TAILIEUCHUNG - Báo cáo khóa học: Cloning and expression of murine enzymes involved in the salvage pathway of GDP-L-fucose

In the salvage pathway of GDP-L-fucose, free cytosolic fucose is phosphorylated by L-fucokinase to form L-fu-cose-1-phosphate, which is then further converted to GDP-L-fucose in the reaction catalyzed by GDP-L-fucose pyrophosphorylase. We report here the cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. MurineL-fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant ofL-fucokinase was enzy-matically active when expressed in COS-7 cells | Eur. J. Biochem. 271 78-86 2004 FEBS 2003 doi Cloning and expression of murine enzymes involved in the salvage pathway of GDP-L-fucose L-fucokinase and GDP-L-fucose pyrophosphorylase Jaana Niittvmaki1 Pirkko Mattila2. ChristoDhe Roos2. Laura Huooaniemi1 Solveia Sioblom1 and Risto Renkonen1 3 1 Department of Bacteriology and Immunology Haartman Institute and Biomedicum University of Helsinki 2MediCel Helsinki 3HUCH Laboratory Diagnostics Helsinki University Central Hospital Finland In the salvage pathway of GDP-L-fucose free cytosolic fucose is phosphorylated by L-fucokinase to form L-fu-cose-1-phosphate which is then further converted to GDP-L-fucose in the reaction catalyzed by GDP-L-fucose pyrophosphorylase. We report here the cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. Murine L-fucokinase is expressed as two transcripts of 3057 and 3270 base pairs encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of L-fucokinase was enzymatically active when expressed in COS-7 cells. Murine GDP-L-fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of kDa. GDP- L-fucose the reaction product of GDP-L-pyrophosphory-lase was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine L-fucokinase and GDP-L-fucose pyrophosphorylase was investigated by quantitative real time PCR which revealed high expression of L-fucokinase and GDP-L-fucose pyrophosphorylase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected froma number of expressed sequence tag libraries which indicate that not only the de novo pathway alone but also the salvage pathway could have a significant role in the synthesis of GDP-L-fucose in the cytosol. Keywords GDP-L-fucose .

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