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 neu rovirulent determinant associated with neonatal meningitis in humans. A functionally similar polyST in human neuroblastomas polysialylates n eural cell adhesion molecules. While bacteria do not synthesize glycos phingolipids (GSLs), we report here that the E. coil K1 polyST can sel ectively polysialylate several structurally related GSLs, when added a s exogenous sialyl accepters. A structural feature common to the prefe rred sialyl accepters (G(D3) > G(T1a) > G(Q1b) = G(T1b) > G(D2) = G(D1 b) = G(D1a) > G(M1)) was the disialyl glycotope, Sia alpha 2,8Sia, alp ha 2,3-linked to galactose (Sia is sialic acid). A linear tetrasacchar ide with a terminal Sia residue (e,g., G(D3)) was the minimum length o ligosaccharide recognized by the polyST. Endo-N-acylneuraminidase was used to confirm the alpha 2,8-specific polysialylation of GSL. Ceramid e glycanase was used to release the polysialyllactose chains from the ceramide moiety. Size analysis of these chains showed that 60-80 Sis r esidues were transferred to the disialyllactose moiety of G(D3). The s ignificance 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 ''de signer'' GSLs may have potentially important biological and pharmacolo gical properties. (ii) The use of GSLs as exogenous sialyl accepters i ncreases the sensitivity of detecting polyST activity. The practical a dvantage of this finding is that polyST activity can be identified and studied in those eukaryotic cells that express low levels of this dev elopmentally regulated enzyme and/or its acceptor.