Characterization and role of fucose mutarotase in mammalian cells.
L-Fucose for mammalian glycosylation contains an anomeric carbon atom generating alpha- and beta-L-fucoses. Based on sequence comparison of mouse and human homologs with the prokaryotic fucose mutarotases (FucU) characterized previously, we investigated their function in mammalian cells. By nuclear magnetic resonance (NMR) measurement with saturation difference analysis, the purified mammalian mutarotases were demonstrated to be involved in an interconversion between the two anomeric forms with comparable efficiency as that of the Escherichia coli FucU. The mouse gene was widely expressed in various tissues and cell lines, including kidney, liver, and pancreas, although expression was marginal in muscle and testis. By generating stably expressed cell lines for mutarotase genes in HepG2, it was shown that fucose incorporations into cellular proteins were increased as demonstrated by an incorporation of radiolabeled fucose into the cells. Furthermore, intracellular levels of GDP-L-fucose, measured with high performance liquid chromatography (HPLC), were enhanced by an overproduction of cellular mutarotase, which was reversed by gene silencing of mutarotase based on RNA interference. The results suggest that the mammalian mutarotase is functional in facilitated incorporation of fucose through the salvage pathway.
Park
Dongkyu
D
Department of Life Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-Dong, Yuseong-Gu, Daejeon 305-701, Korea.
Ryu
Kyoung-Seok
KS
Choi
Dongwook
D
Kwak
Jaechan
J
Park
Chankyu
C
D000595:Amino Acid Sequence; D000818:Animals; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000502:physiology; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000378:metabolism; D005643:Fucose / Q000737:chemistry* / Q000502:physiology; D006154:Guanosine Diphosphate Fucose / Q000737:chemistry*; D006168:Guinea Pigs; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D006154:Guanosine Diphosphate Fucose; D005643:Fucose; D002238:Carbohydrate Epimerases; C552683:fucose mutarotase, mouse; C019646:aldose 1-epimerase