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PubMed1984

Detection of the granulocyte-specific antigen 3-fucosyl-N-acetyl-lactosamine on leukemic cells after neuraminidase treatment.

The carbohydrate antigen 3-fucosyl-N-acetyl-lactosamine (FAL) is expressed on human granulocytes and is detected by a monoclonal antibody B4.3. After neuraminidase treatment, this structure can also be detected on monocytes and on the cells of nearly all acute myeloid leukemia patients (38/39). It is then also present on the cells of a number of CALLA-positive lymphatic leukemias (8/18), but not on T-ALL and B-ALL cells. On cells of patients with AUL, the antigen is then detected in many TdT+ cases, but not in TdT- cases.

Antibodies, Monoclonal↗

PubMed1978

Red cell aging. II. Anomalous electrophoretic properties of neuraminidase treated human erythrocytes.

Desialylation of human red blood cells (RBC) by Vibrio cholerae neuraminidase (VCN) was found to produce cells with electrophoretic properties which were inconsistent with the view of simple loss of N-acetylneuraminic acid (NANA) as the sole effect of VCN treatment. Modification of human RBC with 50--350 U VCN/10(10) RBC for one hour at 37 degrees C releases 90-100% of the NANA and produces a progressive decrease towards zero in their electrophoretic mobilities when measured in 0.15 M NaCl (pH 7.2) at 25 degrees C. The appearance of positive groups on the desialylated cells was indicated by the VCN-treated cells displaying positive mobilities below approximately pH 5.5 and increased negative mobilities at approximately pH 9 as well as substantial increases in their mobility at neutral pH following treatment with formaldehyde. Adsorption of about 95% of the VCN activity at 0 degrees C to the RBC did not produce any significant change in their electrophoretic mobilities thus indicating that the observed changes in the electrophoretic properties of the RBC following VCN treatment could not be attributable to adsorption of VCN. These studies indicate that the cationic charge groups which appear at the electrophoretic surface of the RBC after VCN treatment are probably of endogenous origin. It is suggested that this alteration rather than simple NANA release may operate to shorten the in vivo survival time of desialylated red cells.

Adult↗

PubMed1991

Neuraminidase-induced thrombocytopenia in mice: effects on thrombopoiesis.

Previous studies to examine the effects of thrombocytopenia on thrombopoiesis have generally utilized immune-mediated platelet depletion. We have developed a nonimmune model to exclude the possibility that adverse immune-mediated effects have been misinterpreted as the physiological response to stimulation of thrombopoiesis. Thrombopoiesis was examined in mice after induction of thrombocytopenia with a single injection of the nonimmunologic agent neuraminidase (Ndase). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 8, 12, 24, 48, 72, 96, and 120 hr after administration of Ndase. Eight to 48 hr after induction of acute, severe thrombocytopenia (mean platelet count less than 50,000/microliters), the medians of the platelet sectional area distributions, as measured morphometrically, were significantly greater than the median platelet sectional area of pooled controls. The maximum median value for platelet sectional area was observed at 24 hr. The largest platelets in these samples contained more profiles of endoplasmic reticulum and Golgi cisternae, and a lower concentration of surface-connected canalicular system, as compared with normal platelets. By 72 hr post-injection of Ndase, virtually all platelets exhibited normal size and organelle complement. Mean platelet volumes, determined by electrical impedance analysis, paralleled the serial changes in platelet sectional areas. MK frequency and ploidy, measured by two-color fluorescence activated flow cytometry, were unchanged 12 and 24 hr following Ndase. At 48 hr, total MK frequency increased significantly (P less than 0.01) from 0.11% to 0.17%, and MK ploidy distribution shifted with a reduction in 16N MK (P less than 0.005) and an increase in 32N MK (P less than 0.01). MK ploidy was maximally altered from normal at 72 hr with increased 32N MK frequency (32.0%, P less than 0.001) and increased 64N MK frequency (2.4%, P less than 0.005). Morphologic and morphometric examination of MK at all time points did not reveal detectable changes from normal in cytoplasmic appearance or size, respectively. Therefore, we have demonstrated marked alterations of morphology and size of platelets, and of MK ploidy, using this nonimmunologic model. These studies further support our previous observations that megakaryocyte ploidy and platelet volume are independently regulated in response to depletion of the circulating platelet mass, and they show that these changes are not dependent upon the mechanism of thrombocytopenia.

Animals↗

PubMed2002

Divergent evolution of hemagglutinin and neuraminidase genes in recent influenza A:H3N2 viruses isolated in Canada.

Limited information is available concerning the molecular drift of the influenza neuraminidase (NA) genes. We report on the genetic variability of the NA gene from 31 influenza A:H3N2 viruses isolated in the Province of Québec (Canada) during the last three flu seasons (1997-2000). Amino acid substitutions within the NA protein were observed at rates of 1.01% and 0.45% between strains of the 1997-1998 and 1998-1999 seasons and between those of the 1998-1999 and 1999-2000 seasons, respectively. In most strains (28/31), amino acid changes occurred within at least one of four codons (197, 339, 370, and 401) previously implicated as antigenic sites. The 8 functional and 10 framework residues that compose the catalytic site of the NA enzyme were completely conserved over the study period. All isolates contained the seven conserved asparagine-linked glycosylation sites found in the NA of the progenitor A/Hong Kong/8/68 strain. In addition, most strains (30/31) had an eighth potential glycosylation site at position 329, whereas a ninth one was found at position 93 in 16 strains. The NA of all strains in this study was related to that of the vaccine strain A/Beijing/353/89, whereas the HA genes of these strains were related to the A/Beijing/32/92 vaccine strain. Thus, it appears that recent influenza strains continue to evolve from a reassortant produced during the cocirculation of the two above variants. Interestingly, some strains whose HA genes were closely related showed significant differences in their NA genes and conversely. This study confirms the importance of analyzing both the NA and the HA genes to assess the evolution of recent influenza epidemic strains.

Canada↗

PubMed2004

Changes in the hemagglutinins and neuraminidases of human influenza B viruses isolated in Italy during the 2001-02, 2002-03, and 2003-04 seasons.

Throughout most of the last decade, B/Yamagata/16/88-lineage influenza viruses were predominant among the B isolates circulating worldwide, whereas B/Victoria/2/87-lineage viruses were isolated infrequently and restricted geographically to eastern Asia. During the 2001-02 influenza season, B/Victoria/2/87-lineage viruses re-emerged in North America and Europe and spread worldwide. Virological surveillance in Italy during that season showed wide circulation of influenza B viruses, of which most were antigenically related to the B/Sichuan/379/99 (Yamagata-lineage) vaccine strain, together with a smaller number of B viruses antigenically similar to B/HongKong/330/01, a recent B/Victoria/2/87-lineage antigenic variant. In the subsequent 2002-03 epidemic season, B viruses with a Victoria-lineage hemagglutinin (HA), more closely related to that of B/Shandong/7/97, were isolated exclusively. Similar strains have continued to predominate among the few B viruses isolated in Italy during last season (2003-04), although most influenza B viruses, isolated sporadically elsewhere in Europe, again belong to the Yamagata-lineage. In the present study, phylogenetic analyses of the HA and neuraminidase (NA) genes of representative B strains, isolated throughout Italy during 2001-04, showed that during the first influenza season the NA genes, as well as the HA genes, separated into the two distinct clades, the Yamagata- and Victoria-lineages, and showed no evidence of genetic reassortment. On the contrary, all the B viruses isolated in the 2002-03 and most of those isolated in the 2003-04 epidemic season were "Victoria HA-Yamagata NA" reassortants similar to those isolated in other parts of the world, showing that these reassortants became established in the human population. The frequency of reassortment between HA and NA of distinct lineages and sublineages highlights again the importance of detailed molecular analyses of both surface glycoproteins in understanding the evolution of influenza B viruses.

Genetic Variation↗

PubMed1976

Cytotoxicity reactions during immunotherapy of melanoma with neuraminidase altered autologous tumor cells.

Lymphocyte cytotoxicity, serum cytotoxicity, and the ability of the serum to inhibit lymphocyte cytotoxicity (blocking effect) were studied in a melanoma patient treated with six monthly injections of her own (autologous) tumor cells incubated with neuraminidase to increase their antigenicity. The same tumor cells grown in tissue culture were used as target cells for the cytotoxicity test. Large fluctuations of blocking effect in the serum were found, which correlated with the clinical course of tumor removal, recurrence, and regression. After the fifth injection of autologous tumor cells, the blocking effect disappeared from the serum (unblocking). In general, changes in serum cytotoxicity corresponded with changes in the amount of blocking effect produced by the serum. The results suggest that active immunotherapy may play a role in the prevention of metastases and, that when used within the autologous system, the cytotoxicity test is valuable in studying response to this type of therapy.

Antigens, Neoplasm↗

PubMed1976

Neuraminidase immunotherapy: serum potentiation of lymphocyte cytotoxicity related to immunoglobulin levels.

Seven patients completed couses of immunotherapy using neuraminidase-altered autochthonous cells. Their response to therapy was monitored by a cytotoxicity assay using 3H-proline-tagged tumor cells from the patient's own cultured tumor in a strictly autologous system. Serum effects were measured by exposing the tumor target cells to serum to see whether this impeded (blocked) or augmented (potentiated) lymphocyte cytotoxicity. Three of the seven patients developed increasing degrees of serum blocking effect and all died within six months of completing therapy. Four of the seven showed rapidly decreasing blocking and three eventual potentiation. Three patients are living, improved, and free of evidence of tumor. There was an increase in average serum immunoglobulins in patients developing potentiation, and a decrease in those showing blocking. In any immunotherapy program attention must be given to in vitro monitoring studies, and such studies should include attention to the serum factors influencing host response.

Adult↗

PubMed1977

The combined effect of radiotherapy and neuraminidase-treated tumor cells on 3-methylcholanthrene-induced fibrosarcoma.

Active immunotherapy with tumor cells treated in vitro with Vibrio cholerae neuraminidase (VCN) plus mitomycin C augments the antitumor effects of local x irradiation in the treatment of firmly established methylcholanthrene-induced fibrosarcoma, MC-43, in syngeneic C3H/HeJ female mice. In most experiments, the inhibition of tumor growth was greater when VCN-treated tumor cells were combined with local irradiation than could be achieved with VCN-treated tumor cells or local irradiation alone. Even in those experiments in which the immunotherapeutic effect of VCN-treated cells was negligible, the combination of radiotherapy and immunotherapy appeared to be greater than irradiation alone. Similarly, total permanent regression of established tumors occurred more frequently after combined therapy than after immunotherapy or radiation therapy alone.

Animals↗

PubMed1989

Controlled clinical trial of adjuvant immunotherapy with BCG and neuraminidase-treated autologous tumour cells in large bowel cancer.

A controlled, randomised clinical trial of immunotherapy was performed in 301 patients with stage B or C colorectal cancer. The immunotherapy treatment consisted of 18 vaccinations over a 2 year period following surgery with a combination of BCG given by scarification plus subcutaneous injection of Vibrio cholera neuraminidase (VCN)-modified autologous tumour cells. Five year follow-up has now been completed in all patients. The immunotherapy did not alter either the disease-free interval or the overall survival of patients in comparison with a control group of patients not receiving immunotherapy.

Adenocarcinoma↗

PubMed1994

Influenza virus neuraminidase: structure, antibodies, and inhibitors.

The determination of the 3-dimensional structure of the influenza virus neuraminidase in 1983 has served as a platform for understanding interactions between antibodies and protein antigens, for investigating antigenic variation in influenza viruses, and for devising new inhibitors of the enzyme. That work is reviewed here, together with more recent developments that have resulted in one of the inhibitors entering clinical trials as an anti-influenza virus drug.

Antibodies↗

PubMed1992

The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor.

Crystallographic studies of neuraminidase-sialic acid complexes indicate that sialic acid is distorted on binding the enzyme. Three arginine residues on the enzyme interact with the carboxylate group of the sugar which is observed to be equatorial to the saccharide ring as a consequence of its distorted geometry. The glycosidic oxygen is positioned within hydrogen-bonding distance of Asp-151, implicating this residue in catalysis.

Fourier Analysis↗

PubMed1993

Identification of critical contact residues in the NC41 epitope of a subtype N9 influenza virus neuraminidase.

We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab1 shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild-type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild-type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment.

Amino Acid Sequence↗

PubMed2000

Development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir.

Determination of the sensitivity of influenza viruses to neuraminidase (NA) inhibitors is presently based on assays of NA function because, unlike available cell culture methods, the results of such assays are predictive of susceptibility in vivo. At present the most widely used substrate in assays of NA function is the fluorogenic reagent 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (MUN). A rapid assay with improved sensitivity is required because a proportion of clinical isolates has insufficient NA to be detectable in the current fluorogenic assay, and because some mutations associated with resistance to NA inhibitors reduce the activity of the enzyme. A chemiluminescence-based assay of NA activity has been developed that uses a 1,2-dioxetane derivative of sialic acid (NA-STAR) as the substrate. When compared with the fluorogenic assay, use of the NA-STAR substrate results in a 67-fold reduction in the limit of detection of the NA assay, from 200 pM (11 fmol) NA to 3 pM (0.16 fmol) NA. A panel of isolates from phase 2 clinical studies of zanamivir, which were undetectable in the fluorogenic assay, was tested for activity using the NA-STAR substrate. Of these 12 isolates with undetectable NA activity, 10 (83%) were found to have detectable NA activity using the NA-STAR substrate. A comparison of sensitivity to zanamivir of a panel of influenza A and B viruses using the two NA assay methods has been performed. IC(50) values for zanamivir using the NA-STAR were in the range 1.0-7.5 nM and those for the fluorogenic assay in the range 1. 0-5.7 nM (n = 6). The NA-STAR assay is a highly sensitive, rapid assay of influenza virus NA activity that is applicable to monitoring the susceptibility of influenza virus clinical isolates to NA inhibitors.

Adamantane↗

PubMed2000

Determination of N-acetyl- and N-glycolylneuraminic acids in gangliosides by combination of neuraminidase hydrolysis and fluorometric high-performance liquid chromatography using a GM3 derivative as an internal standard.

A highly sensitive method for quantification of sialic acids in gangliosides was developed. The sialic acids, released by hydrolysis of gangliosides, were converted to fluorescent derivatives with 1,2-diamino-4,5-(methylenedioxy)benzene (DMB) and separated on a reversed-phase C18 column with an isocratic elution. As little as 0.1-1.0 nmol of sialic acid in ganglioside was quantified. The use of acetate buffer instead of water in the mobile phase could prevent damage on the column and reduce background peaks derived from the reagents. When gangliosides were subjected to acid hydrolysis, the velocity of hydrolysis varied depending on their structures and a part of the sialic acid liberated decomposed with prolonged heating time. Therefore gangliosides were hydrolyzed by Arthrobacter ureafaciens neuraminidase in the presence of sodium cholate after addition of an internal standard. For the internal standard, GM3 with N-propionylneuraminic acid (GM3(NeuPr)) was synthesized from GM3(NeuAc) by N-deacylation followed by N-propionylation. Folch partition was used to decrease lipophilic materials included in the sample, and the sialic acids released were recovered from the upper phase. The present method has a satisfactory sensitivity in the simultaneous quantification of NeuAc and NeuGc in purified gangliosides as well as in crude lipid fractions containing a variety of gangliosides.

Carbohydrate Sequence↗

PubMed1993

Crystallization and preliminary X-ray studies of influenza A virus neuraminidase of subtypes N5, N6, N8 and N9.

Neuraminidase heads from the following subtypes of influenza A virus have been crystallized: N5, N6, N8 and whale virus N9. The last three yielded crystals of X-ray diffraction quality; the N5 crystals obtained so far are not suitable for high resolution studies. The cell dimensions and space groups of crystals grown from the four subtypes have been determined.

Crystallography↗

PubMed1994

Defining the requirements for an antibody epitope on influenza virus neuraminidase: how tolerant are protein epitopes?

To determine the conformational requirements for antibody recognition and extent of flexibility within a protein epitope, a chimeric influenza A virus neuraminidase (NA) has been constructed in which five discontinuous polypeptide segments from a subtype N9 NA, which comprise the monoclonal antibody NC41 epitope, have been grafted onto a subtype N2 NA. The resulting chimeric NA was expressed, assembled as a tetramer, and transported to the cell surface, but was not recognized by NC41 in immunoprecipitation experiments or by surface immunofluorescence. Although the N2 and N9 protein folds are identical and this chimera contains all the antibody contacts as defined by the crystal structure of the complex, NC41 binding was not achieved. Modeling studies suggest that at least one polypeptide segment is displaced from its normal position which would account for the observed lack of enzyme activity as well as lack of antibody binding. This implies that in addition to the specific critical interactions between NA and Fab residues required for antibody binding, the overall arrangement of amino acids within an epitope must be in a specific orientation that is necessary for initial antibody recognition.

Amino Acid Sequence↗

PubMed1994

Protein hydration observed by X-ray diffraction. Solvation properties of penicillopepsin and neuraminidase crystal structures.

Solvation in macromolecular crystal structures was studied by analyzing X-ray diffraction data of two proteins, penicillopepsin and neuraminidase. The quality of several solvent models was assessed by complete cross-validation in order to prevent overfitting the diffraction data. Radial solvent distribution functions were computed from electron density maps using phases obtained from multiple isomorphous replacement and from the protein's atomic model combined with the best solvent model. Distribution functions were computed around hydrophilic and hydrophobic groups on the protein's surface. Averaging of the distribution functions was performed in order to reduce the influence of noise. The first solvation shell is characterized by a peak in the average distribution functions. At 1.8 A resolution, polar groups show a sharp peak while non-polar groups show a broad one. The distinction between hydrophobic and hydrophilic solvation sites is lost when using lower resolution (2.8 A) diffraction data. Higher-order solvation shells are not observed in the average distribution functions. We hope that site-specific radial distribution functions obtained from high-quality diffraction data will produce a picture of macromolecular solvation consistent with available experimental data and computational results.

Algorithms↗

PubMed1997

Guanidinobenzoic acid inhibitors of influenza virus neuraminidase.

The active site of influenza virus neuraminidase (NA) is formed by 11 universally conserved residues. A guanidino group incorporated into two unrelated NA inhibitors was previously reported to occupy different negatively charged sites in the NA active site, A new inhibitor containing two guanidino groups was synthesized in order to utilize both sites in an attempt to acquire a combined increase in affinity. The X-ray crystal structures of the complexes show that the expected increase in affinity could not be achieved even though the added guanidino group binds to the negatively charged site as designed. This suggests that the ligand affinity to the target protein is contributed both from ligand-protein interactions and solvation/conformation energy of the ligand.

Binding Sites↗