Sialylation Is Dispensable for Early Murine Embryonic Development in Vitro

Abstract

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.

Sialic acid is a critical fetal defense against maternal complement attack

“…The Sia specificity of the lectin was again demonstrated by complete loss of SNA reactivity and an increase in PNA epitopes upon enzymatic removal of Sia by neuraminidase treatment prior to lectin incubation. The simultaneous lack of MAA and SNA reactivity and the presence of PNA binding sites in knockout embryos confirmed that loss of CMAS activity resulted in a complete lack of cell surface sialylation, referred to as an asialo phenotype, which is identical to the situation in Cmas -/-mESCs (9).…”

“…Depletion of CMAS leads to loss of sialoglycoconjugates in Cmas -/embryos and is embryonic lethal. Genetic inactivation of Cmas in mice was accomplished as described by excision of exon 4, which encodes residues essential for enzymatic activity (9). All genotypes of offspring from Cmas +/intercrosses were discovered in a Mendelian ratio until E8.5 (see Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/ JCI99945DS1).…”

“…showed neuraminidase-sensitive intracellular SNA staining. Since previous studies clearly demonstrated that loss of CMAS causes an asialo phenotype in ESC in vitro, and trophoblast cells are known to endocytose certain maternal proteins (e.g., immunoglobulins), the observed intracellular SNA reactivity in TGCs most likely represented internalized maternal material (9,45). Most notably, asialo Cmas -/embryos established neuroepithelia, head mesenchyme, as well as heart primordia and somites, promoting the hypothesis that sialoglycans are mostly dispensable for development of the embryo proper until E8.5.…”

“…All sialyltransferases strictly depend on the preceding activation of Sia to CMP-Sia, which is catalyzed by the nuclear enzyme CMP-Sia synthase (CMAS) (8). We have recently shown that genetic inactivation of the Cmas gene results in loss of CMAS protein and a complete lack of sialoglycans on the cell surface of murine embryonic stem cells (mESC) (9). Unexpectedly, the asialo mESC were equivalent to control mESC in terms of differentiation, indicating that Sia is dispensable for germ layer formation and early embryonic development in vitro.…”

Enzymatic Cascade Synthesis Provides Novel Linear Human Milk Oligosaccharides as Reference Standards for xCGE-LIF Based High-Throughput Analysis

“…xCGE‐LIF has been demonstrated as versatile HTS method for glycan analysis of complex biological samples to decipher even minor changes in N ‐glycan/oligosaccharide fingerprints . We here utilize xCGE‐LIF as an ideal method for high throughput monitoring of LNT and LNnT type oligomers from synthetic enzyme reactions.…”