Eur J Biochem 1998 Feb 01;2513:874-84. doi: 10.1046/j.1432-1327.1998.2510874.x.

The biosynthesis of 8-O-methylated sialic acids in the starfish Asterias rubens--isolation and characterisation of S-adenosyl-L-methionine:sialate-8-O-methyltransferase.

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The unusual, 8-O-methylated sialic acids have only been found in glycoconjugates of certain species of starfish. Using a detergent-solubilised extract of a particulate fraction from gonads of Asterias rubens, a specific methylation of endogenous and exogenous glycoconjugate-bound sialic acids was detected with [14C]-S-adenosyl-L-methionine ([14C]AdoMet) as the methyl donor. For this test, a filtration assay was developed using glutardialdehyde-fixed horse erythrocyte membranes as methyl acceptor. The enzyme catalysing this reaction, the sialate-8-O-methyltransferase, was purified 22000-fold in a yield of 13% by ion-exchange chromatography and two cycles of affinity chromatography on S-adenosyl-L-homocysteine-Sepharose. Upon SDS/PAGE under reducing conditions, the purified sialate-8-O-methyltransferase revealed two bands with apparent molecular masses of 58 kDa and 62 kDa. Since no evidence for the presence of subunits was found, the relationship between these two species is unknown. The enzyme was optimally active over a broad range of pH (7.4-8.3) and at 37 degrees C. After EDTA treatment, restoration of the activity depended, in contrast to most methyltransferases, on Mn2+ or Co2+, the latter to a lesser extent. Although both, free N-acetylneuraminic acid and N-glycoloylneuraminic acid were methylated, sialic acids alpha-glycosidically bound to a number of oligosaccharides and glycoproteins were better substrates. In the presence of 20 microM AdoMet, apparent Km values of 299 microM and 44 microM were found for N-acetylneuraminic acid and N-acetylneuraminyl alpha2,3-lactose, respectively. Using N-acetylneuraminyl alpha2,3-lactose as acceptor, an apparent Km of 7.1 microM was found for S-adenosyl-L-methionine. Therefore, the sialate-8-O-methyltransferase is the first enzyme known to modify both free and glycoconjugate-bound sialic acids.

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Genes referenced: LOC586122 LOC594261