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.

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 [C-14]-S-adenosyl-L-methionine ([C-14]AdoMet) as the methyl donor. For this rest, a filtration assay was developed using glutardialdehyde-fixed horse erythrocyle 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 chromatograhy 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 370 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 sub-strates, in the presence of 20 mu M AdoMet, apparent K-m values of 299 mu M and 44 mu M were found for N-acetylneuraminic acid and N-acetylneuraminyl alpha 2,3-lactose, respectively. Using N-acetylneuraminyl alpha 2,3-lactose as acceptor, an apparent K-m of 7.1 mu M 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.