The differential glycosylation of human pro-urokinase from various recombinant mammalian cell lines does not affect activity and binding to PAI-1.
Human chromosomal DNA encoding single-chain urokinase-type Plasminogen Activator (scu-PA, or pro-urokinase) was inserted in an expression plasmid and transfected in human A431, mouse LB6 and CHO cells. LB6 cells were also transfected with a Bovine Papilloma Virus derivative containing the scu-PA gene. Human scu-PA was purified from cell supernatants of recombinant clones and characterized for structure and function. All recombinant scu-PAs are undistinguishable from human urine-derived scu-PA for peptide backbone, but possess a higher sugar content, as revealed by SDS-PAGE analysis after digestion with glycopeptidase F. This difference is partly due to an increased sialic acid content, as shown by analysis of neuraminidase-treated scu-PAs. No difference was found, however, among recombinant and natural scu-PAs in the kinetics of conversion into two-chain active forms (tcu-PAs) by human plasmin, and in the KM and kcat values of tcu-PA activity on the chromogenic substrate S-2444 and on human plasminogen. Also, recombinant and non-recombinant tcu-PAs displayed similar dose-response curves for binding to the endothelial inhibitor PAI-1. In conclusion, the glycosylation pattern of u-PA does not affect its interaction with the plasma proteins directly involved in its fibrinolytic function.
Sarubbi
E
E
Merrell Dow Research Institute, Lepetit Research Center, Gerenzano (Varese), Italy.
Nolli
M L
ML
Robbiati
F
F
Soffientini
A
A
Parenti
F
F
Cassani
G
G
D002460:Cell Line; D005343:Fibrinolytic Agents / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000494:pharmacology; D010960:Plasminogen Activators / Q000032:analysis / Q000378:metabolism*; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis; D013329:Structure-Activity Relationship; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis / Q000378:metabolism*
D005343:Fibrinolytic Agents; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D012794:Sialic Acids; D009439:Neuraminidase; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D019158:N-Acetylneuraminic Acid