Sialylation Is Dispensable for Early Murine Embryonic Development in Vitro.

The negatively charged nonulose sialic acid (Sia) is essential for murine development in vivo. In order to elucidate the impact of sialylation on differentiation processes in the absence of maternal influences, we generated mouse embryonic stem cell (mESC) lines that lack CMP-Sia synthetase (CMAS) and thereby the ability to activate Sia to CMP-Sia. Loss of CMAS activity resulted in an asialo cell surface accompanied by an increase in glycoconjugates with terminal galactosyl and oligo-LacNAc residues, as well as intracellular accumulation of free Sia. Remarkably, these changes did not impact intracellular metabolites or the morphology and transcriptome of pluripotent mESC lines. Moreover, the capacity of Cmas-/- mESCs for undirected differentiation into embryoid bodies, germ layer formation and even the generation of beating cardiomyocytes provides first and conclusive evidence that pluripotency and differentiation of mESC in vitro can proceed in the absence of (poly)sialoglycans.

Lec32 is a new mutation in Chinese hamster ovary cells that essentially abrogates CMP-N-acetylneuraminic acid synthetase activity.

LEC29.Lec32 is a glycosylation mutant that was isolated from a selection of mutagenized Chinese hamster ovary (CHO) cells for lectin resistance. Compared with LEC29 CHO cells, the double mutant exhibited an unusually high sensitivity to the toxic lectin, ricin, indicating increased exposure of galactose residues on cell surface carbohydrates. Structural analysis of LEC29.Lec32 cellular glycoproteins showed a nearly complete lack of sialic acid residues. Genetic analysis demonstrated that the lec32 mutation is recessive and novel. Biochemical analysis showed that the mutant cells contained less than 5% of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) present in parental CHO cells (1.6 nmol/mg of cell protein). A sensitive radiochemical assay used to measure CMP-NeuAc synthetase activity showed that the properties of this enzyme in parental CHO cells were essentially identical to those of CMP-NeuAc synthetase in various mammalian tissues. However, no CMP-NeuAc synthetase activity was detected in LEC29.Lec32 extracts. Mixing experiments provided no evidence for an inhibitor in the mutant CHO cells, and two revertants, which expressed only the LEC29 phenotype, had normal CMP-NeuAc synthetase levels. The combined evidence indicates that the lec32 mutation resides in either the structural gene encoding CMP-NeuAc synthetase or in a gene that regulates the production of active enzyme.