Sialylation is essential for early development in mice

Abstract

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. Sialylation is crucial for a variety of cellular functions, such as cell adhesion or signal recognition, and regulates the biological stability of glycoproteins. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. We report that inactivation of the UDP-GlcNAc 2-epimerase by gene targeting causes early embryonic lethality in mice, thereby emphasizing the fundamental role of this bifunctional enzyme and sialylation during development. The need of UDP-GlcNAc 2-epimerase for a defined sialylation process is exemplified with the polysialylation of the neural cell adhesion molecule in embryonic stem cells.

Figure 1

Scheme representing the biosynthesis of sialic acid

Figure 2

(A) Strategy of constructing the UDP-GlcNAc 2-epimerase-targeting vector. A gene-targeting vector was used to generate murine 129 ES cells deficient in one copy of UDP-GlcNAc 2-epimerase by homologous recombination. The murine UDP-GlcNAc 2-epimerase gene was inactivated by replacing the second exon by a neo cassette. Homologous recombinant clones were used to create 129/Ola-C57BL/6J chimeric mice that transmitted the mutated UDP-GlcNAc 2-epimerase gene through the germ line (B = BamHI, E = EcoRI, H = HincII, n = NcoII). (B) Identification of homologous recombination events. Ten days after transfection, genomic DNA was isolated from individual cell clones. Homologous recombination was identified by Southern blot analysis as an additional 3-kb fragment in heterozygous cells (+/−). In contrast, in wild-type cells (+/+) only the 5-kb fragment is detectable.

Figure 3

(A) Reverse transcription–PCR of +/+, +/−, and −/− ES cell clones. Total RNA of wild-type (+/+), heterozygous (+/−), and homozygous deficient (−/−) ES cells were transcribed to cDNA and amplified by using specific primers to UDP-GlcNAc 2-epimerase or glyceraldehyde-3-phosphate dehydrogenase. (B) Western blot analysis of UDP-GlcNAc 2-epimerase-deficient ES cells. UDP-GlcNAc 2-epimerase of wild-type (+/+), heterozygous (+/−), and homozygous deficient (−/−) ES cells was immunoprecipitated and detected by Western blot analysis by using specific polyclonal antibodies to UDP-GlcNAc 2-epimerase. (C) Determination of the enzyme activity in UDP-GlcNAc 2-epimerase-deficient ES-cells. Cytosolic fractions of wild-type (+/+), heterozygous (+/−), and homozygous deficient (−/−) ES cells were prepared by ultracentrifugation, and UDP-GlcNAc 2-epimerase activity was determined. Bars represent mean values of three independent experiments.

Figure 4

Cell surface sialylation of UDP-GlcNAc 2-epimerase-deficient ES cells. Wild-type (+/+) and homozygous deficient ES cell lines (−/−) were grown for 5 days in serum-replacement medium without serum (no serum, open bars), or in serum-containing medium (serum, filled bars). Cell surface expression of sialic acids was analyzed by fluorescence-activated cell sorter analysis with L. flavus agglutinin. Bars represent mean values of three independent experiments.

Figure 5

PSA expression of UDP-GlcNAc 2-epimerase-deficient ES cells. Solubilisates of wild-type (+/+), heterozygous (+/−), or UDP-GlcNAc 2-epimerase-deficient ES cells (−/−) cultured in FCS (no additions), FCS plus 10 mM GlcNAc (GlcNAc) or FCS plus 10 mM ManNAc (ManNAc) were separated on SDS-polyacrylamide gels and transferred to nitrocellulose filters. PSA was detected with a PSA-specific monoclonal antibody.