Sialylation is essential for early development in mice

Martina Schwarzkopf

Klaus Knobeloch

Elvira Rohde

Stephan Hinderlich

^{Institut für Molekularbiologie und Biochemie, Fachbereich Humanmedizin, Freie Universität Berlin, Arnimallee 22, D-14195 Berlin–Dahlem, Germany; and}^{Forschungsinstitut für Molekulare Pharmakologie, Freie Universität Berlin, Krahmerstrasse 6, D-12207 Berlin–Steglitz, Germany}

^{M.S. and K.-P.K. contributed equally to this work.}

^{To whom reprint requests should be addressed. E-mail:}ed.nilreb-uf.tadez@oktsrohr.

Communicated by William J. Lennarz, State University of New York, Stony Brook, NY

Received 2001 Dec 11; Accepted 2002 Feb 4.

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.

Abstract

Sialic acid is the most abundant terminal monosaccharide of glycoconjugates of the eukaryotic cell surface (1, 2). It is involved in a variety of cellular functions, such as cell–cell interaction including metastasis formation and progression of a variety of tumors, virus infection, and the biological stability of glycoproteins (3, 4). Of particular interest is the unique polysialylation of the neural cell adhesion molecule (NCAM), which is down-regulated during development. The expression of the polysialylated form of NCAM is known to be involved in learning and memory in the adult hippocampus. In addition, polysialylation of NCAM often correlates with tumorigenicity (5–9).

Sialic acids are synthesized in the cytosol from UDP-N-acetylglucosamine by four consecutive reactions. The first two steps in sialic acid biosynthesis (Fig. (Fig.1)1) are catalyzed by the bifunctional enzyme, UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase), which has been cloned and characterized in rodents and humans (10–13). It catalyzes both enzymatic reactions as a hexamer only (14). Recently, protein kinase C has been associated with UDP-GlcNAc 2-epimerase, regulating its enzymatic activity (15). Northern-blot analysis and in situ hybridization revealed the highest expression in liver. To a lesser extent the enzyme is also expressed in all other organs investigated. UDP-GlcNAc 2-epimerase is fully expressed at all stages during mouse development that have been investigated so far (11).

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Figure 1

Scheme representing the biosynthesis of sialic acid

The clinical relevance of the UDP-GlcNAc 2-epimerase was demonstrated by the detection of a binding defect of the feedback inhibitor CMP-sialic acid (16, 17) leading to sialuria (13, 18, 19). In this sialic acid storage disease, free sialic acid accumulates in the cytoplasm, which results in severe mental retardation of the surviving patients. The significance of the enzyme is further illustrated by the observation that, in a variant of HL60 cells, the low expression of sialic acids is correlated with dramatically reduced enzyme activity (20). Recently, it was proposed that mutations in the human UDP-GlcNAc 2-epimerase gene are responsible for hereditary inclusion body myopathy, a unique group of neuromuscular disorders characterized by adult onset, slowly progressive weakness, and a typical muscle pathology (21).

Acknowledgments

We thank Dr. R. Gerardy-Schahn for providing monoclonal antibody 735 to polysialic acid. These studies were supported by the Deutsche Forschungsgemeinschaft (Ho 1959/1-1,1-2, SFB 366), the Fonds der Chemischen Industrie, and the Sonnenfeld Stiftung.

Acknowledgments

Abbreviations

NCAM	neural cell adhesion molecule
UDP-GlcNAc 2-epimerase	UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase
ES	embryonic stem
PSA	polysialic acid
ManNAc	N-acetylmannosamine

Abbreviations

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