Neuraminidase induces capacitation and acrosome reaction in mammalian spermatozoa

The treatment of epididymal spermatozoa of guinea pig and ejaculated spermatozoa of rabbit with neuraminidase from Arthrobacter ureafaciens induced significant acrosome reaction while the neuraminidase from Cl. perfringens failed to do so. The addition of the neuraminidase inhibitors kept the enzyme induced acrosome reaction to the control level. The zona-free hamster ova test showed that the treatment of spermatozoa with Arthrobacter neuraminidase rendered 82% of the guinea pig and 69% of the rabbit spermatozoa capable of fertilization. Thus, neuraminidase seems to enhance the rate of acrosome reaction by first capacitating spermatozoa in vitro.     

Coupled-Enzyme System for Measuring Viral Neuraminidase Activity

A coupled-enzyme assay for determining viral neuraminidase activity is described. All reactants-viral neuraminidase, the initial substrate (fetuin), N-acetylneuraminic acid aldolase, lactic acid dehydrogenase, and reduced nicotinamide adenine dinucleotide-are combined in a single cuvette. Thus, in a single coupled system neuraminidase releases N-acetylneuraminic acid, which is cleaved to N-acetyl-D-mannosamine and pyruvic acid; finally, pyruvate is reduced to lactate as reduced nicotinamide adenine dinucleotide is oxidized. The rate of change of absorbance at 340 nm, as reduced nicotinamide adenine dinucleotide is oxidized, is a measure of the rate of reaction of the coupled system. This procedure, which measures the rate of release of N-acetylneuraminic acid by neuraminidase, is an alternate method for those procedures which require multistep, colorimetric determinations.     

Continuous assay of neuraminidase activity by a bienzyme system immobilized in an artificial membrane

Lactate dehydrogenase and NANA-lyase were immobilized in an artificial gelantine membrane. This bienzyme system was used for continuous assay of neuraminidase activity. The K'(m) of the active membrane for lactate dehydrogenase and NANA-lyase using NADH, pyruvic acid, and N-acetylneuraminic acid as substrates were found to be 0.25mM, 0.75mM, and 2.1mM, respectively. The K(m) of soluble neuraminidase using sialyllactose as substrate was found to be 0.13 mM. The pH optimum for neuraminidase activity was 6.0. At 45 degrees C the reaction rate was higher, and no denaturation phenomena of the immobolized enzymes have been observed. This bienzyme membrane was stable for several weeks stored in the reaction buffer at 4 degrees C.     

Measuring neuraminidase activity by the thiobarbiturate method

A number of reducing agents used for removing the excess of periodate in the reaction of the neuraminidase (EC 3.2.1.18) activity measuring by the thiobarbituric acid technique were compared. The toxic reagent sodium arsenite may be replaced by a 20% solution of ascorbic acid. The modified technique of the neuraminidase activity determining can be used in case of both high-molecular weight substrate ovomucin and low-molecular weight substrate-glycomacropeptide from milk whey.     

Characterization of Influenza Virus Neuraminidases: Peptide Changes Associated with Antigenic Divergence Between Early and Late N2 Neuraminidases

Neuraminidases (EC 3.2.1.18) of 1957, 1960, and 1969 influenza virus strains were isolated after proteolytic digestion of viral hemagglutinin. Each neuraminidase was recovered with a final yield of about 15% and had similar specific activities. Immunization of rabbits with the neuraminidases elicited monospecific neuraminidase antibodies, with no antibodies to viral hemagglutinin. Further evidence of purity was the existence of only a single component, about 50,000 daltons in size, when reduced neuraminidase preparations were examined by sodium dodecyl sulfate acrylamide gel electrophoresis. However, storage of neuraminidase in solution resulted in the appearance of slightly smaller degradation products. Preparations of each neuraminidase were denatured under reducing conditions, and exposed sulfhydryl residues were blocked by reaction with (14)C-iodoacetamide. After tryptic digestion, peptide maps were prepared for the neuraminidases, and the (14)C-labeled cysteinyl peptides were then identified by autoradiography. About 20 peptides were present, in agreement with the number predicted from amino acid analysis for neuraminidase subunits of only one type. The 1957 and 1960 neuraminidases exhibited a small antigenic divergence from each other, and maps of their cysteinyl peptides appeared to be identical. The 1969 neuraminidase exhibited considerable antigenic divergence from the other two neuraminidases, and maps of 1969 neuraminidase peptides revealed two major and several minor differences from the other maps. Thus, antigenic divergence between the neuraminidases of Asian and Hong Kong influenza viruses is associated with a small number of changes in the primary structure of the neuraminidase subunit.     

Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidases

A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycone-focused library of 2-difluoromethylphenyl α-sialosides is an effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases. The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-terminated compound library by a click reaction. The focused library showed different inhibition patterns for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and the most potent inhibitors for each neuraminidase were selected. A kinetic analysis of the selected inhibitors demonstrated that the modification of the aglycone moiety improved the K(I) value with little change in the t(1/2) value of the enzyme activity relative to the basic skeleton (2).     

Structural and functional studies of Streptococcus pneumoniae neuraminidase B: An intramolecular trans-sialidase

The human pathogen Streptococcus pneumoniae expresses neuraminidase proteins that cleave sialic acids from complex carbohydrates. The pneumococcus genome encodes up to three neuraminidase proteins that have been shown to be important virulence factors. Here, we report the first structure of a neuraminidase from S. pneumoniae: the crystal structure of NanB in complex with its reaction product 2,7-anhydro-Neu5Ac. Our structural data, together with biochemical analysis, establish NanB as an intramolecular trans-sialidase with strict specificity towards alpha2-3 linked sialic acid substrates. In addition, we show that NanB differs in its substrate specificity from the other pneumococcal neuraminidase NanA.     

Synthesis and evaluation of 1,4,5,6-tetrahydropyridazine derivatives as influenza neuraminidase inhibitors.

1,4,5,6-Tetrahydropyridazine derivative 15 and its C-5 epimer 19, which possessed side chains similar to GS4071, were synthesized via a hetero Diels-Alder reaction, and evaluated as influenza neuraminidase inhibitors. Compounds 15 and 19 exhibited a microM range of influenza neuraminidase inhibitory activity.     

Monomer-micelle transition of the ganglioside GM1 and the hydrolysis by Clostridium perfringens neuraminidase.

The action of Clostridium perfringens neuraminidase on the ganglioside Gm1 tritiated in the ceramide moiety was studied. The rates of hydrolysis of the Gm1 ganglioside were determined from radioactivity in the neutral glycolipid product, which was separated from the substrate on DEAE-Sephadex columns. In order to study the physical state of the substrate in the conditions used in the neuraminidase treatment, the critical micelle concentrations of the Gm1 ganglioside were determined using formation of the triiodide anion in aqueous iodine solution as an indicator. The critical micelle concentrations were also obtained by determining the non-sedimenting radioactivity at different concentrations of the labeled ganglioside per total volume used in ultracentrifugation experiments. In addition, the concentrations of the monomeric ganglioside were concluded from the results of the ultra-centrifugation studies. The increase in the reaction rate of the Gm1 hydrolysis as the function of the substrate concentration was leveled off at 25-28 microM ganglioside. The abrupt change at this concentration is interpreted as reflecting the monomer-micelle transition of the ganglioside in the conditions used (50mM sodium acetate buffer, pH 4.6). The critical micelle concentration was 29 microM on the basis of the triiodide test, and ultracentrifugation revealed the critical micelle concentration 28 microM. The reaction velocity of the hydrolysis was decreased immediately above the critical micelle concentration, and became constant at higher concentrations of the ganglioside. A close correlation to these changes in the reaction rate is suggested to exist in the concentrations of the monomeric Gm1 ganglioside. Saturation of the buffer used in the neuraminidase assays with butanol effected a striking change in the plot of reaction rate versus ganglioside concentration. The reaction rate increased up to 100-110 microM Gm1 ganglioside. The shift of the inflexion point in the rate plot from 25-28 microM to 100-110 microM ganglioside concentration is suggested to be due to a respective change in the critical micelle concentration effected by butanol. N-Acetylneuraminyllactosyl ceramide, lactosyl ceramide and asialo-Gm1 ganglioside had an inhibitory effect on the reaction. In contrast, N-acetylneuraminyllactose, lactose and some other free saccharides were not inhibitory. The results demonstrate that factors other than the saccharide structure must be taken into account when substrate specificity of a glycosidase is studied using competition experiments. It is suggested that the inhibition effected by the glycolipids is due to an increase in the micellar state of the Gm1 ganglioside.     

The effext of papain, neuraminidase and AET on the behavior of lymphocytes in cytotoxicity tests]

The influence of papain, neuraminidase and AET to lymphocytes is reported. All three substances strengthen the lymphocytotoxic reaction in low concentrations or by short incubation time and lower it in higher concentrations or longer incubation since. Membrane changes by these three substances are reversible under defined conditions.     

Metabolism of 4-O-methyl-N-acetylneuraminic acid a synthetic sialic acid

1. 4-O-Methyl-N-acetylneuraminic acid shows a strong positive periodate-thiobarbiturate reaction. The mechanism of dye formation in this test for sialic acids is discussed in view of the studies already published. 2. An efficient preparation of a tritium-labelled 4-O-methyl-N-acetylneuraminic acid, with high specific radioactivity, by an oxymercuration-demercuration procedure is presented. 3. Sialytransferase activities in microsomal fractions of equine liver using desialylated fetuin are studied. The enzyme activity, assayed in a radioactive procedure, shows an apparent Km value for CMP-N-acetylneuraminic acid of 0.7 mM, whereas this value is 3.4 mM for CMP-4-O-methyl-N-acetylneuraminic acid. Differences are also observed in the maximal velocity for the two substrates. 4. The equine liver system can be used to prepare substantial amounts of fetuin containing radioactive N-acetylneuraminic acid or 4-O-methyl-N-acetylneuraminic acid. The isolated reaction products show similar sialic acid release by treatments with acid or fowl-plague virus neuraminidase. In contrast, 4-O-methyl-N-acetylneuraminic acid-fetuin displays a marked resistance to desialylation by Vibrio cholerae neuraminidase. 5. Free 4-O-methyl-N-acetylneuraminic acid is completely resistant to the action of acylneuraminate pyruvate-lyase. It does not inhibit the enzymic cleavage reaction of N-acetylneuraminic acid. 6. The influence of a substitution at C-4 neuraminic acid on the enzymatic reaction mechanisms is discussed.     

Studies on brain cytosol neuraminadase. I. Isolation and partial characterization of two forms of the enzyme from pig brain.

1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B.     

Lysosomal neuraminidase. Catalytic activation in insect cells is controlled by the protective protein/cathepsin A

Lysosomal N-Acetyl-alpha-neuraminidase is active in complex with the protective protein/cathepsin A (PPCA) and beta-galactosidase. The interaction with PPCA is essential for the correct intracellular routing and lysosomal localization of neuraminidase, but the mechanism of its catalytic activation is unclear. To investigate this process, we have used the baculovirus expression system to co-express neuraminidase and PPCA precursors in insect cells, which resulted in high enzymatic activity of neuraminidase. Both the 34- and 20-kDa PPCA subunits were required for the activation. We further demonstrated that when expressed alone, the neuraminidase precursor remained dimeric (114 kDa) and had low enzymatic activity, but when co-expressed with PPCA and beta-galactosidase, it multimerized in a complex of approximately 1350 kDa, together with the other two proteins. The fully active neuraminidase co-precipitated with full-length PPCA and beta-galactosidase precursors. However, when co-expressed with the individual PPCA subunits, neuraminidase co-precipitated only with the small 20-kDa polypeptide, which therefore must contain a neuraminidase-binding site. Our finding suggests a model of activation of neuraminidase dependent on its oligomerization at acidic pH that is mediated by interaction with PPCA.     

Distribution of neuraminidase in Arthrobacter and its purification by affinity chromatography

Neuraminidase [sialidase, EC 3.2.1.18] was found to be widely distributed in bacteria belonging to Arthrobacter. Among these bacteria, Arthrobacter ureafaciens, A. oxydans, and A. aurescens produced relatively potent neuraminidase activities. For the production of this enzyme, not only colominic acid, a homopolymer of N-acetylneuraminic acid, but also N-acetylneuraminic acid, the reaction product of this enzyme, are effective as sources of carbon. An affinity adsorbent specific for neuraminidase was prepared by cross-linking colominic acid with soluble starch by means of epichlorohydrin. Neuraminidase from A. ureafaciens could be purified on this affinity column. The purified neuraminidase was shown to be free from protease, N-acetylneuraminic acid aldolase, phospholipase C, and glycosidases. Aminoff's assay procedure for sialic acid was modified to avoid the centrifugation step. The modified procedure gave a higher molecular extinction coefficient.     

Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki approximately 21 microM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuraminidase activity result in specifically and non-specifically labeled polypeptides. Only labeling in a 55 kDa polypeptide is found to be specific, since it could be prevented by the competitive neuraminidase inhibitor NeuAc2en. We conclude that the 55 kDa polypeptide in the bovine testis beta-galactosidase/neuraminidase/protective protein complex contains the catalytic site of neuraminidase.     

Effect of neuraminidase treatment on the lipid fluidity of the intestinal brush-border membranes

The effect of neuraminidase treatment on the lipid fluidity of the porcine intestinal brush-border membranes was studied using two fluorescence dyes, pyrene and 1,6-diphenyl-1,3,5-hexatriene. By treatment of the membranes with neuraminidase, the fluorescence parameters of pyrene-labeled membranes changed; i.e., a shift of thermal transition temperature, an increase in the fluorescence quenching rate for Tl+ and a decrease in the fluorescence lifetime. These results suggest that the environmental properties around the dye molecules in the membranes change sensitively upon neuraminidase treatment. Perturbation of the lipid domain in the membranes associated with neuraminidase treatment is also demonstrated by a stimulated solubilization of diphenylhexatriene molecules in the membrane lipids, an increased quenching efficiency with Tl+ and a decreased rotational correlation time of diphenylhexatriene-labeled membranes. Based on these results, we conclude that the lipid organization of the membranes is susceptible to neuraminidase treatment and that the membrane lipid fluidity increases by desialylation by the enzyme treatment.     

In vitro activation of neuraminidase in the beta-galactosidase-neuraminidase-protective protein complex by cathepsin C

Neuraminidase can be activated by incubation of crude glycoprotein fractions at acidic pH for 90 minutes at physiological temperature. This activation is inhibited by leupeptin. Incubation of the purified neuraminidase-beta-galactosidase-protective protein complex under the same conditions used for crude glycoprotein fractions did not lead to enhanced neuraminidase activity, but incubation in the presence of exogenous Cathepsin C at 4 degrees C resulted in marked enhancement of neuraminidase activity. This activation was again inhibited by leupeptin. Cathepsin D treatment resulted in destruction of neuraminidase under the same conditions and this effect was again inhibited by leupeptin. beta-galactosidase in crude glycoprotein fractions and in the complex was resistant to both Cathepsin C and D, while homogeneous beta-galactosidase was inactivated by these enzymes. We suggest that in vitro activation of neuraminidase may mimic the in vivo intralysosomal conversion of the neuraminidase precursor into the mature form of the enzyme.     

Budding capability of the influenza virus neuraminidase can be modulated by tetherin

We have determined that, in addition to its receptor-destroying activity, the influenza virus neuraminidase is capable of efficiently forming virus-like particles (VLPs) when expressed individually from plasmid DNA. This observation applies to both human subtypes of neuraminidase, N1 and N2. However, it is not found with every strain of influenza virus. Through gain-of-function and loss-of-function analyses, a critical determinant within the neuraminidase ectodomain was identified that contributes to VLP formation but is not sufficient to accomplish release of plasmid-derived VLPs. This sequence lies on the plasma membrane-proximal side of the neuraminidase globular head. Most importantly, we demonstrate that the antiviral restriction factor tetherin plays a role in determining the strain-specific limitations of release competency. If tetherin is counteracted by small interfering RNA knockdown or expression of the HIV anti-tetherin factor vpu, budding and release capability is bestowed upon an otherwise budding-deficient neuraminidase. These data suggest that budding-competent neuraminidase proteins possess an as-yet-unidentified means of counteracting the antiviral restriction factor tetherin and identify a novel way in which the influenza virus neuraminidase can contribute to virus release.     

Relative susceptibilities to neuraminidase of the glycoproteins of the human erythrorcyte

Release of sialic acid from the glycoproteins of the normal human erythrocyte surface by neuraminidase was investigated. The glycoproteins of the membrane were separated by electrophoresis in sodium dodecylsulfate polyacrylamide gels. Sialic acid was determined in the sliced gel by a modification of the 2-thiobarbituric acid method, revealing three sialic acid-containing glycoproteins. Treatment of intact erythrocytes with neuraminidase to remove varying amounts of sialic acid indicates that all the glycoproteins are essentially equally accessible to the neuraminidase when 20%–60% of the sialic acid is removed. Similar but not quite identical results were obtained with isolated erythrocyte membranes.Treatment of intact cells with the lectins concanavalin A or phytohemagglutinin-P resulted in shielding of about 25% and 50%, respectively, of the sialic acid from neuraminidase. Concanavalin A blocked sialic acid release over long time periods and with high concentrations of neuraminidase. In contrast, the sialic acid shielding by phytohemagglutinin-P can be overcome by high concentrations of neuraminidase. Both lectins were found to shield the various glycoproteins selectively, with different patterns of shielding. Wheat germ agglutinin exhibited no detectable effect on the susceptibility of the erythrocyte sialic acid to neuraminidase.     

Neuraminidase assay utilizing sialyl-oligosaccharide substrates with tritium-labeled aglycone.

A new method for the radioisotopic assay of neuraminidase activity has been developed. The substrate utilized, α-d-N-acetylneuraminosyl-(2 → 3′)-lactit[³H]ol, was prepared by reduction of α-d-N-acetylneuraminosyl-(2 → 3′)-lactose with tritiated borohydride and purified by ion-exchange chromatography. After incubation with neuraminidase, the reaction mixtures were applied to small columns of AG 1-X2 (formate) in order to remove free sialic acid and unhydrolyzed substrate. The lactit[³H]ol released by neuraminidase action was then recovered by washing the columns with distilled water and quantitated by utilizing a liquid scintillation spectrometer. Studies with bacterial, avian, and mammalian neuraminidases are described.