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, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. A subclone of the human B lymphoma cell line BJA-B K20, lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains N-acetylmannosamine kinase activity. A gel filtration analysis of BJA-B K88 control cells, which express UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, revealed two N-acetylmannosamine kinase activity peaks, one co-eluting with UDP-N-acetylglucosamine 2-epimerase activity and one co-eluting with N-acetylglucosamine kinase. For this enzyme previous studies already showed a ManNAc kinase activity in vitro. In contrast, the hyposialylated BJA-B K20 subclone displayed only the N-acetylmannosamine kinase peak, co-migrating with N-acetylglucosamine kinase. The CMP-N-acetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supplementing the medium with N-acetylmannosamine. This N-acetylmannosamine-induced increase was drastically reduced by co-supplementation with N-acetylglucosamine only in K20 cells. We therefore propose the phosphorylation of N-acetylmannosamine as a hitherto unrecognized role of N-acetylglucosamine kinase in living cells.
Hinderlich
S
S
Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Berlin-Dahlem, Germany.
Berger
M
M
Keppler
O T
OT
Pawlita
M
M
Reutter
W
W
D000116:Acetylgalactosamine / Q000378:metabolism / Q000494:pharmacology; D000117:Acetylglucosamine / Q000378:metabolism / Q000494:pharmacology; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002999:Clone Cells; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D029968:Escherichia coli Proteins; D006595:Hexosamines / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D016393:Lymphoma, B-Cell / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D006595:Hexosamines; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C018765:N-acylmannosamine kinase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; C083982:wecB protein, E coli; D019158:N-Acetylneuraminic Acid; D000116:Acetylgalactosamine; D000117:Acetylglucosamine; C002022:N-acetylmannosamine