2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN)- and N-acetylneuraminic acid-cleaving sialidase (KDN-sialidase) and KDN-cleaving hydrolase (KDNase) from the hepatopancreas of oyster, Crassostrea virginica.

KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a sialic acid analog, has been found to be widely distributed in nature. Despite the structural similarity between KDN and Neu5Ac, alpha-ketosides of KDN are refractory to conventional sialidases. We found that the hepatopancreas of the oyster, Crassostrea virginica, contains two KDN-cleaving sialidases but is devoid of conventional sialidase. The major sialidase, KDN-sialidase, effectively cleaves alpha-ketosidically linked KDN and also slowly cleaves the alpha-ketosides of Neu5Ac. The minor sialidase, KDNase, is specific for alpha-ketosides of KDN. We were able to separate these two KDN-cleaving enzymes using hydrophobic interaction and cation-exchange chromatographies. The rate of hydrolysis of 4-methylumbelliferyl-alpha-KDN (MU-KDN) by KDN-sialidase is 30 times faster than that of MU-Neu5Ac in the presence of 0.2 M NaCl, whereas in the absence of NaCl this ratio is only 8. KDNase hydrolyzes MU-KDN over 500 times faster than MU-Neu5Ac and is not affected by NaCl. KDN-sialidase purified to electrophoretically homogeneous form was found to have a molecular mass of 25 kDa and an isoelectric point of 8.4. One of the three tryptic peptides derived from KDN-sialidase contains the consensus motif, SXDXGXTW, that has been found in all conventional sialidases. Kinetic analysis of the inhibition of the hydrolysis of MU-KDN and MU-Neu5Ac by 2, 3-dehydro-2-deoxy-KDN (KDN2-en) and 2,3-dehydro-2-deoxy-(Neu5Ac2-en) suggests that KDN-sialidase contains two separate active sites for the hydrolysis of KDN and Neu5Ac. Both KDN-sialidase and KDNase effectively hydrolyze KDN-G(M3), KDNalpha2-->3Gal beta1-->4Glc, KDNalpha2-->6Galbeta1-->4Glc, KDNalpha2-->6-N-acetylgalactosaminitol, KDNalpha2-->6(KDNalpha2-->3)N-acetylgalactosaminitol, and KDNalpha2-->6(GlcNAcbeta1-->3)N-acetylgalactosaminitol. However, only KDN-sialidase also slowly hydrolyzes G(M3), Neu5Acalpha2-->3Galbeta1-->4Glc, and Neu5Acalpha2-->6Galbeta1-->4Glc. These two KDN-cleaving sialidases should be useful for studying the structure and function of KDN-containing glycoconjugates.     

Evaluation of high-performance anion-exchange chromatography with pulsed electrochemical and fluorometric detection for extensive application to the analysisof homologous series of oligo- and polysialic acids in bioactive molecules.

Our previous studies have shown extensively diverse structures in oligo/polymers of sialic acid (oligo/polySia) that are expressed often in developmentally regulated manner on animal glycoconjugates. The aim of this study was to establish highlysensitive and specific methods that can be used to identify diverse types of oligo/polySia and thus can be applied to studies of biological phenomena associated with the differential expression of oligo/polySia chains with different degree of polymerization (DP). As model compounds, we analyzed five different homologous series of oligo/polySia, (-->8Neu5Acalpha2-->)(n), (-->9Neu 5Acalpha2-->)(n), (-->8Neu5Gcalpha2-->)(n), (-->5-O(glycolyl)-Neu5Gcalpha2-->)(n), and Neu5Gc9SO(4)alpha2-->(-->5-O(glycolyl)-Neu5Gcalpha2--> )(n), ()expressed in various biopolymers. The latter two structures have recently been identified in sea urchin egg receptor for sperm. First we examined application of high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-100 column with pulsed electrochemical detection (PED) to new types of oligo/polySiacompounds and confirmed that resolution of high polymers (DP >70) of sialic acids was remarkable as reported previously. However, there are limitations in sensitivity and selectivity in PED that become significant when material is available only in a minute amount or material contained a large proportion of protein. These limitations can be circumvented by fluorometric detection of oligo/polySia tagged with 1,2-diamino-4, 5-methyl-enedioxybenzene (DMB) at the reducing terminal residues after separation on a MonoQ HR5/5 column. The latter method can be applied to any type of oligo/polySia we examined if we choose the derivatization conditions and is more sensitive and specific than the method with PED for analysis of oligo/polySia with DP up to 25.     

Synthesis of delta4-beta-D-glucopyranosiduronic acids as mimetics of 2,3-unsaturated sialic acids for sialidase inhibition.

Mimetics of Neu5Ac2en and KDN2en, based on delta4-beta-delta-glucopyranosiduronic acids, have been synthesised. The Neu5Ac2en mimetic 5 showed inhibition of both bacterial and viral sialidases, with inhibition of the viral sialidase being comparable to that of Neu5Ac2en itself.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: RESISTANCE OF α2-8-LINKED N-GLYCOLYLNEURAMINIC ACID TO ENZYMATIC CLEAVAGE

The sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative N-glycolylneuraminic acid (Neu5Gc) differ by one oxygen atom. CMP-Neu5Gc is synthesized from CMP-Neu5Ac, with Neu5Gc representing a highly variable fraction of total Sias in various tissues and among different species. The exception may be the brain, where Neu5Ac is abundant and Neu5Gc is reported to be rare. Here, we confirm this unusual pattern and its evolutionary conservation in additional samples from various species, concluding that brain Neu5Gc expression has been maintained at extremely low levels over hundreds of millions of years of vertebrate evolution. Most explanations for this pattern do not require maintaining neural Neu5Gc at such low levels. We hypothesized that resistance of α2-8-linked Neu5Gc to vertebrate sialidases is the detrimental effect requiring the relative absence of Neu5Gc from brain. This linkage is prominent in polysialic acid (polySia), a molecule with critical roles in vertebrate neural development. We show that Neu5Gc is incorporated into neural polySia and does not cause in vitro toxicity. Synthetic polymers of Neu5Ac and Neu5Gc showed that mammalian and bacterial sialidases are much less able to hydrolyze α2-8-linked Neu5Gc at the nonreducing terminus. Notably, this difference was not seen with acid-catalyzed hydrolysis of polySias. Molecular dynamics modeling indicates that differences in the three-dimensional conformation of terminal saccharides may partly explain reduced enzymatic activity. In keeping with this, polymers of N-propionylneuraminic acid are sensitive to sialidases. Resistance of Neu5Gc-containing polySia to sialidases provides a potential explanation for the rarity of Neu5Gc in the vertebrate brain.     

The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-Å resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3-dideoxy-d-glycero-d-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.     

Specificity of the N1 and N2 sialidase subtypes of human influenza A virus for natural and synthetic gangliosides

Sialyl-linkage specificity of sialidases of the human influenza A virus strains, A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1) were studied using natural and synthetic gangliosides. The sialidase of the A/Aichi/2/68 strain hydrolyzed the terminal Neu5Acalpha2-3Gal sequence but not the Neu5Acalpha2-3 linkage on the inner Gal of GM1a, which is a ganglioside that has the gangliotetraose chain (Galbeta1-3GalNAcbeta1-4- (Neu5Acalpha2-3)Galbeta1++ +-4Glcbeta1-Cer). The sialidase hydrolyzed the Neu5Ac on the inner Gal of GM2, which had a shorter gangliotriose chain. GM4, which had the shortest chain (Neu5Acalpha2-3Galbeta1-Cer) of the gangliosides, had a lower substrate specificity. The N1 and N2 sialidase subtypes of the human influenza A virus had no significant variation in their substrate specificity for the gangliosides. Analysis of 11 synthetic gangliosides, which contained various ceramide or sialic acid moieties, demonstrated that A/Aichi/2/68 (H3N2) sialidase recognized the ceramide and sialic acid moiety and the length and structure of the sialyl sugar chain.      

Detection, isolation, and characterization of oligo/poly(sialic acid) and oligo/poly(deaminoneuraminic acid) units in glycoconjugates.

We have evaluated methods for separation, preparation, and characterization of alpha-2----8-linked oligomers of sialic acids (Neu5Ac and Neu5Gc) and deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) recently found as a naturally occurring novel type of sialic acid analogue. (A) We examined preparative anion-exchange chromatography for fractionation and preparation of oligo(Neu5Ac), oligo(Neu5Gc), and oligo(KDN). (B) We also examined the TLC method for separation and differentiation of the partial acid hydrolysates of colominic acid, as well as polysialoglycoproteins (PSGP) and poly(KDN)-glycoproteins (KDN-gp) isolated from rainbow trout eggs, and for discrimination of lower oligomers of Neu5Ac, Neu5Gc, and KDN. (C) We developed the high-performance adsorption-partition chromatographic method for (a) separation of monomers and oligomers of three nonulosonates according to the difference in substituents at C-5 and the presence or absence of 9-O-acetyl groups in oligo(KDN) and (b) separation of three homologous series of lower oligomers according to the degree of polymerization. (D) We examined and compared high-performance anion-exchange chromatographic separation of 3H-labeled oligo(Neu5Ac), oligo(Neu5Gc), and oligo(KDN) alditols by using Mono-Q HR 5/5 resin. (E) We examined a method of selective and quantitative microprecipitation for separation and purification of oligomers and polymers of Neu5Ac by treating them with cetylpyridinium chloride. We also used PSGP and KDN-gp to test both the sensitivity and the selectivity of this method.     

Rapid Trimming of Cell Surface Polysialic Acid (PolySia) by Exovesicular Sialidase Triggers Release of Preexisting Surface Neurotrophin

As acidic glycocalyx on primary mouse microglial cells and a mouse microglial cell line Ra2, expression of polysialic acid (polySia/PSA), a polymer of the sialic acid Neu5Ac (N-acetylneuraminic acid), was demonstrated. PolySia is known to modulate cell adhesion, migration, and localization of neurotrophins mainly on neural cells. PolySia on Ra2 cells disappeared very rapidly after an inflammatory stimulus. Results of knockdown and inhibitor studies indicated that rapid surface clearance of polySia was achieved by secretion of endogenous sialidase Neu1 as an exovesicular component. Neu1-mediated polySia turnover was accompanied by the release of brain-derived neurotrophic factor normally retained by polySia molecules. Introduction of a single oxygen atom change into polySia by exogenous feeding of the non-neural sialic acid Neu5Gc (N-glycolylneuraminic acid) caused resistance to Neu1-induced polySia turnover and also inhibited the associated release of brain-derived neurotrophic factor. These results indicate the importance of rapid turnover of the polySia glycocalyx by exovesicular sialidases in neurotrophin regulation.     

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.     

Sialidase specificity determined by chemoselective modification of complex sialylated glycans

Sialidases hydrolytically remove sialic acids from sialylated glycoproteins and glycolipids. Sialidases are widely distributed in nature and sialidase-mediated desialylation is implicated in normal and pathological processes. However, mechanisms by which sialidases exert their biological effects remain obscure, in part because sialidase substrate preferences are poorly defined. Here we report the design and implementation of a sialidase substrate specificity assay based on chemoselective labeling of sialosides. We show that this assay identifies components of glycosylated substrates that contribute to sialidase specificity. We demonstrate that specificity of sialidases can depend on structure of the underlying glycan, a characteristic difficult to discern using typical sialidase assays. Moreover, we discovered that Streptococcus pneumoniae sialidase NanC strongly prefers sialosides containing the Neu5Ac form of sialic acid versus those that contain Neu5Gc. We propose using this approach to evaluate sialidase preferences for diverse potential substrates.     

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin,Paracentrotus lividus,using capillary LC-ESI-MS/MS

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac₂, Neu5,7Ac₂, Neu5,8Ac₂, Neu5,7,9Ac₃, Neu5Gc7,9Ac₂, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).     

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.     

STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core

The VP8* subunit of rotavirus spike protein VP4 contains a sialic acid (Sia)-binding domain important for host cell attachment and infection. In this study, the binding epitope of the N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding epitope in both methyl alpha-D-N-acetylneuraminide (Neu5Acalpha2Me) and the disaccharide methyl S-(alpha-D-N-acetylneuraminosyl)-(2-->6)-6-thio-beta-D-galactopyranoside (Neu5Ac-alpha(2,6)-S-Galbeta1Me). In the VP8*-disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the protein, whereas the galactose moiety is solvent-exposed. Molecular dynamics calculations of the VP8*-disaccharide complex indicated that the galactose moiety is unable to adopt a conformation that is in close proximity to the protein surface. STD NMR experiments with methyl 9-O-acetyl-alpha-D-N-acetylneuraminide (Neu5,9Ac(2)alpha2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-acetamide's methyl group being saturated to a larger extent, indicating a closer association with the protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en)]. Molecular modeling of the protein-Neu5Ac2en complex indicates that key interactions between the protein and the unsaturated Neu5Ac derivative when compared with Neu5Acalpha2Me would not be sustained. Neu5Acalpha2Me, Neu5Ac-alpha(2,6)-S-Galbeta1Me, Neu5,9Ac(2)alpha2Me, and Neu5Ac2en inhibited rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively, at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of rotavirus infection.     

Separation of Oligo/Polymers of 5-N-Acetylneuraminic Acid, 5-N-Glycolylneuraminic Acid, and 2-Keto-3-deoxy-d-glycero-d-galacto-nononic Acid by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detector

A sensitive and efficient method to analyze oligo/poly-sialic acids containing α2–8-linked 5-N-acetylneuraminic acid (Neu5Ac), 5-N-glycolylneuraminic acid (Neu5Gc), and deaminated neuraminic acid (KDN) using high-performance anion-exchange chromatography (HPAEC) with a pulsed amperometric detector (PAD-2) has been developed. Using a CarboPac PA-100 column and sodium nitrate as the pushing agent, polymers in colominic acid with degree of polymerization (DP) up to 80 were separated in 68 min. A similar DP-based resolution was also obtained on a CarboPac PA-1 column. The elution ladders of the Neu5Ac, Neu5Gc, and KDN series were sufficiently different to be used as diagnostic indices. This technique was applied to identification of the sialic acid components in a polysialoglycoprotein (PSGP) sample as well as monitoring the oligo/poly-KDN-containing fractions during the purification of KDN-containing glycoprotein (KDN-gp). The maximum DPs of oligo-Neu5Gc and oligo-KDN that can be detected in PSGP and KDN-gp hydrolysates were 11 and 8, respectively. The high sensitivity of this method was demonstrated by the quantification of Neu5Ac oligomers. Distributions of the monomer and oligo/polymers in the acid and enzymatic hydrolysates of colominic acid and PSGP under different conditions were also studied.     

The action of sialidases on substrates containing O-acetylsialic acids

O-Acetyl substitution of sialic acids in glycoconjugates reduces the rate of action of sialidases on these substrates. A plasma glycoprotein fraction and an erythrocyte ganglioside containing 4-O-acetylsialic acids were isolated and characterized from equine blood, and a sialyllactose preparation with Neu5,9Ac2 was purified from rat urine. Using the novel substrates II3Neu4Ac5Gc-LacCer and II3Neu5,9Ac2-Lac the influence of individual mono-O-acetylated sialic acids on bacterial and viral sialidases could be clearly shown. This extends and clarifies observations with glycoproteins containing mixtures of mono-, di- and higher O-acetylated sialic acids with substitution at the hydroxyls on carbons 4, 7, 8 and 9. A 4-O-acetyl substitution in sialic acids blocks the action of bacterial sialidases for substrates containing these derivatives, while viral enzymes show low but significant activity, reflected in Km and Vmax values. A small reduction in bacterial sialidase activity was observed for II3Neu5,9Ac2-Lac relative to II3Neu5Ac-Lac in agreement with kinetic analysis. Newcastle disease virus sialidase showed a 50% reduction in hydrolysis rate for the 9-O-acetylated substrate and ten-fold reductions of both Km and Vmax values.     

Identification of sialic acids on Leishmania donovani amastigotes

The presence of Neu5Ac on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, has been reported recently. Here we report the occurrence of Neu5Ac as a major component on amastigotes, as well as Neu5Gc, Neu5,9Ac2 and Neu9Ac5Gc as indicated by fluorimetric high performance liquid chromatography and gas liquid chromatography/electron impact mass spectrometry. Furthermore, binding studies with Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and various Siglecs, showed the presence of both (alpha2 --> 6)- and (alpha2 --> 3)-linked sialic acids; their binding was reduced after sialidase pretreatment. Western blotting of amastigote membrane glycoproteins with SNA demonstrated the presence of two sialoglycoconjugates of Mr values of 164000 and 150000. Similarly, binding of MAA demonstrated the presence of five distinct sialoglycans corresponding to molecular masses of 188, 162, 136, 137 and 124 kDa. Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid (alpha2 --> 6)-linked to GalNAc, demonstrated the occurrence of two 9-O-acetylated sialoglycans with Mr 158000 and 150000, and was corroborated by flow cytometry; this binding was abolished by recombinant 9-O-acetylesterase pretreatment. Our results indicate that Neu5Ac [(alpha2 --> 6)- and (alpha2 --> 3)-linked], as well as Neu5Gc and their 9-O-acetyl derivatives, constitute components of the amastigote cell surface of L. donovani.     

New sialic acids from biological sources identified by a comprehensive and sensitive approach: liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones

Sialic acids are a family of 9-carbon carboxylated sugars, wheredifferent substitutions of the backbone define over 30 members.Biological roles of these substitutions have been missed untilrecently because of their low abundance and lability to conventionalisolation/purification methods. This new approach characterizessialic acids using electrospray ionization-mass spectrometry(ESI-MS) to monitor the HPLC separation of their DMB (1,2-diamino-4,5-methylenedioxy-benzene)derivatives (quinoxalinones). A combination of retention timesand spectra characteristics allows definition of the type andposition of the various substituents. This approach requiresno previous purification, involving a simple derivatizationreaction followed by direct injection on the microbore HPLCcolumn. A complete spectrum, including molecular ions and CADfragments of a sialic acid qainoxalinone, is obtained by injecting10–20 pmol of the compound. Individual quinoxalinonescan be purified by regular RP-HPLC and analyzed by direct-injectionESI-MS or LSIMS. Using this approach, we identified 28 differentsialic acids, including the following new species: Neu5Gc9Lt(BSM), anhydro derivatives of Neu5Ac other than the 4,8-anhydro(horse serum hydrolyzates), KDNS(7)Ac and KDN5(7),9Ac₂ (amphibianPleurodeles waltl), four isomers of Neu5Gc8MexAc and three anhydroderivatives of Neu5Gc8MS (glycolipids of the starfish Pisasterbrevirpinus), and Neu5Ac8S (in addition to Neu5Gc8S, in theglycolipids of the sea urchin Lovenia cordiformis). Resultsshow the usefulness of LC-ESIMS to study sialic acid diversity,and identification of small amounts of unexpected sialic acidsor new members of their family. sialic acid purification electrospray ionizationmass spectrometry quinoxalinone     

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d- galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 &mgr;m recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.      

Triazole-linked transition state analogs as selective inhibitors against V. cholerae sialidase

Sialidases or neuraminidases are enzymes that catalyze the cleavage of terminal sialic acids from oligosaccharides and glycoconjugates. They play important roles in bacterial and viral infection and have been attractive targets for drug development. Structure-based drug design has led to potent inhibitors against neuraminidases of influenza A viruses that have been used successfully as approved therapeutics. However, selective and effective inhibitors against bacterial and human sialidases are still being actively pursued. Guided by crystal structural analysis, several derivatives of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en or DANA) were designed and synthesized as triazole-linked transition state analogs. Inhibition studies revealed that glycopeptide analog E-(TriazoleNeu5Ac2en)-AKE and compound (TriazoleNeu5Ac2en)-A were selective inhibitors against Vibrio cholerae sialidase, while glycopeptide analog (TriazoleNeu5Ac2en)-AdE selectively inhibited Vibrio cholerae and A. ureafaciens sialidases.     

A new sialic acid analogue, 9-O-acetyl-deaminated neuraminic acid, and alpha -2,8-linked O-acetylated poly(N-glycolylneuraminyl) chains in a novel polysialoglycoprotein from salmon eggs

A new polysialoglycoprotein, designated PSGP(On), was isolated from the unfertilized eggs of the kokanee salmon, Oncorhynchus nerka adonis. 400-MHz 1H NMR analyses showed the O. nerka adonis PSGP contained alpha -2,8-linked oligo- and polysialic acid (polySia) chains that were made up of 4-O-Ac-, 7-O-Ac-, and 9-O-Ac esters of N-glycolylneuraminic acid (Neu5Gc) residues. The presence of a new sialic acid derivative, identified by 1H NMR as 9-O-acetyl-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (trivial name, 9-O-acetyldeaminated neuraminic acid; 9-O-Ac-KDN), was also shown to be present as a minor component. The O-acetylated KDN residues appear to cap the nonreducing termini of the O-acetylated poly(Neu5Gc) chains. The O-acetylated polySia chains were resistant to depolymerization by bacterial exosialidases and a bacteriophage-derived endo-N-acylneuraminidase that is specific for catalyzing the hydrolysis of alpha -2,8-linkages in polySia containing either N-acetylneuraminic acid or Neu5Gc residues. After de-O-acetylation by mild alkali, the polySia chains were sensitive to digestion by endo-N-acylneuraminidase, yet partially resistant to exosialidase. These data confirm the alpha -2,8-ketosidic linkage in these chains and the nonreducing terminal location of the KDN residues. These results extend further the range of structural diversity in polySia-containing glycoconjugates, and in the family of naturally occurring sialic acids. They also suggest that the O-acetylated Neu5Gc and 9-O-Ac-KDN residues may have an important role during oogenesis.