Biosynthesis of N-acetylneuraminic acid in cells lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

The first two steps in mammalian biosynthesis of N-acetylneuraminic acid, a n important carbohydrate moiety in biological recognition systems, are perf ormed by the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acet ylmannosamine kinase. A subclone of the human B lymphoma cell line BJA-B K2 0, lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase m RNA as well as epimerase activity, displayed hyposialylated, functionally i mpaired cell surface glycoconjugates, Here we show that this cell line surp risingly still retains N-acetylmannosamine kinase activity. A gel filtratio n analysis of BJA-B K88 control cells, which express UDP-N-acetylglucosamin e 2-epimerase/N-acetylmannosamine kinase, revealed two N-acetylmannosamine kinase activity peaks, one co-eluting with UDP-N-acetylglucosamine 2-epimer ase activity and one co-eluting with N-acetylglucosamine kinase. For this e nzyme previous studies already showed a ManNAc kinase activity in vitro. In contrast, the hyposialylated BJA-B K20 subclone displayed only the N-acety lmannosamine kinase peak, co-migrating with N-acetylglucosamine kinase. The CMP-N-acetylneuraminic acid content of both K88 and K20 cells and the sial ylation of cell surface glycoconjugates of K20 cells could be significantly increased by supplementing the medium with N-acetylmannosamine. This N-ace tylmannosamine-induced increase was drastically reduced by co-supplementati on with Nacetylglucosamine only in K20 cells. We therefore propose the phos phorylation of N-acetylmannosamine as a hitherto unrecognized role of N-ace tylglucosamine kinase in living cells.