Rüdiger Horstkorte

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The physiological precursor of all sialic acids is N-acetyl-D-mannosamine. The N-acyl side chain of sialic acid can be modified by exposure of cells to synthetic N-acyl-modified D-mannosamines. In a new experimental approach cells were cultivated in the presence of N-propanoyl-D-mannosamine. This unnatural precursor of sialic acid is taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acid in vitro and in vivo. Here we report on the biological consequences of the incorporation of the unnatural N-propanoylneuraminic acid into glycoconjugates of HL60 cells. Biochemical engineering of the acyl side chain of neuraminic acids activates β₁-integrins (VLA4 or VLA5), resulting in an increased adhesion of HL60 cells to fibronectin.

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.

UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase (UDP‐GlcNAc 2‐epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP‐GlcNAc 2‐epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP‐N‐acetylglucosamine to ManAc and the phosphorylation to ManAc‐6‐phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP‐N‐acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP‐GlcNAc 2‐epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP‐GlcNAc 2‐epimerase and this phosphorylation results in an upregulation of the UDP‐GlcNAc 2‐epimerase enzyme activity.

Neuraminic acids are widely expressed as terminal carbohydrates on glycoconjugates and are involved in a variety of biological functions. The key enzyme of N‐acetylneuraminic acid synthesis is UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase, which catalyses the first two steps of neuraminic acid biosynthesis in the cytosol. In this study we report the complete amino acid sequence of the mouse UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase. The ORF of 2166 bp encodes 722 amino acids and a protein with a predicted molecular mass of 79.2 kDa. Northern blot analysis and in situ hybridization revealed that UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase is expressed at early stages during development and in all tissues investigated with a maximal expression in the liver.