The nature of sialic acids in human lymphocytes

Analysis of the sialic acids obtained by mild acid hydrolysis of B lymphocytes reveals the presence of N-acetylneuraminic acid and 9-O-acetyl-N-acetylneuraminic acid. For T lymphocytes only N-acetylneuraminic acid has been demonstrated to occur. The applied methods include quantitative colorimetry, thin-layer chromatography and combined gas-liquid chromatography-mass spectrometry.     

Chemoenzymatic synthesis of C8-modified sialic acids and related α2-3- and α2-6-linked sialosides

Naturally occurring 8-O-methylated sialic acids, including 8-O-methyl-N-acetylneuraminic acid and 8-O-methyl-N-glycolylneuraminic acid, along with 8-O-methyl-2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (Kdn8Me) and 8-deoxy-Kdn were synthesized from corresponding 5-O-modified six-carbon monosaccharides and pyruvate using a sialic acid aldolase cloned from Pasteurella multocida strain P-1059 (PmNanA). In addition, α2-3- and α2-6-linked sialyltrisaccharides containing Neu5Ac8Me and Kdn8Deoxy were also synthesized using a one-pot multienzyme approach. The strategy reported here provides an efficient approach to produce glycans containing various C8-modified sialic acids for biological evaluations.     

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.     

Binding specificity of influenza C-virus to variablyO-acetylated glycoconjugates and its use for histochemical detection ofN-acetyl-9-O-acetylneuraminic acid in mammalian tissues

The specificity of influenza C-virus binding to sialoglycoconjugates was tested with various naturallyO-acetylated gangliosides or syntheticallyO-acetylated sialic acid thioketosides, which revealed binding to 9-O-acetylatedN-acetylneuraminic acid. Binding was also observed with a sample of Neu5,7Ac₂-GD3, however at a lower degree. Sialic acids with two or threeO-acetyl groups in the side chain of synthetic sialic acid derivatives are not recognized by the virus. In these experiments, bound viruses were detected with esterase substrates. Influenza C-virus was also used for the histological identification of mono-O-acetylated sialic acids in combination with an immunological visualization of the virus bound to thin-sections. The occurrence of these sialic acids was demonstrated in bovine submandibular gland, rat liver, human normal adult and fetal colon and diseased colon, as well as in human sweat gland. Submandibular gland and colon also contain significant amounts of glycoconjugates with two or three acetyl esters in the sialic acid side chain, demonstrating the value of the virus in discriminating between mono- and higherO-acetylation at the same site. The patterns of staining showed differences between healthy persons and patients with colon carcinoma, ulcerative colitis or Crohn's disease. Remarkably, some human colon samples did not showO-acetyl sialic acid-specific staining. The histochemical observations were controlled by chemical analysis of tissue sialic acids.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - HAU haemagglutination units - HPLC high-performance liquid chromatography - HPTLC high-performance thin-layer chromatography - Neu5Ac N-acetylneuraminic acid - Neu5,9Ac₂ N-acetyl-9-O-acetylneuraminic acid - Neu5,7,9Ac₃ N-acetyl-7,9-di-O-acetylneuraminic acid - Neu5,7,8,9Ac₄ N-acetyl-7,8,9-tri-O-acetylneuraminic acid - PBS phosphate-buffered saline - TLC thin-layer chromatography Dedicated to Prof. Dr Nathan Sharon on the occasion of his 70th birthday.     

Indications for the enzymatic synthesis of 9-O-lactoyl-N-acetylneuraminic acid in equine liver

Fractionation of horse liver homogenate by centrifugation into heavy membranes at 10 000 × g, microsomal fraction at 105 000 × g, and the supernatant revealed sialate 9-O-lactoyltransferase activity only in the latter fraction. For the enzyme assay, the various fractions were incubated with¹⁴C labelled CMP-N-acetylneuraminic acid,N-acetylneuraminic acid and glycoconjugate-boundN-acetylneuraminic acid. Lactoylation was identified in three different TLC systems after acid hydrolysis and purification of the sialic acids in the incubation mixtures. Enzyme activity was found only in the supernatant fraction. Glycoconjugate-boundN-acetylneuraminic acid was the best substrate tested, although some lactoylation was also found when using CMP-N-acetylneuraminic acid.     

Fucopyranosyl-(1 → 4)-N-glycolylneuraminic acid,a constituent of glycoproteins of the cuvierian tubules of the sea cucumber Holothuria forskali Della Chiaje

Analysis of the sialic acid fraction obtained by mild acid hydrolysis of the Cuvierian tubules of the sea cucumber Holothuria forskali Della Chiaje revealed the presence of F-glycolylneuraminic acid and fucopyranosyl-(1 → 4)-N-glycolylneuraminic acid. Furthermore, methylation analysis of a pronase-digest of the tubules demonstrated that all N-glycolylneuraminic acid residues are substituted at C-4, explaining the earlier reported resistance of sialic acid in tubules to cleavage by neuraminidase.     

Mass spectrometry of pertrimethylsilyl nueraminic acid derivatives

The mass spectra of the trimethylsilyl (TMS) derivatives of the methyl and trideuteriomethyl esters of N-acetylneuraminic acid, the methyl ester of N-glycolylneuraminic acid, the methyl ester methyl β-glycoside of N-acetylneuraminic acid, the trideuteriomethyl ester trideuteriomethyl β-glycoside of N-acetylneuraminic acid, and the methyl esters of the (2→3)- and (2→6)-linked isomers of N-acetylneuraminic acid—lactose are discussed. The characteristic fragmentation patterns of the sialic acid derivatives can be used for the identification of this type of carbohydrate. The (2→3)- and (2→6)-linked isomers of N-acetylneuraminic acid—lactose can be differentiated.     

High-performance liquid chromatography of N,O-acylated sialic acids

A rapid, isocratic high-performance liquid chromatographic method for the analysis of N-acetylneuraminic acid, N-glycolylneuraminic acid, and their O-acetylated derivatives is described. Separation of sialic acids and of other monosaccharides as sugar-borate complexes is achieved on an anion-exchange resin. The sialic acids elute as individual peaks after the other sugars tested. The method allows quantitative determination, for example, of amounts of N-acetylneuraminic acid as small as 10 nmol. On cation-exchange resin sialic acids cannot be differentiated, but can be separated from neutral and amino sugars, allowing the determination of as little as 3 nmol of total sialic acids.     

Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus

Influenza C virus spike glycoprotein HEF specifically recognizesglycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid.The same protein also contains an esterase activity. Takingadvantage of these two properties, influenza C virus was usedas a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminicacid in human leucocytes. The binding of influenza C virus toleucocyte glycoproteins and gangliosides separated by sodiumdodecyl sulphate–polyacrylamide gel electrophoresis andthin-layer chromatography, respectively, was assayed using achromogenic esterase substrate. In this way, glycoproteins ofB-lymphocytes and T-lymphocytes were found to contain 9-O-acetylatedsialic acids. Of the various 9-O-acetylated gangliosides detected,one had the characteristics of 9-O-acetylated G^D3. The identificationof 9-O-acetylated sialic acids on distinct glycoproteins andglycolipids should be helpful in assigning a physiological roleto this sugar. O-acetylation gangliosides influenza C virus lymphocytes sialic acids     

Cloning and expression of human sialic acid pathway genes to generate CMP-sialic acids in insect cells

The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. For sialylation to occur, the donor sugar nucleotide cytidine monophospho-sialic acid (CMP-SA) must be generated and enzymatically transferred to an acceptor oligosaccharide. However, examination of insect cells grown in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac). To increase substrate levels, the enzymes of the metabolic pathway for CMP-SA synthesis have been engineered into insect cells using the baculovirus expression system. In this study, a human CMP-sialic acid synthase cDNA was identified and found to encode a protein with 94% identity to the murine homologue. The human CMP-sialic acid synthase (Cmp-Sas) is ubiquitously expressed in human cells from multiple tissues. When expressed in insect cells using the baculovirus vector, the encoded protein is functional and localizes to the nucleus as in mammalian cells. In addition, co-expression of Cmp-Sas with the recently cloned sialic acid phosphate synthase with N-acetylmannosamine feeding yields intracellular CMP-Neu5Ac levels 30 times higher than those observed in unsupplemented CHO cells. The absence of any one of these three components abolishes CMP-Neu5Ac production in vivo. However, when N-acetylmannosamine feeding is omitted, the sugar nucleotide form of deaminated Neu5Ac, CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (CMP-KDN), is produced instead, indicating that alternative sialic acid glycoforms may eventually be possible in insect cells. The human CMP-SAS enzyme is also capable of CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) synthesis when provided with the proper substrate. Engineering the CMP-SA metabolic pathway may be beneficial in various cell lines in which CMP-Neu5Ac production limits sialylation of glycoproteins or other glycans.     

Carbohydrate receptor specificity of K99 fimbriae of enterotoxigenicEscherichia coli

K99 Fimbriae from enterotoxigenicEscherichia coli (ETEC) were found to bind specifically to sialic acid, as measured in a haemagglutination inhibition assay using the intact bacteria and human erythrocytes. The affinity forN-glycolylneuraminic acid was about twice that ofN-acetylneuraminic acid (NeuAc), and other monosaccharides were found to be at least ten-fold less effective as inhibitors. The specificity was found to depend on electrostatic interaction where the carboxyl group and its orientation plays an important role. 2--Benzyl-NeuAc was a better inhibitor than 2--methyl-NeuAc suggesting a hydrophobic patch near the binding site on the protein. Axially oriented hydroxyl groups as in 4-epi-NeuAc and 3-hydroxy-NeuAc seemed to participate in binding since these derivatives were better inhibitors thanN-acetylneuraminic acid. K99 was found to have a higher affinity for 4-O-acetyl-NeuAc and lower affinity forN-acetylneuraminic acid withO-substituents at C7-C9 as compared toN-acetylneuraminic acid. Hence, the degree ofO-acetylation of sialic acid in the mucosa of the small intestine may influence colonization and determine susceptibility to infection.     

Mass spectra of acetylated derivatives of sialic acids

The fragmentation pattern in electron-impact mass spectrometry has been established for the peracetylated methyl ester methyl glycoside derivative of N-acetylneuraminic acid. The resulting, data allow the interpretation of the mass spectrum of the corresponding derivative of a new sialic acid isolated from the starfish Distolasterias nipon which is shown to be 8-O-methyl-N-acetylneuraminic acid.     

Identification of amino acid residues of influenza A virus H3 HA contributing to the recognition of molecular species of sialic acid

To identify a determinant of human H3 hemagglutinin (HA) amino acid residues linked to the recognition of molecular species of sialic acid, we generated six mutant viruses possessing either the wild-type HA gene from A/Memphis/1/71 (H3N2) or a genetically single-mutated HA gene at position 137, 144, 155, 158 or 193 from a genetic backbone of A/WSN/33 (H1N1) by reverse genetics. We evaluated the binding ability with four types of synthetic sialylglycolipids. The results indicate that the amino acid substitutions Thr155 to Tyr and Glu158 to Gly in H3 HA facilitate virus binding to N-glycolylneuraminic acid.     

Sialic acid concentrations in plants are in the range of inadvertent contamination

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC–electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-d-manno-octulosonic acid and trace amounts (3–18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.     

Sialic acids as receptor determinants for coronaviruses

Among coronaviruses, several members are able to interact with sialic acids. For bovine coronavirus (BCoV) and related viruses, binding to cell surface components containing N-acetyl-9- O-acetylneuraminic acid is essential for initiation of an infection. These viruses resemble influenza C viruses because they share not only the receptor determinant, but also the presence of an acetylesterase that releases the 9- O-acetyl group from sialic acid and thus abolishes the ability of the respective sialoglycoconjugate to function as a receptor for BCoV. As in the case of influenza viruses, the receptor-destroying enzyme of BCoV is believed to facilitate the spread of virus infection by removing receptor determinants from the surface of infected cells and by preventing the formation of virus aggregates. Another coronavirus, porcine transmissible gastroenteritis virus (TGEV) preferentially recognizes N-glycolylneuraminic acid. TGEV does not contain a receptor-destroying enzyme and does not depend on the sialic acid binding activity for infection of cultured cells. However, binding to sialic acids is required for the enteropathogenicity of TGEV. Interaction with sialoglycoconjugates may help the virus to pass through the sialic acid-rich mucus layer that covers the viral target cells in the epithelium of the small intestine. We discuss that the BCoV group of viruses may have evolved from a TGEV-like ancestor by acquiring an acetylesterase gene through heterologous recombination.     

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.     

Low incidence of N-glycolylneuraminic acid in birds and reptiles and its absence in the platypus

The sialic acids of the platypus, birds, and reptiles were investigated with regard to the occurrence of N-glycolylneuraminic (Neu5Gc) acid. They were released from tissues, eggs, or salivary mucin samples by acid hydrolysis, and purified and analyzed by thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry. In muscle and liver of the platypus only N-acetylneuraminic (Neu5Ac) acid was found. The nine bird species studied also did not express N-glycolylneuraminic acid with the exception of an egg, but not tissues, from the budgerigar and traces in poultry. Among nine reptiles, including one turtle, N-glycolylneuraminic acid was only found in the egg and an adult basilisk, but not in a freshly hatched animal. BLAST analysis of the genomes of the platypus, the chicken, and zebra finch against the CMP-N-acetylneuraminic acid hydroxylase did not reveal the existence of a similar protein structure. Apparently monotremes (platypus) and sauropsids (birds and reptiles) cannot synthesize Neu5Gc. The few animals where Neu5Gc was found, especially in eggs, may have acquired this from the diet or by an alternative pathway. Since Neu5Gc is antigenic to man, the observation that this monosaccharide does not or at least only rarely occur in birds and reptiles, may be of nutritional and clinical significance.     

Isolation and properties of two sialate-<Emphasis Type="Italic">O</Emphasis>-acetylesterases from horse liver with 4- and 9-<Emphasis Type="Italic">O</Emphasis>-acetyl specificities

Sialate-O-acetylesterase was purified almost 900-fold from particle-free supernatants of horse liver by gel filtration, ion-exchange chromatography and isoelectric focussing. The native enzyme on gel filtration exhibits a molecular weight of 54,000 Da. It was separated by isoelectric focussing into two forms with pI values of 4.8 and 5.7, respectively. The esterase with a lower pI hydrolyses only 9-O-acetyl groups from sialic acids (K_M 1.1 mM), while that with the higher pI esterifies both 4- and 9-O-acetylated monosaccharides at similar rates (K_M 0.3 M and 1.3 mM, respectively). Both forms are inactive with 7-O-acetylated N-acetylneuraminic acid. Enzyme assays were carried out at the pH optimum (pH 8.4–8.6) using free O-acetylated sialic acids followed by direct analysis of the reaction products by isocratic anion-exchange HPLC. Glycosidically bound sialic acids can also be de-O-acetylated. Horse liver esterase seems to be an essential enzyme for the catabolism of 4-O-acetylated sialoglycoconjugates, since sialidase from this tissue cannot act on 4-O-acetylated sialic acids.     

Investigation into an efficient synthesis of 2,3-dehydro-N-acetyl neuraminic acid leads to three decarboxylated sialic acid dimers

Sialic acid, an important carbohydrate found incorporated on the cell surface of many organisms, has been modified for use in a wide range of biological and pharmaceutical applications. We hypothesized that 4,7,8,9-tetra-O-acetyl-2-deoxy-2,3-dehydro-N-acetyl neuraminic acid methyl ester (4) could be efficiently synthesized in a one-pot reaction by heating peracetylated sialic acid (2) in pyridine and acetic anhydride to induce beta-elimination. When reduced to practice, this reaction produced only modest yields of 4. Six compounds, including three new decarboxylated sialic acid dimers, were also found to have been synthesized in the reaction. In an effort to better understand the chemistry and the mechanisms of this reaction, all of the side products were isolated and fully characterized.     

Engineering the sialic acid in organs of mice using N-propanoylmannosamine

Sialic acids play an important role during development, regeneration and pathogenesis. The precursor of most physiological sialic acids, such as N-acetylneuraminic acid is N-acetyl-d-mannosamine. Application of the novel N-propanoylmannosamine leads to the incorporation of the new sialic acid N-propanoylneuraminic acid into cell surface glycoconjugates. Here we analyzed the modified sialylation of several organs with N-propanoylneuraminic acid in mice. By using peracetylated N-propanoylmannosamine, we were able to replace in vivo between 1% (brain) and 68% (heart) of physiological sialic acids by N-propanoylneuraminic acid. The possibility to modify cell surfaces with engineered sialic acids in vivo offers the opportunity to target therapeutic agents to sites of high sialic acid concentration in a variety of tumors. Furthermore, we demonstrated that application of N-propanoylmannosamine leads to a decrease in the polysialylation of the neural cell adhesion molecule in vivo, which is a marker of poor prognosis for some tumors with high metastatic potential.