Abstract The O-acetylation of sialic acids turns out to be one of the most important modifications that influence the diverse biological and pathophysiological properties of glycoconjugates in animals and microorganisms. To understand the functions of this esterification, knowledge of the properties, structures and regulation of expression of the enzymes involved is essential. Attempts to solubilise, purify or clone the gene of one of the sialate-O-acetyltransferases have failed so far. Here we report on the solubilisation of the sialate 4-O-acetyltransferase from guinea pig liver, the first and essential step in the purification and molecular characterisation of this enzyme, by the zwitterionic detergent CHAPS. This enzyme Oacetylates sialic acids at C-4 both free and bound to oligosaccharides, glycoproteins and glycolipids with varying activity, however, gangliosides proved to be the best substrates. Correspondingly, a rapid enzyme test was elaborated using the ganglioside GD3. The soluble Oacetyltransferase maximally operated at 30C, pH 5.6, and 50-70 mM KCl and K2HPO4 concentrations. The Km values were 3.6 M for AcCoA and 1.2 M for GD3. CoA inhibits the enzyme with a Ki value of 14.8 M. A most important discovery enabling further enzyme purification is its need for an unknown low molecular mass and heat-stable cofactor that can be separated from the crude enzyme preparation by 30 kDa ultrafiltration.

Sialic acids present on human colonic mucins are highly O-acetylated, however, little is known about the underlying enzymatic activity required for O-acetylation in this tissue. Here we report on the substrate specificity, subcellular localization and characterization of the sialate-7(9)-O-acetyltransferase in normal human colonic mucosa. Using CMP-Neu5Ac, the most efficient acceptor substrate of all those tested, the enzymatic activity was found to be optimal at 37 degreesC, with a pH optimum of 7.0. Activity was also found to be dependent on protein, CMP-Neu5Ac (K-m: 59.2 mum) and AcCoA (K-m: 6.1 muM) concentrations, as well as membrane integrity. The enzyme's activity could be inhibited by CoA with a K-i of 11.9 muM. In addition, enzymatic activity was found to be localized in the Golgi-enriched membrane fraction. The nature of the O-acetylated products formed were verified with the aid of chromatographic and enzymatic techniques. The main product was 9-O-acetylated Neu5Ac, with a significant amount of oligo-O-acetylated Neu5Ac also being detected. The utilization of CMP-Neu5Ac as the acceptor substrate was confirmed by the isolation and characterization of the putative product, CMP-Neu5,9Ac(2), using ion-exchange chromatography. The ability of CMP-Neu5,9Ac(2) to act as a sialic acid donor for sialyl-transferases represents the conclusive demonstration for the formation of CMP-Neu5,9Ac(2).

In the still growing family of sialic acids with more than 40 different derivatives of neuraminic acid, the interest of the scientific community is increasingly directed towards the O-acetylated species. In the past two decades it evolved that O-acetylated sialic acids play fundamental roles in the development of organisms, in the regulation of the immune system, in cancer processes and many other biological and pathophysiological events.

The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.

ABSTRACT By comparative analysis of the hemagglutinin-esterase (HE) protein of mouse hepatitis virus strain S (MHV-S) and the HE protein of influenza C virus, we found major differences in substrate specificities. In striking contrast to the influenza C virus enzyme, the MHV-S esterase was unable to release acetate from bovine submandibulary gland mucin. Furthermore, MHV-S could not remove influenza C virus receptors from erythrocytes. Analysis with free sialic acid derivatives revealed that the MHV-S HE protein specifically de-O-acetylates 5- N -acetyl-4- O -acetyl sialic acid (Neu4,5Ac 2 ) but not 5- N -acetyl-9- O -acetyl sialic acid (Neu5,9Ac 2 ), which is the major substrate for esterases of influenza C virus and bovine coronaviruses. In addition, the MHV-S esterase converted glycosidically bound Neu4,5Ac 2 of guinea pig serum glycoproteins to Neu5Ac. By expression of the MHV esterase with recombinant vaccinia virus and incubation with guinea pig serum, we demonstrated that the viral HE possesses sialate-4- O -acetylesterase activity. In addition to observed enzymatic activity, MHV-S exhibited affinity to guinea pig and horse serum glycoproteins. Binding required sialate-4- O -acetyl groups and was abolished by chemical de-O-acetylation. Since Neu4,5Ac 2 has not been identified in mice, the nature of potential substrates and/or secondary receptors for MHV-S in the natural host remains to be determined. The esterase of MHV-S is the first example of a viral enzyme with high specificity and affinity toward 4-O-acetylated sialic acids.

Helicobacter pylori, a human gastric pathogen causing chronic gastritis and duodenal ulcer disease, has been found in large amounts in gastric mucous gel layer. Mucin preparations, separated from human gastric juices and isolated from different colon regions, were examined for their ability to inhibit haemagglutination of H. pylori with the emphasis on evaluating the role of sialic acid-dependent haemagglutinins of the bacteria in colonisation of the stomach. The mucins showed high inhibitory activity for H. pylori, which was significantly decreased after the removal of sialic acids from the mucins. The inhibitory potencies using high molecular mass mucin-like components from bovine milk were comparable with those obtained for gastric mucins, suggesting their possible role in the prevention of H. pylori infection.

Sialic acids comprise a large family of N- and O-substituted neuraminic acid derivatives as components of glycoconjugates. N-Glycolylneuraminic acid is formed from N-acetylneuraminic acid by the action of the CMP-N-acetylneuraminic acid hydroxylase studied in various animals. O-Methylated sialic acids originate from the action of S-adenosylmethionine-8-O-methyltransferase studied in starfish. Sialic acids are O-acetylated at diverse positions by the action of acetyl-CoA-4-O- and -7-O-acetyltransferases found in various animals and, leading to the O-acetylation of sialic acid glycerol side chain, also in man. Some properties of these enzymes are described and biological implications discussed.

Two factors potentially determining the consistent overexpression of sialyl-Le(x) antigen in colon carcinoma and metastases were investigated: (i) the expression of the mucins MUC1 and MUC2, known to carry sialyl-Le(x), by Northern blotting; (ii) the extent of sialic acid O-acetylation, by Western blotting and HPLC. RNA and sialyl-lex-positive mucins were purified from normal colonic mucosa (N), primary carcinomas (T) and their liver metastases (M). Northern blots showed that mRNA expression both of MUC1 and of MUC2 decreases during the progression of the disease, and is lowest in metastatic tissue, The expression of mucin-bound sialyl-Le(x) increased strongly from N to T and, to a lesser extent, to M, After alkali treatment of the mucins these differences disappeared, indicating that the total amount of mucin-bound sialyl-Le(x) is the same in the 3 types of tissues, The O-acetylation of mucin-bound sialyl-Le(x) gradually decreased from N to M, HPLC analysis showed that in N about 70%, in T 45% and in M only 20% of mucin-bound sialic acids are O-acetylated., Thus, the increase of sialyl-Le(x) detectable during colon-carcinoma progression is due to diminished O-acetylation and not to increased expression of mucin protein cores, The decrease of O-acetylation is therefore the primary chemical alteration contributing to colon carcinoma-associated overexpression of sialyl-Le(x). (C) 1997 Wiley-Liss, Inc.