidase.

Gonococci examined directly from urethral exudates are resistant to killing by human serum, but most strains become susceptible on subculture. Previous work with gonococci grown in vitro indicates that resistance in vivo is due to sialylation of gonococcal lipopolysaccharide (LPS) by a host factor, cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) or a related compound present in urogenital secretions and blood cells including phagocytes, which exude during inflammation. This sialylation inhibits the reaction between bactericidal IgM in serum and its target LPS sites. Here, we confirm the indication by using gonococci grown in vivo. Crucial to the above conclusions was the marked reduction of CMP-NANA-conferred serum resistance when gonococci were treated with neuraminidase to remove sialyl groups from their LPS. We now show that the serum resistance of gonococci in urethral exudates was reduced by treatment with neuraminidase from more than 95% (calculated in relation to controls incubated with heated serum) to 2-11% according to sample and incubation time. Subculture of the gonococci also reduced resistance to 9-11% but resistance was restored to more than 95% by incubation with CMP-NANA. This work is the culmination of an investigation that underlines the need to identify specific host factors and the virulence determinants they induce in vivo in future studies of pathogenicity.

Blood Bactericidal Activity↗

PubMed1989

Three-dimensional structures of influenza virus neuraminidase-antibody complexes.

X-ray diffraction analysis of crystals of a monoclonal Fab fragment NC41 bound to a viral antigen, influenza virus neuraminidase, shows an epitope involving five surface loops of the antigen. In addition it reveals an unusual pairing pattern between the domains of light and heavy chains in the variable module of the antibody. We interpret this result to imply that association with antigen can induce changes in the quaternary structure of the Fab, through a sliding of domains at the variable light/variable heavy chains (VL-VH) interface. In addition, Fab binding has altered the conformation of some of the surface loops of the antigen. The structure of the NC10 Fab-neuraminidase complex has now also been solved. It binds an epitope that overlaps the NC41 epitope. In this structure, there is no electron density for the C-module of the Fab fragment, implying it is disordered in the crystal lattice. The implications of these, and other antibody-antigen structures, for immune recognition are discussed.

Antigen-Antibody Complex↗

PubMed1983

A temperature-sensitive mutant of Newcastle disease virus which is affected in both haemagglutinin-neuraminidase and matrix proteins.

Virions prepared from a non-revertible temperature-sensitive (ts) mutant (ts53) of Newcastle disease virus (NDV) grown in ovo at the permissive temperature (34 degrees C) possessed thermolabile haemagglutination and neuraminidase activities compared with parental (ts+) virions. Purified haemagglutinin-neuraminidase (HN) protein from ts53 virions was also more thermolabile than ts+ HN protein. SDS-PAGE analysis of [3H]leucine pulse- and pulse/chase-labelled NDV proteins synthesized in chick embryo fibroblasts following infection with ts+ and ts53 virus revealed that ts53 matrix (M) protein was unstable and disappeared during chase incubations only at the non-permissive temperature (42 degrees C). The non-revertibility of the ts53 mutant may indicate that it is a double mutant affected in both HN and M genes; alternatively this mutant may only be affected in the HN gene, the close physical association of the thermolabile HN with the M protein during virus maturation resulting in the lack of protection of the M protein from the action of cellular proteases at the non-permissive temperature.

Cycloheximide↗