POLYSIALIC ACID ENGINEERING - SYNTHESIS OF POLYSIALYLATED NEOGLYCOSPHINGOLIPIDS BY USING THE POLYSIALYLTRANSFERASE FROM NEUROINVASIVE ESCHERICHIA-COLI K1

The CMP-sialic acid:poly alpha 2,8sialosyl sialyltransferase (polyST) in neurotropic Escherichia coli K1 inner membranes catalyzes synthesis of the alpha 2,8-linked polysialic acid capsule. The capsule is a neurovirulent determinant associated with neonatal meningitis in humans. A functionally similar polyST in human neuroblastomas polysialylates neural cell adhesion molecules. While bacteria do not synthesize glycosphingolipids (GSLs), we report here that the E. coli K1 polyST can selectively polysialylate several structurally related GSLs, when added as exogenous sialyl accepters. A structural feature common to the preferred sialyl accepters (G(D3) > G(T1a) > G(Q1b) = G(T1b) > G(D2) = G(D1b) = G(D1a) > G(M1)) was the disialyl glycotope, Sia alpha 2,8Sia, alpha 2,3-linked to galactose (Sia is sialic acid). A linear tetrasaccharide with a terminal Sia residue (e,g., G(D3)) was the minimum length oligosaccharide recognized by the polyST. Endo-N-acylneuraminidase was used to confirm the alpha 2,8-specific polysialylation of GSL. Ceramide glycanase was used to release the polysialyllactose chains from the ceramide moiety. Size analysis of these chains showed that 60-80 Sis residues were transferred to the disialyllactose moiety of G(D3). The significance of these findings is two-fold. (i) The. coli K1 polyST can be used as a synthetic reagent to enzymatically engineer the glycosyl moiety of GSL, thus cresting oligo- or polysialylated GSLs. Such ''designer'' GSLs may have potentially important biological and pharmacological properties. (ii) The use of GSLs as exogenous sialyl accepters increases the sensitivity of detecting polyST activity. The practical advantage of this finding is that polyST activity can be identified and studied in those eukaryotic cells that express low levels of this developmentally regulated enzyme and/or its acceptor.