Gerd Reuter

Biochemistry and Role of Sialic Acids

Sialic acids mainly occur as terminal components of cell surface glycoproteins and glycolipids, playing as such a major role in the chemical and biological diversity of glycoconjugates. Cell-type-specific expression of glycosyltransferases, particularly of sialyltransferases (Paulson and Colley, 1989; van den Eijnden and Joziasse, 1993), leads to specific sialylation patterns of oligosaccharides which can be considered as key determinants in the makeup of cells. Striking differences have been found in the sialoglycosylation patterns of cells during development, activation, aging, and oncogenesis. Research on the structures, metabolism, and molecular biology, as well as on the biological and clinical importance of sialic acids as components of these glycoconjugates, has therefore intensified during the past several years.

Determination of sialic acids.

Publisher Summary This chapter describes isolation, purification, and characterization of monomeric sialic acids. There are two basic procedures for liberating sialic acids from glycosidic linkages—enzymatic and acid hydrolysis. In the former case, a variety of sialidases may be used, which differ in specificity for sialic acid linkage or species. Quantitation of sialic acids usually requires purified samples because a number of substances are known to interfere with certain tests. Colorimetric sialic acid quantitation is still one of the most important methods to determine the amount of the sugars in a given sample accurately. Two basically different tests are used—the orcinol/Fe 3+ /HCl or resorcinol/Cu 2+ /HCl assay and the periodic acid/thiobarbituric acid test. The procedures for a microadaptation of the orcinol/Fe 3+ /HCl and the periodic acid/thiobarbituric acid assays are discussed. The determination of sialic acids in serum, urine, or tissues or on cells is often used in clinical applications, because it seems to be a valuable marker for certain malignancies.

The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein☆

A developmentally regulated trans-sialidase activity is present on the surface of procyclic Trypanosoma brucei. Bloodstream stages display no trans-sialidase activity. T. brucei trans-sialidase is capable of transferring sialic acids from a variety of glycoconjugates into new glycosidic linkages without requirement for CMP-Neu5Ac. The enzyme is linked to the plasma-membrane via a GPI-PLC-resistant GPI-anchor. The comparison of enzymic and structural features of sialidase and trans-sialidase suggests that the two activities may be catalyzed by the same protein, since highly enriched sialidase fractions display trans-sialidase activity. 2-Deoxy-2,3-didehydro-N-acetylneuraminic acid is only a poor inhibitor for the two enzymic activities. Sialic acids are transferred to alpha (2-3)-positions of terminal beta-galactose residues of oligosaccharides and glycoconjugates at various rates. Neu5Ac-alpha(2-3)-lactose is the best trans-sialylation donor tested. Lewis is a poor sialic acid acceptor. T. brucei trans-sialidase utilizes serum glycoconjugates, human and bovine erythrocytes as sialic acid donors, and resialylates sialidase-treated erythrocytes. The enzyme transfers sialic acids from the GPI-anchor of procyclic acidic repetitive protein (PARP) onto lactose and vice versa. Also structures within a variant surface glycoprotein (sVSG MITat. 1.7.) can be trans-sialylated.

Binding of Plasmodium falciparum 175-kilodalton erythrocyte binding antigen and invasion of murine erythrocytes requires N-acetylneuraminic acid but not its O-acetylated form.

Abstract Sialic acid on human erythrocytes is involved in invasion by the human malaria parasite, Plasmodium falciparum. Mouse erythrocytes were used as a reagent to explore the question of whether erythrocyte sialic acid functions as a nonspecific negative charge or whether the sialic acid is a necessary structural part of the receptor for merozoites. Human erythrocytes contain N-acetylneuraminic acid (Neu5Ac), whereas mouse erythrocytes, which are also invaded by P. falciparum merozoites, contain 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) and N-glycoloylneuraminic acid (Neu5Gc), in addition to Neu5Ac. We compared the effects of sialidase and influenza C virus esterase treatments of mouse erythrocytes on invasion and the binding of a 175-kDa P. falciparum protein (EBA-175), a sialic acid-dependent malaria ligand implicated in the invasion process. Sialidase-treated mouse erythrocytes were refractory to invasion by P. falciparum merozoites and failed to bind EBA-175. Influenza C virus esterase, which converts Neu5,9Ac2 to Neu5Ac, increased both invasion efficiency and EBA-175 binding to mouse erythrocytes. Thus, the parasite and EBA-175 discriminate between Neu5Ac and Neu5,9Ac2, that is, the C-9 acetyl group interferes with EBA-175 binding and invasion by P.falciparum merozoites. This indicates that sialic acid is part of a receptor for invasion.

Purification and characterization of a novel sialidase found in procyclic culture forms of Trypanosoma brucei.

A membrane-bound sialidase (EC 3.2.1.18) was found in procyclic trypomastigotes of Trypanosoma brucei. The mammalian stage bloodstream form, however, displayed no sialidase activity. This sialidase is an integral surface protein, linked to the membrane via a glycosylphosphatidylinositol anchor. After osmotic lysis and solubilization with Triton CF-54, the enzyme was purified 1900-fold by gel filtration and ion exchange chromatography. Its size, as determined by conventional and high-performance liquid gel chromatography, is 67 kDa. The sialidase is active over a broad pH and temperature range with optima at pH 6.9 and 35 degrees C, respectively. No loss of activity is observed after 4 freeze-thaw cycles. T. brucei sialidase activity is inhibited by N-(4-nitrophenyl)oxamic acid and 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, the latter, however, being less effective. N-Acetylneuraminic acid shows no inhibitory effect, whereas a variety of metal ions are potent inhibitors. The sialidase is activated by di- and tricarboxylic acids, but inhibited by chloride. Relative hydrolysis rates of various sialic acid-containing compounds reveal that de-O-acetylated bovine submandibular gland mucin is the preferred substrate and that alpha(2-3)-linkages are hydrolyzed faster than alpha(2-6)-linkages.

Sialate O-acetylesterases: key enzymes in sialic acid catabolism

Sialate 9(4)-O-acetylesterases (EC 3.1.1.53) have been isolated from equine liver, bovine brain and influenza C virus. In this latter case, the esterase represents the receptor-destroying enzyme of the virus. The kinetic properties of these enzymes were determined with Neu5,9Ac2 and in part with 4-methylumbelliferyl acetate and Neu5,9Ac2-lactose. The Km values vary between 0.13 and 24 mM and the Vmax values from 0.55 to 11 U/mg of protein. The pH optima are in the range of 7.4-8.5, the molecular masses at 56,500 and 88,000 Da. In addition to a fast hydrolysis found for aromatic acetates, such as 4-methylumbelliferyl acetate or 4-nitrophenyl acetate, N-acetyl-9-O-acetylneuraminic acid is de-O-acetylated at the highest relative rate. Other substituents at the 9-position, such as lactoyl residues, or acetyl groups at other positions within the side chain are not hydrolyzed. Neu4,5Ac2, however, is a substrate for all 3 enzymes. The hydrolysis rates of this ester function, which renders sialic acids resistant to the action of sialidases, vary from 3 to 100% relative to Neu5,9Ac2. Whereas Neu5,9Ac2-lactose is hydrolyzed by the bovine and viral esterases, other O-acetylated sialic acids in glycoconjugates are only attacked by the enzyme from influenza C virus and not by that from bovine brain. The esterase from horse liver also releases 4-O-acetyl groups from equine submandibular gland mucin. By incubation with appropriate substrates and inhibition studies, carboxylesterase, amidase and choline esterase activities were excluded, as well as the cleavage of other acyls, e.g., butyryl groups. Thus, the enzymes investigated belong to the acetylesterases.(ABSTRACT TRUNCATED AT 250 WORDS)

A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz1H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe3+/HCl assay were determined.

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 recognizes glycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid. The same protein also contains an esterase activity. Taking advantage of these two properties, influenza C virus was used as a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminic acid in human leucocytes. The binding of influenza C virus to leucocyte glycoproteins and gangliosides separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and thin-layer chromatography, respectively, was assayed using a chromogenic esterase substrate. In this way, glycoproteins of B-lymphocytes and T-lymphocytes were found to contain 9-O-acetylated sialic acids. Of the various 9-O-acetylated gangliosides detected, one had the characteristics of 9-O-acetylated GD3. The identification of 9-O-acetylated sialic acids on distinct glycoproteins and glycolipids should be helpful in assigning a physiological role to this sugar.

Isolation and properties of a sialidase fromTrypanosoma rangeli

AbstractIn the culture supernatant ofTrypanosoma rangeli, strain El Salvador, a sialidase was present with an activity of 0.1 U/mg protein as determined with the 4-methylumbelliferyl glycoside of α-N-acetylneuraminic acid as substrate. This enzyme was purified about 700-fold almost to homogeneity by gel chromatography on Sephadex G-100 and Blue Sepharose, and affinity chromatographies on 2-deoxy-2,3-didehydroneuraminic acid and horse submandibular gland mucin, both immobilized on Sepharose. The pH optimum is at 5.4–5.6, and the molecular weight was determined by gel chromatography, high performance liquid chromatography and sodium dodecyl sulphate gel electrophoresis to be 70 000. The substrate specificity of the enzyme is comparable to bacterial, viral and mammalian sialidases with cleavage rates for the following substrates in decreasing order: N-acetylneuraminyl-α(2–3)-lactose> N-glycoloylneuraminy-α(2–3)-lactose> N-acetylneuraminyl-α(2–6)-lactose >sialoglycoproteins>gangliosides>9-O-acetylated sialoglycoproteins.4-O-Acetylated derivatives are resistant towards the action of this sialidase. The enzyme activity can be inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, Hg2+ ions, andp-nitrophenyloxamic acid; it is not dependent on the presence of Ca2+ Mn2+ or Mg2+ ions.

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 Crohns 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.