Analysis of sialidase and N-acetylneuraminate pyruvate-lyase substrate specificity by high-performance liquid chromatography

A rapid and sensitive assay by high-performance liquid chromatography for determination of the activity and substrate specificity of sialidase (EC 3.2.1.18) and N-acetylneuraminate lyase (EC 4.1.3.3) is described. Sialic acids were separated on a strong anion-exchange resin using 0.75 mM sodium sulfate as elution medium. This method allows the determination of a minimum amount of 200 pg (0.6 pmol) of sialic acid. Usually the enzyme mixtures were directly applied to the column without prior purification of substrates and products. The action of sialidase was studied either by the decrease of sialyllactose concentration or by the amount of sialic acid liberated. The relative hydrolysis rates of N-acetylneuraminyl-alpha(2-3)-lactose, N-glycolylneuraminyl-alpha(2-3)-lactose, N-acetylneuraminyl-alpha(2-6)-lactose, N-acetyl-9-O-acetylneuraminyl-alpha(2-3)-lactose, and N-acetyl-4-O-acetylneuraminyl-alpha(2-3)-lactose by Vibrio cholerae sialidase were 100, 88, 25, 12, and 0, respectively. The activity of N-acetylneuraminate lyase from Clostridium perfringens was determined by measuring the rate of disappearance of sialic acids or the formation of acylmannosamines, which is possible in the same chromatogram. Relative cleavage rates of N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, and N-acetyl-4-O-acetylneuraminic acid were found to be 100, 67, 24, 3, and 0, respectively. Comparison of the substrate specificities shows that substituents on the neuraminic acid molecule influence the reactions of both enzymes in a similar way.     

Properties of sialidase isolated from Actinomyces viscosus DSM 43798

The cell-bound sialidase of Actinomyces viscosus DSM 43798 was solubilized by mechanical cell disruption and lysozyme treatment. The enzyme was enriched 30,000-fold by cation-exchange chromatography, gel-filtration, and FPLC ion-exchange chromatography, thus obtaining 10 micrograms sialidase protein from 26 g wet cells with a specific activity of 680 U/mg protein. Since sialidase activity was also found in the culture medium, this enzyme was isolated as well, requiring the additional application of FPLC gel-filtration. Both sialidase preparations were apparently homogenous on SDS-PAGE and have similar properties. The substrate specificity of the A. viscosus sialidase was tested with 16 sialoglycoconjugates: The enzyme showed a higher activity with serum glycoproteins than with gangliosides, mucins or sialyllactoses. 4-O-Acetylated N-acetylneuraminic acid was not cleaved from equine submandibular gland mucins or serum glycoproteins in contrast to N-acetyl- and N-glycoloylneuraminic acid. 9-O-Acetyl-N-acetylneuraminic acid was released from bovine submandibular gland mucin, as confirmed by TLC. The sialidase hydrolyses alpha(2----6)-linkages more rapidly than alpha(2----8)- and alpha(2----3)-bonds. Cations, except Hg2+, or chelating agents have no influence on enzyme activity. The sialidase has a relatively high molecular mass of 150 kDa, but consists of only one unit. The enzyme is labile towards freezing and thawing, but can be stored at 4 degrees C in 0.1 M acetate buffer, pH 5.     

4-Methylumbelliferyl-alpha-glycosides of partially O-acetylated N-acetylneuraminic acids as substrates of bacterial and viral sialidases

4-O-Acetylated, 7-O-acetylated, and 9-O-acetylated 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acids (Neu4,5Ac2-MU, Neu5,7Ac2-MU, Neu5,9Ac2-MU) were tested as substrates of sialidases of Vibrio cholerae and of Clostridium perfringens. Both sialidases were unable to hydrolyse Neu4,5Ac2-MU. This compound at 1 mM concentration did not inhibit significantly the cleavage of Neu5Ac-MU, the best substrate tested. The 4-O-acetylated sialic acid glycoside is hydrolysed slowly by the sialidase from fowl plague virus. The relative substrate specificity, reflected in V/Km of the Vibrio cholerae sialidase is Neu5Ac-MU much greater than Neu5,7Ac2-MU approximately Neu5,9Ac2-MU and of the clostridial enzyme it is Neu5Ac-MU greater than Neu5,9Ac2-MU greater than Neu5,7Ac2-MU. The affinities of both enzymes for the side-chain O-acetylated sialic acid derivatives are higher than for Neu5Ac-MU. The artificial, well-defined substrates, described here, provide the opportunity to quantify the influence of sialic acid O-acetylation on the hydrolysis of sialoglycoconjugates without the side effects introduced by other parts of more complex glycans.     

The synthesis and evaluation of novel sialic acid analogues bound to matrices for the purification of sialic acid-recognising proteins

A novel N-acetylneuraminic acid analogue, 2-S-(5'-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, as well as the thiosialoside 2-S-(2'-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group. The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield.     

Saturation transfer difference (STD) 1H-NMR experiments and in silico docking experiments to probe the binding of N-acetylneuraminic acid and derivatives to Vibrio cholerae sialidase

Saturation transfer difference (STD) (1)H NMR experiments were used to probe the epitope binding characteristics of the sialidase [EC 3.2.1.18] from the bacterium Vibrio cholerae, the causative agent of cholera. Binding preferences were investigated for N-acetylneuraminic acid (Neu5Ac, 1), the product of the sialidase catalytic reaction, for the known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid (Neu5Ac2en, 2), and for the uronic acid-based Neu5Ac2en mimetic iso-propyl 2-acetamido-2,4-dideoxy-alpha-L-threo-hex-4-enopyranosiduronic acid (3), in which the native glycerol side-chain of Neu5Ac2en is replaced with an O-iso-propyl ether. The STD experiments provided evidence, supporting previous studies, that Neu5Ac (1) binds to the sialidase as the alpha-anomer. Docking experiments using DOCK (version 4.0.1) revealed further information regarding the binding characteristics of the enzyme active site in complex with Neu5Ac2en (2) and the Neu5Ac2en mimetic (3), indicating an expected dominant interaction of the acetamide moiety with the protein.     

Neuraminidase inhibition by chemically sulphated glycopeptides.

Chemically sulphated glycopeptides (derived from pig duodenal mucosa) inhibited Clostridium perfringens neuraminidase (EC 3.2.1.18) activity in a pH-dependent manner. Analysis of inhibition kinetics data indicated that, although the enzyme inhibition could not be categorized into any of the classical types of inhibition, it could be interpreted as a function of the size and shape of the substrates used. The enzyme activity was inhibited by 86% and 40% when tested with bovine submaxillary-gland mucin (mol. wt. 4 x 10(5)-40 x 10(5) and N-acetylneuraminyl-lactose (mol. wt. 633) as substrates respectively. Presence of sulphated glycopeptide did not affect the binding of N-acetylneuraminic acid (mol. wt. 309), a competitive inhibitor of Vibrio cholerae neuraminidase, to the enzyme active site. The enzyme inhibition was thus considered to be due to steric hindrance as a consequence of the non-specific interactions between the enzyme molecule and polyanionic sulphated glycopeptide affecting the differential accessibility of the substrate molecules to the enzyme active site. The enzyme-inhibitor interaction could be suppressed by rapid and many-fold dilution of the reaction mixture, by concurrent addition of the inactive enzyme or by partial removal of the sulphate esters from the sulphated glycopeptide molecule by the action of Helix pomatia arylsulphatase (EC 3.1.6.1).     

Characteristics of human intestinal acid sialidase

With methylumbelliferyl-N-acetyl-neuraminic acid (MU-NANA) as substrate, acid sialidase was determined in intestinal biopsies of children. The enzyme has an acid pH optimum, a Km value of 4 mmol/l and a pronounced thermal lability which can be partially prevented by the addition of albumin. N-acetyl-neuraminic acid (NANA) and derivatives as well as other glycoprotein and oligosaccharide sialidase substrates inhibit sialidase whereas gangliosides have no effect. This could be an indication that intestinal MU-NANA sialidase is different from ganglioside sialidase as has been reported for many other tissues.     

Studies on the interaction of Clostridium perfringens sialidase with sialic acid linked to the internal galactose in monosialogangliotetraosyl ceramide

Investigation of the action of highly purified Clostridium perfringens sialidase on ganglioside II3Neu5Ac-Gg4Cer and its oligosaccharide II3Neu5Ac-Gg4, in the presence and absence of sodium cholate, extend earlier results obtained with impure enzyme fractions. Sialidase labeled with 125I was found to bind to various ganglioside substrate micelles, including II3Neu5Ac-Gg4Cer, and to mixed ganglioside-sodium cholate micelles. No binding occurred between the enzyme and the ganglioside-derived oligosaccharide II3Neu5Ac-Gg4, even when radioactive II3Neu5Ac-Gg4-[3H]ol was used. The binding of sialidase to micellar substrate is a condition for enzymic hydrolysis. Correspondingly, II3Neu5Ac-Gg4Cer and II3Neu5Ac-Gg4Cer-sodium cholate micelles were hydrolyzed by the enzyme but II3Neu5Ac-Gg4 was not. Ganglioside oligosaccharide analogues containing an amino function at the reducing terminus or between two oligosaccharide chains, II3Neu5Ac-Gg4-NH2 and (II3Neu5Ac-Gg4)2NH, were hydrolyzed in the absence of cholate. A synthetic analogue of II3Neu5Ac-Gg4Cer containing only the fatty acid moiety and not the sphingosine residue (I1-deoxy-I1-stearamido-II3-monosialo-gangliotetraitol ) behaved as the ganglioside in the presence and absence of sodium cholate.     

Fluorescent cytochemical detection of sialidase activity using 5-bromo-4-chloroindol-3-yl-α-D-N-acetylneuraminic acid as the substrate

A novel fluorescent cytochemical method for sialidase activity was developed using 5-bromo-4-chloroindol-3-yl-alpha- D- N-acetylneuraminic acid (X-Neu5Ac) as the substrate. Intact nuclei were isolated from porcine liver and incubated at 37 degrees C for 3 h with 1 mM X-Neu5Ac at pH 4.8. The nuclei were stained with blue color that was derived from the oxidized compound of the reaction product X (5-bromo-4-chloro-3-hydroxyindole). A specific sialidase inhibitor, 2,3-dehydro-2-deoxy- N-acetylneuraminic acid, suppressed the staining in a dose-dependent manner. Despite the specificity of the cytochemical reaction, the staining was too weak to analyze the staining distribution and pattern of individual nuclei. To attain more sensitive detection of sialidase activity, the nuclei were incubated with X-Neu5Ac in the presence of Fast Red Violet LB. Individual nuclei of porcine liver were clearly stained with fluorescence that was produced by the conjugated compound of product X with Fast Red Violet LB. This fluorescent cytochemical method was also employed successfully for detection of sialidase activity of intact GOTO neuroblastoma cells in culture. The present method should provide a useful tool for investigating the localization and stage-specific expression of sialidase activity in tissues and cells.     

N-Acetyl-4-deoxy-d-neuraminic acid is activated and transferred on to asialoglycoprotein

The sialic acid analogue,N-acetyl-4-deoxy-neuraminic acid, is readily activated by CMP-sialic acid synthase from bovine brain. We also show that sialyl-transfer from CMP-N-acetyl-4-deoxy-neuraminic acid to asialo- ₁-acid glycoprotein is achieved at a high rate using Gal1-4GlcNAc (2.6)-sialyltransferase from rat liver.In contrast toVibrio cholerae sialidase, fowl plague virus sialidase liberates boundN-acetyl-4-deoxy-neuraminic acid from the glycoprotein. Thus, as opposed to the general view, the action of neither synthase nor transferase depends on the presence of the hydroxy group at C-4 ofN-acetylneuraminic acid.Abbrevations BSA bovine serum albumin - DTE dithioerythritol - HPLC high performance liquid chromatography - NeuAc N-acetyl-d-neuraminic acid - 4-deoxy-NeuAc N-acetyl-4-deoxy-d-neuraminic acid - 4-epi-NeuAc 4-acetamido-3,5-dideoxy-d-glycero-d-talononulosonic acid - CMP-NeuAc Cytidine-5-monophospho-N-acetylneuraminic acid - CMP-4-deoxy-NeuAc Cytidine-5-monophospho-N-acetyl-4-deoxy-neuraminic acid - FPV-sialidase Fowl plague virus sialidase - VCN Vibrio cholerae neuraminidase     

Effects of strong electrolyte upon the activity of Clostridium perfringens sialidase toward sialyllactose and sialoglycolipids.

Clostridium perfringens sialidase was purified by affinity chromatography. Kinetic properties of the enzyme were examined with sialyllactose and with mixed sialoglycolipids (gangliosides) as substrates. With the latter substrate in 0.01 M Tris-acete in the absence of strong electrolyte, the pH optimum for enzymatic activity was 6.8. Addition of strong electrolyte (0.01 to 0.10 M Nac1) to the reaction medium caused an acidic shift and a broadening of the pH optimum, Enzymatic activity at pH 5.8 rose approximately 2.5-fold; a concomitant loss of activity at pH 6.8 was also observed. The alteration of enzymatic activity caused by strong electrolyte were dependent upon changes in Vmax. Km remained nearly invariant. Thus, a reversible transition of the enzyme from a relatively inactive to a highly active form occurred as a function of strong electrolyte concentration. Determination of the pK values of the active functional groups of C. perfringens sialidase revealed that the effects of strong electrolyte were exerted upon the pKa group of the enzyme. Strong electrolyte appeared to shield unfavorable electrostatic interactions between polyanionic sialoglycolipid micelles and the enzyme molecule, thus protecting the pKa group from inactivation. In comparision with the effects of strong electrolyte upon enzymatic activity toward the sialoglycolipid substrate, those observed with the monovalent substrate, sialyllacthose, were minor. Collectively, these findings indicate that ionic environment may effectively control the activity and relative substrate specificity of C. perfringens sialidase at a given pH. Furthermore, they explain the low pH optima and skewed pH profiles previously reported for enzymatic activity toward high molecular weight substrates.     

Sialidase assay by luminescence in the low picomole-range of sialic acid. Its application to the measurement of this activity in influenza virus

A new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.     

On the specificity of sialidase. Synthesis and properties of N5-acetyl-beta-D-neuraminoylpeptides - AcNeu-Gly-OH, AcNeu-Glu-OH, AcNeu-Phe-OH - and the corresponding alpha-ketosides

By means of the mixed anhydride procedure the benzyl alpha-ketoside of N5-acetyl-D-neuraminic acid was linked to L-glycine, L-glutamic acid and L-phenylalanine. Hydrogenolytic cleavage of the benzyl group resulted in the corresponding free N5-acetyl-beta-D-neuraminoylpeptides. This new class of compounds is no substrate for Vibrio cholerae sialidase. The enzyme does not split the benzyl alpha-ketosides of N5-acetyl-D-neuraminoylpeptides nor is its activity inhibited by these compounds. The results strongly support the assumption that in sialidase substrates the carboxy group must be located close to the ketosidic oxygen. N-(N5-acetyl-beta-D-neuraminoyl)-L-phenylalanine was readily hydrolysed by carboxypeptidase A from bovine pancreas.     

Peroxidase-amplified assay of sialidase activity toward gangliosides.

Sialidase assays were carried out with the substrate, ganglioside GD1a, coated onto enzyme immunoassay plate wells. Following the incubation of GD1a with sialidase from V. cholerae, the amount of ganglioside GM1 produced was measured as follows: cholera toxin B subunit conjugated to horseradish peroxidase was added to specifically bind to GM1, and then the amount of bound peroxidase was determined in a colorimetric enzymatic assay. In the absence of detergent, linearity for the detection of GM1 was 0 to 0.5 pmol per well, and the sensitivity for sialidase detection was about 3 fmol of product formed per minute. The addition of detergent (Triton CF-54) to the assay reduced the sensitivity and increased the amount of substrate required. Application of this assay for the detection of cell-derived neutral (pH 6.5) sialidase activities in the conditioned medium of human skin fibroblasts is described.     

Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic preference for sialyl alpha 2----3 linkages.

Subclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Overproduced enzyme was purified to near electrophoretic homogeneity after 65-fold enrichment using conventional preparative techniques. Unlike all previously investigated sialidases, S. typhimurium sialidase was positively charged (pI greater than or equal to 9.0). Km, Vmax, and turnover number of the purified sialidase, measured using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5,200 nmol min-1, and 2,700 s-1, respectively. These values are the highest yet reported for a sialidase. Sialidase was inhibited by 2-deoxy-2,3-didehydro-N-acetyl-neuraminic acid at unusually high concentrations (Ki = 0.38 mM), but not by 20 mM N-acetylneuraminic acid. Divalent cations were not required for activity. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. Substrate specificity measurements using natural sialoglycoconjugates showed a 260-fold kinetic preference for sialyl alpha 2----3 linkages when compared with alpha 2----6 bound sialic acids. The enzyme also efficiently cleaved residues from glycoproteins and gangliosides, but not from mucin or sialohomopolysaccharides. S. typhimurium sialidase is thus the first bacterial enzyme to be described with influenza A virus sialidase-like kinetic preference for sialyl alpha 2----3 linkages and to have a basic pI.     

Modelling, synthesis and biological evaluation of novel glucuronide-based probes of Vibrio cholerae sialidase

The development of sialidase inhibitors is an area of continuing interest due to their potential use as therapeutic agents to combat viral and bacterial infections. Herein, we report our studies involving the sialidase from the pathogen Vibrio cholerae, through the modelling, synthesis and biological evaluation of mimetics of 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic acid (Neu5Ac2en, 1), a naturally occurring sialidase inhibitor. These mimetics are O- and S-glycosides of N-acetyl-D-glucosaminuronic acid in which the aglycone portion effectively replaces the C-6 glycerol side chain of Neu5Ac2en (1). The choice of aglycones was aided by use of the X-ray crystal structure of V. cholerae sialidase complexed with Neu5Ac2en (1). All Neu5Ac2en mimetics tested were found to inhibit V. cholerae sialidase as determined using a standard fluorometric assay.     

Purification and characterization of neuraminidase from Clostridium perfringens

Neuraminidase (EC 3.2.1.18) has been purified from the culture medium of Clostridium perfringens ATCC 10543, through steps of gel filtration on Sephadex G-75 column, DEAE-cellulose DE 23 anion exchange chromatography, and isochromatofocusing. A homogeneous enzyme was obtained with a 7552-fold increase in specific activity to 295 units/mg protein. The yield was about 25%. The enzyme consists of a single polypeptide with a molecular weight of 69,000 as determined by SDS-polyacrylamide gel electrophoresis. Kinetic studies showed that Km is 1.5 mM for sialyllactose and Vmax is 0.41 mumole/min/ml at the enzyme concentration of 0.14 microgram/ml. The enzyme is stable at pH 5.2-8.0 with an optimal pH of 6.0. A concentrated solution of the purified enzyme was stable over one year at 4 degrees C. The purified enzyme hydrolyzed human alpha 1-acid glycoprotein completely; thus, it can be used in the clinical assay of N-acetylneuraminic acid in the serum.     

Inhibitory effect of taurolipids on Clostridium perfringens sialidase

Taurolipids A and B, which are detergent-type compounds isolated from protozoan Tetrahymena cells, were demonstrated to inhibit strongly the activity of Clostridium perfringens sialidase. On addition of 280 pmol of taurolipid B to 20 mU of the enzyme, the sialidase activity was decreased to 7% of the original activity at pH 5.1 as the optimum pH. The inhibition was non-competitive. Effective inhibition was observed at the acidic region from the isoelectric point of the sialidase, and at a low ionic strength. Both the long chain acyl and sulfonic acid groups of taurolipids were required for the inhibition of the sialidase activity. A mechanism is postulated for the inhibition.     

Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis.

A procedure for the synthesis of the fluorogenic substrate analogue 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid for the human acid neuraminidase has been developed. The substrate was employed for the characterization of the enzyme in sonicates of cultured human skin fibroblasts and for enzymatic detection of the neuraminidase deficiency in the neurological storage disorder, sialidosis. Synthesis was accomplished by reacting 2-deoxy-2-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in acetonitrile at room temperature. The coupled product was purified on silicic acid chromatography, followed by base-catalyzed removal of the O-acetyl and methoxy blocking groups, and with additional purification of the hydrolyzed product on silicic acid. The overall yield, based on N-acetylneuraminic acid, was 37%. Under linear assay conditions, at pH 4.3, the apparent maximal velocities (nmol (mg of protein)-1 h-1) for normal fibroblasts were 58--115, 0.2--1.8 for sialidosis fibroblasts, and 28--38 for obligate heterozygotes. The apparent Km for normals was 0.13 mM.     

A transgenic insect cell line engineered to produce CMP-sialic acid and sialylated glycoproteins

We have previously engineered transgenic insect cell lines to express mammalian glycosyltransferases and showed that these cells can sialylate N-glycoproteins, despite the fact that they have little intracellular sialic acid and no detectable CMP-sialic acid. In the accompanying study, we presented evidence that these cell lines can salvage sialic acids for de novo glycoprotein sialylation from extracellular sialoglycoproteins, such as fetuin, found in fetal bovine serum. This finding led us to create a new transgenic insect cell line designed to synthesize its own sialic acid and CMP-sialic acid. SfSWT-1 cells, which encode five mammalian glycosyltransferases, were transformed with two additional mammalian genes that encode sialic acid synthase and CMP-sialic acid synthetase. The resulting cell line expressed all seven mammalian genes, produced CMP-sialic acid, and sialylated a recombinant glycoprotein when cultured in a serum-free growth medium supplemented with N-acetylmannosamine. Thus the addition of mammalian genes encoding two enzymes involved in CMP-sialic acid biosynthesis yielded a new transgenic insect cell line, SfSWT-3, that can sialylate recombinant glycoproteins in the absence of fetal bovine serum. This new cell line will be widely useful as an improved host for baculovirus-mediated recombinant glycoprotein production.