Characterization of the enzymatic 7-O-acetylation of sialic acids and evidence for enzymatic O-acetyl migration from C-7 to C-9 in bovine submandibular gland.

Microsomes prepared from bovine submandibular glands incubated with radioactive AcCoA incorporated acid-insoluble radioactivity, which was dependent on time, and the concentrations of AcCoA and proteins, and was inhibited by CoA in a concentration-dependent manner. Under the conditions used, the apparent Km for AcCoA was 1.63 microM with a Vmax of 21.9 pmol/mg protein.min. The radioactivity incorporated was mainly due to the O-acetylation of glycosidically bound Neu5Ac. The primary attachment site of O-acetyl groups was exclusively the hydroxyl at C-7 of Neu5Ac, the presence of an AcCoA:Neu5Ac 7-O-acetyltransferase thus being demonstrated. After longer incubation 9-O-acetylated Neu5Ac also appeared, suggesting the migration of an ester group from C-7 to C-9. This isomerisation was inhibited by heat-inactivation of the microsomal protein, enzymatic isomerisation by a "migrase" thus being suggested. Data are presented which lead to the assumption that this 7-O-acetylation involves at least two reactions: the transport by a translocase of acetyl groups from AcCoA from the cytosol across the Golgi membrane, followed by the enzymatic transfer of these acetyl groups onto sialic acids in the Golgi lumen.

Enzymatic 4-O-acetylation of N-acetylneuraminic acid in guinea-pig liver.

Sialic acids from the liver and serum of guinea-pig are composed of N-acetylneuraminic acid (Neu5Ac; 85% and 61%, respectively), N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2; 10% and 32%, respectively) and N-glycolylneuraminic acid (Neu5Gc; 5% and 7%, respectively), besides traces of N-glycolyl-4-O-acetylneuraminic acid in serum. The analysis was carried out using thin-layer chromatography, high-performance liquid chromatography, electron impact ionization mass spectrometry, and different enzymes (sialidase, sialate esterase, and sialate-pyruvate lyase after hydrolysis and purification of the sialic acids by ion-exchange chromatography). We showed that this O-acetylation of sialic acids is due to the activity of an acetyl-coenzyme A:sialate-4-O-acetyltransferase (EC 2.3.1.44), which occurs together with sialyltransferase activity in Golgi-enriched membrane fractions of guinea-pig liver. The enzyme operates optimally at 30 degrees C in 70 mM potassium phosphate buffer at pH 6.7 and in the presence of 90 mM KCI with an apparent KM for AcCoA of 0.6 1microM and a Vmax of 20 pmol/mg protein x min. The enzyme is inhibited by coenzyme A in a mixed-competitive manner (Ki = 4.2 microM), as well as by parachloromercuribenzoate, MnCl2, saponin and Triton X-100.

Low O-acetylation of sialyl-Le(x) contributes to its overexpression in colon carcinoma metastases.

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-Le(x)-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).

Functional and biosynthetic aspects of sialic acid diversity.

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.