Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UK delta GS1) by changing culture conditions of a lymphoblastoid cell line Namalwa KJM-1 adapted to serum-free medium.
Pro-UK delta GS1 was designed as a long-life and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKDGS1, pIH1UK delta GS1SEd1-5 was constructed and introduced into Namalwa KJM-1, a lymphoblastoid cell line adapted to serum-free medium, and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UK delta GS1 producer (resistant to 200 nM of MTX), clone 2-9, was selected and used for further studies. Under the conventional conditions, i.e. at 37 degrees C in serum-free ITPSGF medium (based on RPMI-1640 medium), the oligosaccharide structure of pro-UK delta GS1 produced by clone 2-9 mainly consisted of fucose (Fuc)-containing biantennary complex-type oligosaccharide. Addition of dexamethasone (Dex), changed the carbohydrate contents in the media, and a shift down of incubation temperature caused a change in oligosaccharide structure of pro-UK delta GS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri- and tetraantennary complex-type oligosaccharide. The modulated pro-UK delta GS1 showed superior in vivo activity for a canine femoral thrombosis formed by inserting a copper-coil.
Hosoi
S
S
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Japan.
Satoh
M
M
Higo
K
K
Sugimoto
S
S
Miyaji
H
H
Karasawa
A
A
Yamaguchi
K
K
Hasegawa
M
M
Tamaoki
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002051:Burkitt Lymphoma / Q000473:pathology*; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D003907:Dexamethasone / Q000494:pharmacology; D004285:Dogs; D019008:Drug Resistance, Neoplasm; D005263:Femoral Artery; D005343:Fibrinolytic Agents / Q000378:metabolism* / Q000627:therapeutic use; D006031:Glycosylation / Q000187:drug effects; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use; D013696:Temperature; D015912:Thrombolytic Therapy; D013927:Thrombosis / Q000188:drug therapy; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use
D016895:Culture Media, Serum-Free; D005343:Fibrinolytic Agents; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003907:Dexamethasone; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase; D008727:Methotrexate