Lothar Lucka

A Bifunctional Enzyme Catalyzes the First Two Steps in N-Acetylneuraminic Acid Biosynthesis of Rat Liver MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF UDP-N-ACETYL-GLUCOSAMINE 2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE

N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a group of important molecules in biological recognition systems. Biosynthesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC5.1.3.14)/N-acetylmannosamine kinase (ManNAc kinase, EC2.7.1.60) in rat liver (Hinderlich, S., Stäsche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 272, 24313–24318). In the present paper we report the isolation and characterization of a cDNA clone encoding this bifunctional enzyme. An open reading frame of 2166 base pairs encodes 722 amino acids with a predicted molecular mass of 79 kDa. The deduced amino acid sequence contains exact matches of the sequences of five peptides derived from tryptic cleavage of the enzyme. The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both epimerase and kinase activity. Distribution of UDP-GlcNAc 2-epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining both specific enzyme activities. Secreting organs (liver, salivary glands, and intestinal mucosa) showed high specific activities of UDP-GlcNAc 2-epimerase/ManNAc kinase, whereas significant levels of these activities were absent from other organs (lung, kidney, spleen, brain, heart, skeletal muscle, and testis). Northern blot analysis revealed no UDP-GlcNAc 2-epimerase/ManNAc kinase mRNA in the non-secreting tissues.

Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase

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.

Carcinoembryonic antigen-related cell-cell adhesion molecule C-CAM is greatly increased in serum and urine of rats with liver diseases

C‐CAM (rat cell CAM/human CD66a) is ubiquitous and multifunctional. It is involved in intercellular adhesion, signal transduction and cell growth inhibition. Structurally, it is related to the carcinoembryonic antigen. In the present study serum, bile and urine of rats with liver diseases were analyzed for the presence of cell CAM. After bile duct ligation and during galactosamine (GalN) hepatitis we found that large amounts of liver membrane‐bound C‐CAM are secreted or shed into blood. The serum level of another liver membrane‐bound protein, LI‐cadherin, is not increased. It was shown that C‐CAM is also present in bile fluid, and for the first time that C‐CAM is present in the urine of rats with liver diseases. A particularly high concentration was measured in the urine of rats suffering from GalN hepatitis.