Decrease in antithrombin III fucosylation by expressing GDP-fucose transporter siRNA in Chinese hamster ovary cells.
The full-length cDNA of GDP-fucose transporter (GFT) was cloned from Chinese hamster ovary (CHO) 13D-35D cells. The predicted product consisted of 366 amino acids with 86% and 92% similarities to human GFT and mouse GFT, respectively. When synthetic small interfering RNA (siRNA) against CHO GFT was introduced into human antithrombin III (AT-III)-producing recombinant CHO cells, defucosylated AT-III increased by 30-40% after 3-d incubation. A GFT siRNA expression plasmid was also constructed to defucosylate recombinant AT-III. CHO cells, which were transfected with the siRNA expression plasmid, showed approximately 75% reduction of Golgi-GDP fucose, resulting in 10-40% increase in defucosylated AT-III.
Omasa
Takeshi
T
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan. omasa@bio.eng.osaka-u.ac.jp
Tanaka
Ryo
R
Doi
Tomohiro
T
Ando
Masayuki
M
Kitamoto
Yuka
Y
Honda
Kohsuke
K
Kishimoto
Michimasa
M
Ohtake
Hisao
H
D000818:Animals; D000990:Antithrombin III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005643:Fucose / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D009004:Monosaccharide Transport Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C426650:SLC35C1 protein, human; D005643:Fucose; D000990:Antithrombin III