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PubMed1992

Crystal structures of two mutant neuraminidase-antibody complexes with amino acid substitutions in the interface.

The site on influenza virus N9 neuraminidase recognized by NC41 monoclonal antibody comprises 19 amino acid residues that are in direct contact with 17 residues on the antibody. Single sequence changes in some of the neuraminidase residues in the site markedly reduce antibody binding. However, two mutants have been found within the site, Ile368 to Arg and Asn329 to Asp selected by antibodies other than NC41, and these mutants bind NC41 antibody with only slightly reduced affinity. The three-dimensional structures of the two mutant N9-NC41 antibody complexes as derived from the wild-type complex are presented. Both structures show that some amino acid substitutions can be accommodated within an antigen-antibody interface by local structural rearrangements around the mutation site. In the Ile368 to Arg mutant complex, the side-chain of Arg368 is shifted by 2.9 A from its position in the uncomplexed mutant and a shift of 1.3 A in the position of the light chain residue HisL55 with respect to the wild-type complex is also observed. In the other mutant, the side-chain of Asp329 appears rotated by 150 degrees around C alpha-C beta with respect to the uncomplexed mutant, so that the carboxylate group is moved to the periphery of the antigen-antibody interface. The results provide a basis for understanding some of the potential structural effects of somatic hypermutation on antigen-antibody binding in those cases where the mutation in the antibody occurs at antigen-contacting residues, and demonstrate again the importance of structural context in evaluating the effect of amino acid substitutions on protein structure and function.

Antibodies, Monoclonal↗

PubMed1983

Sialic acid, sialyltransferase and neuraminidase levels in maternal plasma, urine and lymphocytes during pregnancy and post-partum period--a longitudinal study in women.

The total sialic (N-acetylneuraminic acid) levels were serially determined in maternal plasma, urine and lymphocytes during human pregnancy and post-partum period. The enzymes, sialyltransferase and neuraminidase, were also examined in the plasma samples. The sialic acid content of plasma and urine (expressed in terms of the creatinine content in urine) was elevated during pregnancy and increased with advancing gestation (P less than 0.001). A few days (0-6) prior to parturition there was a significant (P less than 0.01) fall in the plasma sialic acid levels, which subsequently increased again in the post-partum period (1-14 days), reaching values even greater than that observed during pregnancy. On the other hand, there was no decrease in the urinary sialic acid levels before delivery, and the post-partum values in urine, though higher than the values obtained in non-pregnant women, were not significantly greater than the levels observed during pregnancy. The sialic acid levels in lymphocytes were not altered during pregnancy. There was also no statistically significant change in serum sialyltransferase activity at any stage of pregnancy or post-partum period, while neuraminidase was not detectable in any of the plasma samples. The results are discussed with reference to the role of circulating sialic acid during pregnancy.

Adult↗

PubMed1986

Loss of enzyme activity in a site-directed mutant of influenza neuraminidase compared to expressed wild-type protein.

Full-length double-stranded DNA copies of the neuraminidase (NA) gene of influenza virus A/Tokyo/3/67 (N2) and a mutant generated in vitro by site-specific, oligonucleotide-directed mutagenesis with a substitution of leucine for tryptophan at position 178 were cloned into an SV40 late replacement expression vector. Indirect immunofluorescence of cells infected with these recombinant vectors showed the presence of NA protein in the cytoplasm and on the surface of infected cells. Cells expressing the wild-type protein showed neuraminidase enzyme activity for both fetuin, a sialated glycoprotein (mol wt = 50,000) and N-acetylneuraminyl lactose, a trisaccharide (mol wt = 600). This enzyme activity was inhibited by 44% toward N-acetylneuraminyl lactose and by 98% toward fetuin by adding anti-NA antibody before substrate. In contrast, cells expressing the mutant NA had no detectable enzyme activity for either substrate. The conserved nature of the tryptophan at position 178 in all known NA strains, its location in the substrate binding pocket in the three-dimensional structure and the lack of activity of the mutant protein indicate that this residue is essential for enzyme activity.

Cloning, Molecular↗

PubMed1986

Monoclonal antibodies reveal extensive antigenic differences between the hemagglutinin-neuraminidase glycoproteins of human and bovine parainfluenza 3 viruses.

A panel of twenty monoclonal antibodies to the hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza 3 (PI3) virus has been obtained and tentatively classified into four different groups based on reactivity in hemagglutination inhibition (HI), neuraminidase inhibition (NI), and plaque neutralization (NT) tests. The antibodies were tested for cross-reactivity with bovine PI3 virus, Sendai virus, and simian virus 5 (SV5). Only two of these antibodies showed similar reactivities with human and bovine PI3 viruses in HI and NT tests; a few other antibodies showed low levels of reactivity with the heterologous viruses in HI tests. A competitive binding assay further suggested that the two cross-reactive antibodies are directed against the same domain of the HN molecule. Therefore, the HN glycoproteins of human and bovine PI3 viruses appear to be antigenically dissimilar, although they share at least one common epitope.

Antibodies, Monoclonal↗

PubMed1987

Expression of biologically active and antigenically authentic parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein by a recombinant baculovirus.

The hemagglutinin-neuraminidase (HN) gene of human type 3 parainfluenza virus has been inserted into a baculovirus expression vector under the control of the polyhedrin promoter. HN protein produced in insect cells by the recombinant baculovirus appeared to be glycosylated, was transported to the cell surface, and was biologically active. All of the HN epitopes previously mapped functionally to a region(s) involved in neuraminidase and/or hemagglutination activities were conformationally unaltered on the recombinant protein. The HN produced in this system also induced a protective immune response in immunized cotton rats. From these studies we conclude that the HN expressed in insect cells represents a source of authentic HN glycoprotein suitable for structural analysis and immunization.

Animals↗

PubMed1987

Crystals of antibodies complexed with influenza virus neuraminidase show isosteric binding of antibody to wild-type and variant antigens.

We describe here, for the first time, crystals of antibodies bound to a viral antigen which diffract X-rays to beyond 3 A. Crystals have been grown of Fab fragments of monoclonal antibody NC41, complexed with influenza virus neuraminidase (NA) of the N9 subtype and with a variant of N9 NA having a sequence change of Asn to Asp at position 331. This reduces, but does not abolish, the binding of NC41 antibody (in the case of another variant, Ser 371 to Leu, binding of NC41 antibody appears to be abolished). We are presenting data on the three-dimensional structure of these two complexes which indicates that NC41 antibody binds isosterically to the wild type and variant neuraminidase molecules.

Antibodies, Monoclonal↗

PubMed1988

Protection against lethal influenza with neuraminidase.

The role of the neuraminidase in eliciting protection against a lethal influenza A virus [A/Ck/Penn/1370/83 (H5N2)] infection was investigated in chickens. Isolated N2 neuraminidase administered in adjuvant did not prevent infection but did prevent systemic spread of virus and death of chickens. N2 expressed in a recombinant vaccinia virus protected chickens when administered in adjuvant but was less effective when allowed to replicate and produce pox on the chicken's comb. Chickens vaccinated with isolated N2 in adjuvant or with inactivated H5N2 influenza virus were protected from clinical signs and death after challenge with A/Ck/Penn/1370/83 influenza virus. However, these animals were completely susceptible and died of infection with a heterologous subtype (H7N7) of influenza virus. The role of the different antigenic determinants of the N2 NA was investigated in chickens by passive transfer of monoclonal antibodies. Antibodies to antigenic determinants rimming the enzyme active center reduced disease signs in approximately half of the birds but did not significantly reduce virus levels. Antibodies to one of the two independent antigenic determinants that are distant from the enzyme active center were most effective at reducing virus replication and disease signs. This is surprising because antigenic variants could not be selected in vitro with these antibodies and suggests that they may facilitate clearance of virus. Antibodies to the other determinant that is located distally to the enzyme site were ineffective at providing protection.

Animals↗

PubMed1989

Identification of amino acid residues important to the neuraminidase activity of the HN glycoprotein of Newcastle disease virus.

Monoclonal antibodies (MAbs) to three overlapping antigenic sites (designated 12, 2, and 23) on the hemagglutinin-neuraminidase glycoprotein (HN) of Newcastle disease virus (NDV) were previously shown to inhibit neuraminidase activity (NA) on neuraminlactose (R. M. Iorio and M. A. Bratt, 1984a, J. Immunol. 133, 2215-2219; R. M. Iorio et al., 1989, Virus Res. 13, 245-262). However, a competitive inhibitor of NA blocks the binding of only MAbs to site 23, suggesting that the domain they recognize may be closely related to the NA site. Antigenic variants selected with site 23 MAbs have single amino acid substitutions at HN residues 192, 193, or 200. Virions of variants, which have a substitution at residue 193 or 200, have alterations in NA which are not attributable to a commensurate change in HN content. A revertant of a temperature-sensitive mutant, which has markedly diminished NA relative to the wild type, has an amino acid substitution at residue 175. A second step revertant having partially restored NA has an additional substitution at residue 192 identical to that in one of the site 23 variants, which, in turn, also makes the revertant resistant to neutralization by site 23 MAbs. Thus, an amino acid substitution at residue 175, 193, or 200 of the HN of NDV can have marked effects on the NA of the protein. The amino acids in the region around residue 175 are highly conserved between the HNs of NDV and other paramyxoviruses, suggesting that this domain is important to the integrity of the NA site in this group of viruses.

Amino Acid Sequence↗

PubMed1990

Cloning and expression of NDV hemagglutinin-neuraminidase cDNA in a baculovirus expression vector system.

The hemagglutinin-neuraminidase (HN) gene of the Hitchner B1 strain of Newcastle disease virus (NDV) was cloned as a cDNA and inserted into a baculovirus expression vector. The recombinant HN (recHN) expressed in Spodoptera frugiperda cells had both hemagglutinating and neuraminidase activities both of which were inhibited by polyclonal anti-NDV sera or a monoclonal antibody (MAb) against HN. Infected insect cells could hemadsorb chicken red blood cells suggesting that the recHN is properly glycosylated and transported to the cell surface. A 67-kDa recHN precursor and a 74-kDa, presumably mature, recHN from infected cells were detected by Western blot analysis and were found to comigrate with similar proteins from NDV-infected chick embryo fibroblast cells. The kinetics of synthesis of recHN was similar to that for polyhedrin and some HN appeared in the extracellular medium. HN was copurified with extracellular virus (ECV) from the extracellular medium and was used to immunize chickens. The anti recHN serum was specific to NDV in both ELISA and Western blot analysis.

Animals↗

PubMed1992

A single amino acid substitution in the hemagglutinin-neuraminidase of Newcastle disease virus results in a protein deficient in both functions.

Sequence determinations of the hemagglutinin-neuraminidase (HN) glycoproteins of a temperature-sensitive mutant of Newcastle disease virus and two sequentially selected revertants had previously shown that substitution at a pair of residues, 129 and 175, resulted in a deficiency in neuraminidase (NA) activity, which was partially restored by a third substitution at residue 193. To evaluate the role of the substitution at residue 175 in diminished NA activity, the mutation was introduced into HN and the protein expressed in COS cells. The mutated HN not only had minimal NA activity but also was unable to absorb chicken erythrocytes, even though it was transported to the cell surface in normal amounts, in an apparently antigenic form. Attachment function was restored to the protein by the introduction of the additional substitution(s) at 129 and/or 193. These results indicate that residue 175 influences not only NA activity but also receptor recognition.

Animals↗

PubMed1992

Neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes.

The interaction of infectious bronchitis virus (IBV) with erythrocytes was analyzed. The binding activity of IBV was not sufficient to agglutinate chicken erythrocytes. However, it acquired hemagglutinating activity after treatment with neuraminidase to remove alpha 2,3-linked N-acetylneuraminic acid from the surface of the virion. Pretreatment of erythrocytes with neuraminidase rendered the cells resistant to agglutination by IBV. Susceptibility to agglutination was restored by resialylation of asialo-erythrocytes to contain alpha 2,3-linked sialic acid. These results indicate that IBV attaches to receptors on erythrocytes, the crucial determinant of which is sialic acid alpha 2,3-linked to galactose. In contrast to other enveloped viruses with such a binding specificity (influenza viruses and paramyxoviruses) IBV lacks a receptor-destroying enzyme.

Animals↗

PubMed1996

Site-directed mutations of the catalytic and conserved amino acids of the neuraminidase gene, nanH, of Clostridium perfringens ATCC 10543.

The small nanH gene encoding the neuraminidase from Clostridium perfringens ATCC 10543 was cloned in JM109 using pUC19 as a vector. Sequence analysis revealed an ORF encoding 382 amino acids without a signal peptide sequence. Four regions of amino-acid sequence, 71-82, 140-151, 208-219, and 255-266 constituted four repeated and conserved sequence motifs-Ser-X-Asp-X-Gly-X-Thr-Trp-, the "Asp boxes." When compared, the nanH polypeptides of C. perfringens ATCC 10543 and Salmonella typhimurium LT12 shared 33% sequence identity and 60% similarity if conservative replacements were included. The homology-modeled structure of C. perfringens NanH showed the same folding topology as the x-ray three-dimensional structure of NanH in S. typhimurium LT12. Amino acid residues Arg37, Arg56, Asp62, His63, Asp100, Glu230, Asp247, Tyr347, and Glu362 located around the pocket of modeled C. perfringens small nanH were superimposed with the active-site pocket of S. typhimurium LT12, nanH. The catalytic amino-acid residues as well as the role of the "Asp boxes" have not been characterized for C. perfringens and S. typhimurium. In this study, Asp100, Glu230, and Asp62 were found to be involved in the catalytic activity of C. perfringens small nanH with immunoreactive properties and site-directed mutagenesis analysis. Four "Asp-box" motifs were found remote from the active-site pocket. Mutational and immunoreactive analysis of the highly conserved amino acids located in the "Asp boxes" suggest that these highly conserved residues are important in maintaining the tertiary structure of NanH. The results of this study provide some knowledge for the design of new inhibitors of small neuraminidase.

Amino Acid Sequence↗

PubMed1985

Differences in carbohydrate specificities and complement-activating capacity of guinea pig and human antibodies to neuraminidase-treated autologous erythrocytes.

Guinea pig erythrocytes desialated by treatment with neuraminidase from Vibrio cholerae were lyzed in autologous serum through a natural-antibody-dependent activation of the classical complement pathway. Lysis was inhibited when a mannose, glucose, galactose or N-acetyl-glucosamine was added to the incubation mixture. Methyl-alpha- or -beta-D-galactopyranosides were poorly effective and N-acetyl-D-galactosamine was not effective at all. Inhibition of lysis by the carbohydrates was due neither to an anti-complementary effect nor to a modification of the osmotic pressure since: (a) they did not alter the total complement haemolytic activity of guinea pig serum, and (b) they did not inhibit lysis of desialated guinea pig erythrocytes in human serum through activation of the alternative complement pathway. The presence of mannose, glucose, galactose or N-acetyl-glucosamine in the incubation mixture resulted in an impaired fixation of natural auto-antibodies on antigenic sites, namely the T-antigen (Thomsen-Friedenreich), which were unmasked following membrane sialic acid removal. When tested under the same conditions, only small percentage of the normal human population showed the phenomenon of lysis of desialated erythrocytes in autologous serum. Lysis was not due to a particular susceptibility of erythrocytes from these individuals to complement-mediated lysis but to the presence in their serum of complement-activating anti-T antibodies. As expected, the activity of human anti-T antibodies was inhibited by galactose and N-acetyl-galactosamine, which are the immunodominant sugars of the human T-antigen. Mannose and glucose had no effect, and methyl- alpha- or - beta-D-galactopyranosides were almost as effective as galactose. The heterogeneity of the human population with regard to the complement-activating capacity of anti-T antibodies could be of significance for the individual response of the host to an infection by a neuraminidase-producing microorganism. That the immunodominant sugars of the T-antigen were different between humans and guinea pigs was further assessed by absorption experiments. We have demonstrated that guinea pig anti-T antibodies were not removed during contact with desialated human red cells which do not have the mannose specificity, whereas human antibodies were almost entirely retained on desialated guinea pig red cells which, beside mannose, express galactose. These results also suggest that guinea pig antibodies are mostly directed towards mannose and glucose.

Agglutination Tests↗

PubMed1985

A single-radial haemolysis technique for measurement of antibody to influenza virus neuraminidase in equine sera.

A modified single-radial-haemolysis (SRH) test for measurement of antibody to influenza virus neuraminidase (NA) is described. The test requires treatment of sheep erythrocytes with butanol to increase sensitivity. In comparative assays, SRH was found to be more sensitive than the conventional neuraminidase-inhibition test. The SRH test was reproducible, specifically measured antibody to influenza NA and was easy to use. SRH antibody responses in ponies vaccinated with bivalent equine influenza vaccine were shown to be vaccine dose-related but were lower in magnitude and shorter in duration than comparable anti-haemagglutinin responses.

Animals↗

PubMed1989

Trichomonas vaginalis and Tritrichomonas foetus secrete neuraminidase into the culture medium.

Supernatants taken from axenic cultures of Trichomonas vaginalis and Tritrichomonas foetus contain a neuraminidase activity, the detection of which is augmented when the trichomonad culture media are supplemented with 30% supernatant of confluent epithelial cultures. The enzyme was active against human erythrocytes, which became highly reactive to peanut agglutinin lectin. The specificity of the enzyme was checked by using a substrate specific to neuraminidase: 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuramic acid.

Animals↗

PubMed1989

Proteins of Newcastle disease virus envelope: interaction between the outer hemagglutinin-neuraminidase glycoprotein and the inner non-glycosylated matrix protein.

Using linear sucrose-density ultracentrifugation analysis of Triton-solubilized Newcastle Disease Virus envelopes, we have evidenced, for the first time, the existence of interactions between the outer hemagglutinin-neuraminidase transmembrane glycoprotein and the inner non-glycosylated peripheral matrix protein. Such interactions seem to be electrostatic. These conclusions are based on the behavior of both proteins at different ionic strengths. When in low ionic strength buffer, hemagglutinin-neuraminidase and matrix proteins band together in the sucrose gradient, whereas at high ionic strength both proteins band at different rates in the gradient. The behavior of the inner matrix protein in our conditions was the expected one for a peripheral protein. The results of these 'in vitro' studies are also discussed in terms of the possible 'in vivo' role of such interactions.

Animals↗

PubMed1985

Aging in vivo and neuraminidase treatment of rat erythrocytes and their sequestration by the isolated perfused rat liver.

The isolated perfused rat liver as a model for phagocytosis leaves the specific arrangement of macrophages in the original tissue structure intact. Therefore phagocytosis may occur under terms closely approaching physiological conditions. Separation of rat erythrocytes according to their density in 13% young, 75% mature and 12% old cells permits the establishment of differences in the mean cellular volume (MCV), mean cellular hemoglobin concentration (MCHC) and content of N-acetylneuraminic acid per red blood cell (NANA/RBC). Perfusion of these fractions does not result in significant differences of sequestration of the isolated liver. Enzymatic release of 90% of membrane-bound sialic acid causes a significant increase of 20% in the elimination of erythrocytes from the perfusate. Treatment with neuraminidase is a trigger for sequestration either by adherence or by complete phagocytosis. Incubation of neuraminidase-treated erythrocytes with plasma does not increase the removal rate of cells during perfusion.

Animals↗

PubMed1984

The carboxyterminus of the hemagglutinin-neuraminidase of Newcastle disease virus is exposed at the surface of the viral envelope.

The amino-terminal and the carboxy-terminal amino acids of the hemagglutinin-neuraminidase glycoprotein of the Ulster strain of Newcastle disease virus have been analyzed before and after proteolytic activation of the precursor HNo (Mr approximately 82K). The amino termini of HNo and of the large cleavage fragment HN (approximately 74K) obtained by in vivo and in vitro proteolysis could not be sequenced by Edman degradation. This indicates that in both instances the amino termini are blocked. The carboxy termini of HNo and HN are different as demonstrated by end-point digestion with carboxypeptidase A. Furthermore, a small cleavage fragment (approximately 9K) of HNo that was removed from the virion after trypsin treatment could be purified by HPLC. In contrast to HN, this fragment displays a free amino terminus susceptible to Edman degradation. These data indicate that conversion of HNo involves removal of a 9K glycopeptide from the carboxy-terminal end. Thus, it has to be concluded that, unlike most other viral glycoproteins, the hemagglutinin-neuraminidase is inserted in the envelope with its carboxy terminus exposed at the surface of the virus particle.

Amino Acid Sequence↗