MAMMALIAN CYTIDINE 5'-MONOPHOSPHATE N-ACETYLNEURAMINIC ACID SYNTHETASE - A NUCLEAR-PROTEIN WITH EVOLUTIONARILY CONSERVED STRUCTURAL MOTIFS

Sialic acids of cell surface glycoproteins and glycolipids play a pivo tal role in the structure and function of animal tissues. The pattern of cell surface sialylation is species- and tissue-specific, is highly regulated during embryonic development, and changes with stages of di fferentiation. A prerequisite for the synthesis of sialylated glycocon jugates is the activated sugar-nucleotide cytidine 5'-monophosphate N- acetylneuraminic acid (CMP-Neu5Ac), which provides a substrate for Gol gi sialyltransferases. Although a mammalian enzymatic activity respons ible for the synthesis of CMP-Neu5Ac has been described and the enzyme has been purified to near homogeneity, sequence information is restri cted to bacterial CMP-Neu5Ac synthetases. Hn this paper, me describe t he molecular characterization, functional expression, and subcellular localization of murine CMP-Neu5Ac synthetase, Cloning was achieved by complementation of the Chinese hamster ovary lec32 mutation that cause s a deficiency in CMP-Neu5Ac synthetase activity. A murine cDNA encodi ng a protein of 432 amino acids rescued the lec32 mutation and also ca used polysialic acid to be expressed in the capsule of the CMP-Neu5Ac synthetase negative Escherichia coli mutant EV5. Three potential nucle ar localization signals mere found in the murine synthetase, and immun ofluorescence studies confirmed predominantly nuclear localization of an N-terminally Flag-tagged molecule. Four stretches of amino acids th at occur in the N-terminal region are highly conserved in bacterial CM P-Neu5Ac synthetases, providing evidence for an ancestral relationship between the sialylation pathways of bacterial and animal cells.