Neuraminidase Specificity Research Articles

Identification of internally sialylated carbohydrate tumor marker candidates, including Sda/CAD antigens, by focused glycomic analyses utilizing the substrate specificity of neuraminidase.

In our previous study, 14 sulfated carbohydrate tumor marker candidates were identified by focused glycomic analyses. Here, glycomic analyses focused on internally sialylated glycans to identify novel marker candidates. Internally sialylated glycans were enriched by digestion of pyridylaminated glycans prepared from sera with α-neuraminidase from Salmonella typhimurium, which did not cleave sialic acids linked to internal residues, followed by anion-exchange chromatography. Next, internally sialylated O-glycan profiles were constructed using two types of high performance liquid chromatography, which were compared between 20 healthy controls and 11 patients with gastric cancer and 9 patients with pancreatic cancer. In all, 17 marker candidates were identified. The structures of glycan candidates were precisely analyzed using enzymatic digestion, glycan synthesis, 2D mapping and mass spectrometry. Among 17 candidates, one was STn, and the other 16 comprised 10 core1, 1 core2 and 5 core3 glycans. The various structures included a α2,6-sialylated reducing terminal GalNAc and α2,6-sialylated type1 N-acetyl-lactosamine. Eight candidates possessed the Sda/CAD antigen. The levels of these candidate glycans in sera from all 40 subjects were quantified using a selected reaction monitoring assay and found to be elevated in at least one or more patients. Although the serum levels of each candidate glycan varied between patients, those candidates having the same backbone or determinant, such as core3 backbone and core1 structures with extended type1 N-acetyl-lactosamine, displayed similar patterns of elevation. These results suggest that analysis of multiple markers may be an effective means of diagnosing various cancers.

Neuraminidase activity of blue eye disease porcine rubulavirus: Specificity, affinity and inhibition studies

Porcine rubulavirus (PorPV), also known as La Piedad Michoacan Virus (LPMV) causes encephalitis and reproductive failure in newborn and adult pigs, respectively. The hemagglutinin–neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PorPV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid releasing) activity has been proposed as a virulence factor.This work describes the purification and characterization of PorPV HN protein (isolate PAC1). The specificity of neuraminidase is restricted to sialyl(α2,3)lactose (3SL). HN showed typical Michaelis-Menten kinetics with fetuin as substrate (km=0.029μM, Vmax=522.8nmolmin−1mg−1). When 3SL was used as substrate, typical cooperative kinetics were found (S50=0.15μM, Vmax=154.3nmolmin−1mg−1). The influenza inhibitor zanamivir inhibited the PorPV neuraminidase with IC50 of 0.24μM. PorPV neuraminidase was activated by Ca2+ and inhibited by nucleoside triphosphates with the level of inhibition depending on phosphorylation level. The present results open possibilities to study the role of neuraminidase in the pathogenicity of PorPV infection and its potential inhibitors.

Quantification of Influenza Neuraminidase Activity by Ultra-High Performance Liquid Chromatography and Isotope Dilution Mass Spectrometry.

Mounting evidence suggests that neuraminidase's functionality extends beyond its classical role in influenza virus infection and that antineuraminidase antibodies offer protective immunity. Therefore, a renewed interest in the development of neuraminidase (NA)-specific methods to characterize the glycoprotein and evaluate potential advantages for NA standardization in influenza vaccines has emerged. NA displays sialidase activity by cleaving off the terminal N-acetylneuraminic acid on α-2,3 or α-2,6 sialic acid containing receptors of host cells. The type and distribution of these sialic acid containing receptors is considered to be an important factor in transmission efficiency of influenza viruses between and among host species. Changes in hemagglutinin (HA) binding and NA specificity in reassortant viruses may be related to the emergence of new and potentially dangerous strains of influenza. Current methods to investigate neuraminidase activity use small derivatized sugars that are poor models for natural glycoprotein receptors and do not provide information on the linkage specificity. Here, a novel approach for rapid and accurate quantification of influenza neuraminidase activity is achieved utilizing ultra-high performance liquid chromatography (UPLC) and isotope dilution mass spectrometry (IDMS). Direct LC-MS/MS quantification of NA-released sialic acid provides precise measurement of influenza neuraminidase activity over a range of substrates. The method provides exceptional sensitivity and specificity with a limit of detection of 0.38 μM for sialic acid and the capacity to obtain accurate measurements of specific enzyme activity preference toward α-2,3-sialyllactose linkages, α-2,6-sialyllactose linkages, or whole glycosylated proteins such as fetuin.

Mismatched hemagglutinin and neuraminidase specificities in recent human H3N2 influenza viruses

The hemagglutinin (HA) of influenza viruses initiates infection by binding to sialic acid on the cell surface via α2,6 (human) or α2,3 (avian) linkage. The influenza neuraminidase (NA) can cleave both α2,3- and α2,6-linked sialic acids, but all influenza NAs have a marked preference for the non-human α2,3 linkage. Recent H3N2 influenza viruses have lost the ability to agglutinate chicken red blood cells. To determine if changes in HA specificity or affinity correlate with NA specificity or activity, we examined red cell binding and elution of a series of H3N2 viruses. We found that the NA activity of many influenza viruses does not release binding by their HA. In some egg-adapted strains, lack of elution correlates with low levels of viral NA activity, and these elute rapidly when bacterial NA is added. However, a Fujian-like virus, A/Oklahoma/323/03, does not elute by its own NA or with Vibrio cholerae sialidase, and it binds to red cells pre-treated with V. cholerae sialidase. It elutes after addition of the broad specificity Micromonospora viridifaciens sialidase. Human glycophorin inhibits A/Oklahoma/323/03 hemagglutination 6-fold better than fetuin. We conclude that specific forms of sialic acid are used as receptor by recent human H3N2 influenza viruses, perhaps involving branched α2,6 sialic acid or α2,8 sialic acid structures on O-linked carbohydrates. The virus itself has no O-linked glycans, so even though the NA is not able to cleave receptors on cells, the viruses will not self-aggregate. It will be important to monitor efficacy of neuraminidase inhibitors in case there are NA-resistant receptors in the human respiratory tract that allow the viruses to be less dependent on NA activity.

Fluorescent assay for studying the substrate specificity of neuraminidase

Toyopearl 650 M was purchased from Tosoh (Japan). The 10-~1 pipette tips with filter (cat. no. FI08-96R-I0) and thin-well 0.2-ml PCR microtubes (cat. no. 430-01XNBZ-Q) with caps were obtained from Porex. Black polystyrene 96-well microtiter plates were purchased from Nunc (Denmark, cat. no. 437111). Vibrio cholerae neuraminidase (3.2.1.18) was purchased from Sigma (USA). 3'SiaLac and 6'SiaLac were purchased from Lectinity (Russia). ro-Aminospacered 3'SiaLac, 3'SiaLacNAc (Neu5Ac Cl2-3GaI131-4GlcNAc), and 6'SiaLacNAc (Neu5Ac Cl2-6GaI131-4GlcNAc) were synthesized previously [5-7]. ro-Aminospacered trisaccharide (5 ~ol), followed by triethylamine (5 ~l), was added to BODIPY FLSE (5.5 ~ol) in dimethylformamide (0.5ml). The reaction mixture was stirred at room temperature for 2-3 h and was subjected to gel filtration on a Sephadex LH-20 column equilibrated with acetonitrile/water (1:1, v/v). The yield of BODIPYlabeled trisaccharides was 80-90%. (Complete lH nuclear magnetic resonance data (500 MHz, DzO) are available from www.carbohydrate.ru/docs/nmrdata_OOl.rtf.) Virus NBulgaria/120/82 (HINl) and virus A!FPV/ Rostock/34 (H7Nl) were obtained from the virus repository of the Ivanovsky Institute of Virology in Moscow, Russia, and were propagated in 10-day-old embryonated chick~n eggs. A Madin-Darby canine kidney (MDCK)derived variant of human influenza virus A/Hongkong/ 1143/99 (H3N2) was obtained from the Institute of Applied Microbiology in Vienna, Austria. Primary isolation, propagation of human influenza virus, and purification of all viruses were done as described previously [8]. A titer of viruses was determined in a hemagglutination reaction with human erythrocytes Neuraminidase, or sialidase, is an exoglycosidase that hydrolyzes tX-ketosidic linkage between sialic acid and adjacent sugar residue. There are several methods of neuraminidase activity determination, usually based on fluorescent or chemiluminescent detection of an aglycon formed due to neuraminic acid cleavage [1-3]. Substrate specificity of neuraminidase is usually defined as the enzyme ability to distinguish the type of bond between neuraminic acid and galactose residues. As a rule, 3'SiaLac (Neu5ActX2-3Galf31-4Glc)! and 6'SiaLac (Neu5Ac tX2-6Galf31-4Glc) are used as substrates, and the amount of released neuraminic acid is measured [4]. In this article, we propose a simple and sensitive fluorescent method for determining the neuraminidase specificity using BODIPY -labeled trisaccharides as substrates. The method is based on the quantitative separation of neutral disaccharide-BODIPY from negatively charged uncleaved substrate using an anion exchange microcartridge, with the reaction product being registered by its fluorescence (Scheme 1). BODIPY FLSE (4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-propionic acid) was purchased from Molecular Probes (The Netherlands). All other chemicals used were obtained from Fluka (Switzerland). All chemicals used were of analytical grade. DEAE-

Specificity of neuraminidase activity from influenza viruses isolated in different hosts tested with novel substrates

Influenza A and B viruses isolated in Vero and Madin Darby canine kidney (MDCK) cells as well as in fertilised hen eggs were tested for the specificity of their neuraminidase (NA) activity. Novel glycoconjugates with variations of terminally bound sialic acid mimicking the three main receptor types for influenza viruses were synthesised. These new substrates together with the lectin from Ricinus communis were used in a solid phase microtitre assay for the detection of NA specificity. Egg or MDCK isolated virus strains tended to exhibit highest NA activity against 3'sialyl-bound sialic acid whereas Vero isolated strains favoured 6'sialyl-(N-acetyllactosamine)-bound sialic acid. Differences were more pronounced for influenza A than for influenza B strains.

Role of neuraminidase in influenza virus-induced apoptosis.

The virulent influenza virus clone 7a produced a greater level of apoptosis in MDCK cells compared with the attenuated strain A/Fiji. In both cases, apoptosis could be partially blocked by treatment with three anti-neuraminidase compounds [4-amino-(GR121158A) and 4-guanidino- (GG167; Zanamivir) 2,3-dehydro-N-acetylneuraminic acid and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA)] when they were given to cells during the virus attachment/entry phase, but not subsequent to this phase. In contrast, GG167, which does not enter cells, did not affect the numbers of infected cells and, in addition, acted late in the infection cycle to inhibit virus yields. Clone 7a neuraminidase was more active than A/Fiji neuraminidase when fetuin was used as the substrate. Similar differences in activity between the two viruses were seen when alpha-2,6 sialyl lactose was used as a substrate, but not with alpha-2,3 sialyl lactose. No sequence differences in the enzyme active site of the two neuraminidases were observed, indicating that differences in neuraminidase specificity and activity may be dictated by other residues. These results suggest that neuraminidase plays some role in the induction of apoptosis and that it acts prior to or during virus entry. However, apoptosis was considerably reduced when UV-irradiated virus, which retains >75% of its neuraminidase activity, was used. In addition, ammonium chloride, used to prevent virus entry, reduced virus-induced apoptosis. Amantadine, which inhibits virus uncoating, also inhibited apoptosis induced by the amantadine-sensitive strain A/Udorn/307/72 (H3N2), but not the amantadine-resistant clone 7a. Hence, one or more intracellular processes are also involved in influenza virus-induced apoptosis.

Two evolutionary strategies of influenza viruses to escape host non-specific inhibitors: alteration of hemagglutinin or neuraminidase specificity

The porcine serum inhibitor α 2-macroglobulin prevents influenza virus from entering host cells by competing for the SAα2,6Gal-binding site of the hemagglutinin (HA). We studied a series of inhibitor-sensitive and inhibitor-resistant human and porcine influenza virus isolates of the H3N2 subtype, all of which contained HAs, which initially bound only to SAα2,6Gal oligosaccharides. When their neuraminidase was inhibited, the naturally resistant viruses, as a result of no longer being able to elute from the inhibitor, became sensitive. Evidently it is the neuraminidase which enabled these viruses to grow in hosts which possess the inhibitor. Escape-mutants selected under laboratory conditions in the presence of porcine serum became inhibitor-resistant by two alternative mechanisms: they changed either their HA-specificity or their neuraminidase-specificity. The study thus disclosed two evolutionary strategies for acquiring resistance to a host neuraminidase-sensitive inhibitor: (i) acquisition of an HA able to bind to oligosaccharides not present on the inhibitor; or (ii) acquisition of a neuraminidase able to cleave the oligosaccharide bound by the HA.

The N2 neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity

A survey of 10 human influenza A viruses of the N2 serotype, isolated between 1957 and 1987, has revealed a drift in neuraminidase linkage specificity. While the earliest N2 strains examined exhibit strict specificity for cleavage of the NeuAcα2,3Gal sequence, N2 isolates from 1967 to 1968 also show limited activity towards the NeuAcα2,6Gal linkage. In strains isolated in 1972 and later, the N2 neuraminidase has approximately equal activity towards both types of linkages. The NeuAcα2,6Gal linkage cleaved by the later N2 neuraminidases is the preferred receptor determinant of human H2 and H3 hemagglutinins. Thus, the acquired neuraminidase specificity of the later isolates allows elution of bound virus from erythrocytes derivatized to contain the NeuAcα2,6Gal linkage, while earlier isolates, which cleave only the NeuAcα2,3Gal sequence, fail to elute from these erythrocytes. These results suggest that the observed drift in N2 neuraminidase specificity in the direction of the preferred H2 and H3 receptor determinant may facilitate release of progeny virus from host cells.

Characterization of a neuraminidase from Corynebacterium aquaticum responsible for Th polyagglutination.

Th polyagglutinability is characterized by the agglutination of the red blood cells (RBC) by Arachis hypogaea, Medicago disciformis, Vicia cretica but, in contrast to the T phenomenon, not by Glycine max (Glycine soja). Because Th transformation of RBC has been obtained in vitro, the mechanism of Th polyagglutinability expression has been studied and reproduced experimentally. An enzyme with neuraminidase specificity has been isolated from the culture supernatant of Corynebacterium aquaticum, and further characterized (MW = 55,600 kDa, pH = 5.5, Km = 0.138 microM, Kcat = 0.22 micrograms). Reversely, Th transformation of RBC could be obtained by using other neuraminidases but in very mild conditions of hydrolysis. From our results, it can be concluded that by the release of less than 20 micrograms of sialic acid per 10(10) RBC, Th reactivity can be induced whereas hydrolysis of greater amounts of sialic acid (greater than 20 micrograms/10(10) RBC) give the classical T polyagglutinability.

Characterization of a Neuraminidase from Corynebacterium Aquaticum Responsible for Th Polyagglutination

Th polyagglutinability is characterized by the agglutination of the red blood cells (RBC) by Arachis hypogaea, Medicago disciformis, Vicia eretica but, in contrast to the T phenomenon, not by Glycine max (Glycine soja). Because Th transformation of RBC has been obtained in vitro, the mechanism of Th polyagglutinability expression has been studied and reproduced experimentally. An enzyme with neuraminidase specificity has been isolated from the culture supernatant of Corynebacterium aquaticum, and further characterized (MW = 55,600 kDa, pH = 5.5, Km = 0.138 µM, Kcat = 0.22 µg). Reversely, Th transformation of RBC could be obtained by using other neuraminidases but in very mild conditions of hydrolysis. From our results, it can be concluded that by the release of less than 20 µg of sialic acid per 10^10 RBC, Th reactivity can be induced whereas hydrolysis of greater amounts of sialic acid (>20 µg/10^10 RBC) give the classical T polyagglutinability.

On the specificity of neuraminidase The carboxymethyl α-ketoside of N-acetyl-D-neuraminic acid, a Vibrio cholerae neuraminidase substrate having two anionic sites

FEBS LettersVolume 22, Issue 1 p. 46-48 Full-length articleFree Access On the specificity of neuraminidase The carboxymethyl α-ketoside of N-acetyl-D-neuraminic acid, aVibrio cholerae neuraminidase substrate having two anionic sites L. Holmquist, L. Holmquist Institut für Biochemie II (Med. Fak.), D-69 Heidelberg, Akademiestrasse 5, W. Germany On leave of absence from Research Institute of National Defence, Department 1, S-17204 Sundbyberg 4, Sweden. Search for more papers by this authorR. Brossmer, R. Brossmer Institut für Biochemie II (Med. Fak.), D-69 Heidelberg, Akademiestrasse 5, W. GermanySearch for more papers by this author L. Holmquist, L. Holmquist Institut für Biochemie II (Med. Fak.), D-69 Heidelberg, Akademiestrasse 5, W. Germany On leave of absence from Research Institute of National Defence, Department 1, S-17204 Sundbyberg 4, Sweden. Search for more papers by this authorR. Brossmer, R. Brossmer Institut für Biochemie II (Med. Fak.), D-69 Heidelberg, Akademiestrasse 5, W. GermanySearch for more papers by this author First published: April 15, 1972 https://doi.org/10.1016/0014-5793(72)80215-1Citations: 8 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume22, Issue1April 15, 1972Pages 46-48 ReferencesRelatedInformation

On the specificity of neuraminidase. Synthesis and properties of the 2-aminoethyl -and the 2-pyridyl -and -ketosides of N-acetyl-D-neuraminic acid.

On the specificity of neuraminidase. Synthesis and properties of the 2-aminoethyl -and the 2-pyridyl -and -ketosides of N-acetyl-D-neuraminic acid.

On the specificity of neuraminidase. Synthesis of 5-N-acetyl-D-nonulosamine (5-acetamido-3,5-dideoxy-D-glycera- -D-galacto-nonulose) and 5-N-acetyl-D-neuraminamide (5-acetamido-3,5-dideoxy-D-glycero- -D-galacto-nonulosonamide) and their methyl - and benzyl -ketosides.

On the specificity of neuraminidase. Synthesis of 5-N-acetyl-D-nonulosamine (5-acetamido-3,5-dideoxy-D-glycera- -D-galacto-nonulose) and 5-N-acetyl-D-neuraminamide (5-acetamido-3,5-dideoxy-D-glycero- -D-galacto-nonulosonamide) and their methyl - and benzyl -ketosides.

Synthesis of N-formyl- and N-succinyl-D-neuraminic acid on the specificity of neuraminidase

Synthesis of N-formyl- and N-succinyl-D-neuraminic acid on the specificity of neuraminidase