On the biosynthesis of alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid catalyzed by the sialyltransferase of Escherichia coli Bos-12

Escherichia coli Bos-la synthesizes a heteropolymer of sialic acids with al ternating alpha-2,9/alpha-2,8 glycosidic linkages (1), In this study, we ha ve shown that the polysialyltransferase of the E. coli Bos-12 recognizes an a-2,8 glycosidic linkage of sialic acid at the nonreducing end of an exoge nous acceptor of either the alpha-2,8 homopolymer of sialic acid or the alt ernating alpha-2,9/alpha-2,8 heteropolymer of sialic acid and catalyzes the transfer of Neu5Ac from CMP-Neu5Ac to this residue. When the exogenous acc eptor is an alpha-a,8-linked oligomer of sialic acid, the main product synt hesized is derived from the addition of a single residue of [C-14]Neu5Ac to form either an alpha-2,8 glycosidic linkage or an alpha-2,9 glycosidic lin kage at the nonreducing end, at an alpha-2,8/alpha-2,9 ratio of approximate ly 2:1, When the acceptor is the alternating alpha-2,9/alpha-2,8 heteropoly mer of sialic acid, chain elongation takes place four to five times more ef ficiently than the alpha-2,8-linked homopolymer of sialic acid as an accept or. It was found that the alpha-2,9-linked homopolymer of sialic acid and t he alpha-2,8/alpha-2,9-linked hetero-oligomer of sialic acid with alpha-2,9 at the nonreducing end not only failed to serve as an acceptor for the E. coli Bos-la polysialyltransferase for the transfer of [C-14]Neu5Ac, but the y inhibited the de novo synthesis of polysialic acid catalyzed by this enzy me. The results obtained in this study favor the proposal that the biosynth esis of the alpha-2,9/alpha-2,8 heteropolymer of sialic acid catalyzed by t he E. coil Bos-la polysialyltransferase involves a successive transfer of a preformed alpha-2,8-linked dimer of sialic acid at the nonreducing terminu s of the acceptor to form an alpha-2,9 glycosidic linkage between the incom ing dimer and the acceptor. The glycosidic linkage at the nonreducing end o f the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid produced by E, coli Bos-la should be an alpha-2,8 glycosidic bond and not an alpha- 2,9 glycosidic linkage.