Identification and partial characterization of soluble and membrane-bound KDN(deaminoneuraminic acid)-glycoproteins in human ovarian teratocarcinoma PA-1, and enhanced expression of free and bound KDN in cells cultured in mannose-rich media

KDN (Deaminoneuraminic acid, or deaminated neuraminic acid) is a minor but biosynthetically independent member of the sialic acid. Human occurrence of KDN has already been established, although its level is so little that it is often undetectable by conventional sialic acid analysis. Elevated expression of KDN in fetal cord blood cells and some malignant tumor cells have been reported. However, in mammalian cells and tissues KDN mostly occurs as the free sugar and little occurred conjugated to glycolipids and/or glycoproteins. A positive correlation between the ratio of free KDN/free Neu5Ac in ovarian adenocarcinomas and the stage of malignancy has been noted for diagnostic use. We hypothesized that elevated expression of KDN in mammalian systems may be closely related to elevated activities of enzymes involved in the formation of sialoglycoconjugates and/or aberrant supply of the precursor sugar, mannose, used in the biosynthesis of KDN. In this study we used human ovarian teratocarcinoma cells PA-1 to further analyze KDN expression in human cells. Major findings reported in this paper are, (i) a 30 kDa KDN-glycoprotein immunostainable with monoclonal antibody, mAb.kdn3G, (specific for the KDNα2 → 3Galβ1→ epitope) and sensitive to KDNase was identified in the membrane fraction of the cell: (ii) a 49 kDa KDN-glycoprotein that is not reactive with mAb.kdn3G but is sensitive to KDNase was identified in the soluble fraction: and (iii) PA-1 cells showed unique response to mannose added to the growth medium in that the levels of both free and bound forms of KDN are elevated. This is the first report on the identification of mammalian KDN-glycoproteins by chemical and biochemical methods.     

The presence of N-acetylneuraminic acid in Malpighian tubules of larvae of the cicada Philaenus spumarius

Sialic acid-containing glycoconjugates are generally considered to be unique to the deuterostomes, a lineage of the animal kingdom which includes animals from the echinoderms up to the vertebrates. There are, however, two isolated reports of sialic acid occurring in the insect species Drosophila melanogaster and Galleria mellonella. Since insects are classified as protostomes, these findings call previous assumption on the phylogenetic distribution and thus on the evolution of sialic acids into question. Here, we report the occurrence of N-acetylneuraminic acid (Neu5Ac) in larvae of the cicada Philaenus spumarius. Cytochemical analysis of larval sections with lectins from Sambucus nigra and Limax flavus suggested the presence of sialic acids in the concrement vacuoles of the Malpighian tubules. The monoclonal antibody MAb 735, which is specific for polysialic acid, labelled the same structures. A chemical analysis performed by HPLC of fluorescent derivatives of sialic acids and by GLC-MS provided sound evidence for the presence of Neu5Ac in the Philaenus spumarius larvae. These data suggest that in this cicada Neu5Ac occurs in 2,8-linked polysialic acid structures and in 2,6-linkages. The results provide further evidence for the existence of sialic acids in insects and in linkages known to occur in glycoconjugates of deuterostomate origin.     

Isolation and properties of the natural and the recombinant sialidase fromClostridium septicum NC 0054714

The natural sialidase ofClostridium septicum was purified and characterized in parallel with the recombinant enzyme expressed byEscherichia coli. The two enzymes exhibit almost identical properties. The maximum hydrolytic activity was measured at 37 °C in 60mm sodium acetate buffer, pH 5.3. Glycoproteins like fetuin and saponified bovine submandibular gland mucin, most of them having (2-6) linked sialic acids, are preferred substrates, while sialic acids from gangliosides, sialyllactoses, or the (2-8) linked sialic acid polymer (colominic acid) are hydrolysed at lower rates. (2-3) Linkages are more rapidly hydrolysed than (2-6) bonds of sialyllactoses. The cleavage rate is markedly reduced by O-acetylation of the sialic acid moiety. These properties are similar to those of other secreted clostridial sialidases. The enzyme exists in mono-, di- and trimeric forms, the monomer exhibiting a molecular mass of 125 kDa, which is close to the protein mass of 111 kDa deduced from the nucleotide sequence of the cloned gene.Abbreviations BSM bovine submandibular gland mucine - CMM cooked meat medium - EDTA ethylenediaminetetraacetic acid - FPLC fast performance liquid chromatography - LB Luria-Bertani - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - Neu4,5Ac₂ N-acetyl-4-O-acetylneuraminic acid - pI isoelectric point - SDS sodium dodecyl sulfate     

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.     

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.     

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.     

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.     

Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability

Sialic acids participate in many important biological recognition events, yet eukaryotic sialic acid biosynthetic genes are not well characterized. In this study, we have identified a novel human gene based on homology to the Escherichia coli sialic acid synthase gene (neuB). The human gene is ubiquitously expressed and encodes a 40-kDa enzyme. The gene partially restores sialic acid synthase activity in a neuB-negative mutant of E. coli and results in N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) production in insect cells upon recombinant baculovirus infection. In vitro the human enzyme uses N-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates to generate phosphorylated forms of Neu5Ac and KDN, respectively, but exhibits much higher activity toward the Neu5Ac phosphate product.     

Preparation and immunological studies of protein conjugates of N-acylneuraminic acids

The overexpression of N-acetylneuraminic acid (Neu5Ac) is closely correlated with malignant transformations. Thus, Neu5Ac is an important target in the design of cancer vaccines. To study the influence of chemical modifications of Neu5Ac on its immunological properties, the α-allyl glycosides of five differently N-acylated neuraminic acid derivatives were prepared. Following selective ozonolysis of their allyl group to form an aldehyde functionality, they were coupled to keyhole limpet hemocyanin (KLH) via reductive amination. Resultant glycoconjugates were studied in C57BL/6 mice. The N-propionyl, N-iso-butanoyl and N-phenylacetyl derivatives of neuraminic acid provoked robust immune responses of various antibody isotypes, including IgM, IgG1, IgG2a and IgG3, whereas N-trifluoropropionylneuraminic acid and natural Neu5Ac were essentially nonimmunogenic. Moreover, the N-phenylacetyl and N-iso-butanoyl derivatives mainly induced IgG responses that are desirable for antitumor applications. These results raise the promise of formulating effective glycoconjugate cancer vaccines via derivatizing sialic acid residues of sialooligosaccharides. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640-2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

Identification,characterization, and developmental expression of a novel α2 → 8-KDN-transferase which terminates elongation of α2 → 8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2–5mm Mg²⁺ or Mn²⁺. Expression of KDN-transferase was developmentally regulated in parallel with expression of the 2 8-polysialytransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in capping of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of 2 8-polysialylation in glycoproteins.Abbreviations KDN 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid or naturally occurring deaminated neuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ge N-glycolylneuraminic acid - CMP-KDN cytidine 5-(3-deoxy-d-glycero-d-galacto-2-nonulosonic phosphate) or cytidine 5-KDN phosphate - CMP-NeuAc cytidine 5-Neu5Ac phosphate; oligo-polySia, oligo- and/or polysialic acid - PSGP rainbow trout egg polysialoglycoprotein comprising 2 8-linked oligo- polyNeu5Gc - PSGP (low Sia) a precursor of PSGP present at early stages of oogenesis which contains mostly the disialyl group, Sia2 8Sia2 6- - *K-PSGP [¹⁴C]KDN-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]KDN with the immature cortical vesicle fraction P1 containing KDN-transferase - *A-PSGP [¹⁴C]Neu5Ac-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]Neu5Ac with the P1 fraction - A-*K-PSGP andK-*K-PSGP the products obtained after incubating *K-PSGP with P1 fraction and unlabelled CMP-Neu5Ac or CMP-KDN, respectively - *K-PSGP _cho ,A-*K-PSGP _cho , andK-*K-PSGP _cho mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of *K-PSGP,A-*K-PSGP, and K-*K-PSGP, respectively - *A-PSGP _cho a mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of [¹⁴C]Neu5Ac-labelled PSGP - Endo-N endo-N-acylneuraminidase - DP degree of polymerization - GLC gas-liquid chromatography - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Inhibition of human parainfluenza virus type 1 sialidase by analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid

Eleven novel analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) modified at the C-4 and C-9 positions were designed and tested for their ability to inhibit sialidase of human parainfluenza virus type 1 (hPIV-1). The analogs modified by the cyanomethyl, amidinomethyl, and thiocarbamoylmethyl groups at the C-4 position exhibited potent inhibition against hPIV-1 sialidase compared with Neu5Ac2en. The most effective compound was thiocarbamoylmethyl analog (4-O-thiocarbamoylmethyl-Neu5Ac2en). The activity of 4-O-thiocarbamoylmethyl-Neu5Ac2en causing 50% enzyme inhibition at a concentration of approximately 1.0×10^–5M was 30-fold larger than Neu5Ac2en. While, the analogs of Neu5Ac2en modified by the azido and N-acetyl groups at the C-9 showed a decrease in inhibition of sialidase compared with the 9-hydroxy analogs. In addition, 4-O-thiocarbamoylmethyl-Neu5Ac2en strongly inhibited hPIV-1 infections of Lewis lung carcinoma-monkey kidney cells in comparison with Neu5Ac2en. The present findings would provide useful information for the development of anti-human parainfluenza virus compounds.     

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.      

Structure of the O-antigen and characterization of the O-antigen gene cluster of Escherichia coli O108 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic (8-epilegionaminic) acid

On mild acid degradation of the lipopolysaccharide of Escherichia coli O108, the O-polysaccharide was isolated and studied by sugar analysis and one- and two-dimensional ¹H- and ¹³C-NMR spectroscopy. The polysaccharide was found to contain an unusual higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). The following structure of the tetrasaccharide repeating unit of the polysac-charide was established: →4)-α-8eLegp5Ac7Ac-(2→6)-α-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-α-D-GlcpNAc-(1→. Functions of the E. coli O108 antigen biosynthetic genes, including seven putative genes for synthesis of 8eLeg5Ac7Ac, were assigned by sequencing the O-antigen gene cluster along with comparison with gene databases and known biosynthetic pathways for related nonulosonic acids.     

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.     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

On the biosynthesis of alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid catalyzed by the sialyltransferase of Escherichia coli Bos-12.

Escherichia coli Bos-12 synthesizes a heteropolymer of sialic acids with alternating alpha-2,9/alpha-2,8 glycosidic linkages (1). In this study, we have shown that the polysialyltransferase of the E. coli Bos-12 recognizes an alpha-2,8 glycosidic linkage of sialic acid at the nonreducing end of an exogenous acceptor of either the alpha-2,8 homopolymer of sialic acid or the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid and catalyzes the transfer of Neu5Ac from CMP-Neu5Ac to this residue. When the exogenous acceptor is an alpha-2,8-linked oligomer of sialic acid, the main product synthesized is derived from the addition of a single residue of [14C]Neu5Ac to form either an alpha-2,8 glycosidic linkage or an alpha-2,9 glycosidic linkage at the nonreducing end, at an alpha-2, 8/alpha-2,9 ratio of approximately 2:1. When the acceptor is the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid, chain elongation takes place four to five times more efficiently than the alpha-2,8-linked homopolymer of sialic acid as an acceptor. It was found that the alpha-2,9-linked homopolymer of sialic acid and the alpha-2,8/alpha-2,9-linked hetero-oligomer of sialic acid with alpha-2,9 at the nonreducing end not only failed to serve as an acceptor for the E. coli Bos-12 polysialyltransferase for the transfer of [14C]Neu5Ac, but they inhibited the de novo synthesis of polysialic acid catalyzed by this enzyme. The results obtained in this study favor the proposal that the biosynthesis of the alpha-2, 9/alpha-2,8 heteropolymer of sialic acid catalyzed by the E. coli Bos-12 polysialyltransferase involves a successive transfer of a preformed alpha-2,8-linked dimer of sialic acid at the nonreducing terminus of the acceptor to form an alpha-2,9 glycosidic linkage between the incoming dimer and the acceptor. The glycosidic linkage at the nonreducing end of the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid produced by E. coli Bos-12 should be an alpha-2,8 glycosidic bond and not an alpha-2,9 glycosidic linkage.     

Adhesion ofHelicobacter pylori strains toα-2,3-linked sialic acids

Helicobacter pylori is a human pathogen associated with gastritis and peptic ulcer. Adhesion properties ofH. pylori to various structures have been described in the literature, including evidence for sialic acid-binding. To study the specificity and frequency of sialic acid-binding, fourteenH. pylori strains were investigated using haemagglutination with derivatized erythrocytes carrying sialic acids only on defined glycans and using haemagglutination inhibition assays. From these studiesH. pylori strains can be grouped into sialic acid-dependent and sialic acid-independent classes. The sialic acid-dependent strains require -2,3-linked sialic acid for haemagglutination. The potential roles of sialic acid-dependent adhesions forH. pylori-related infections are discussed.Abbreviations Sia sialic acids - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolylneuraminic acid - Neu5Fm N-formylneuraminic acid - Neu5TFA N-trifluoroacetylneuraminic acid - RBC human red blood cells (erythrocytes)     

An efficient method for N-acetyl-d-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system

N-Acetyl-d-neuraminic acid (Neu5Ac) is a precursor for producing many pharmaceutical drugs such as zanamivir which have been used in clinical trials to treat and prevent the infection with influenza virus, such as the avian influenza virus H5N1 and the current 2009 H1N1. Two recombinant Escherichia coli strains capable of expressing N-acetyl-d-glucosamine 2-epimerase and N-acetyl-d-neuraminic acid aldolase were constructed based on a highly efficient temperature-responsive expression system which is safe compared to chemical-induced systems and coupled in Neu5Ac production. Carbon sources were optimized for Neu5Ac production, and the concentration effects of carbon sources on the production were investigated. With 2,200 mM pyruvate as carbon source and substrate, 61.9 mM (19.1 g l⁻¹) Neu5Ac was produced from 200 mM N-acetyl-d-glucosamine (GlcNAc) in 36 h by the coupled cells. Our Neu5Ac biosynthetic process is favorable compared with natural product extraction, chemical synthesis, or even many other biocatalysis processes.