The influence of glycosylation on the catalytic and fibrinolytic properties of pro-urokinase.
We previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-)(302)pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (approximately 2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK. These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.
Lenich
C
C
Vascular Research Laboratory, Institute for the Prevention of Cardiovascular Disease, New England Deaconess Hospital, Harvard Medical School, Boston, MA.
Pannell
R
R
Henkin
J
J
Gurewich
V
V
D002384:Catalysis; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005341:Fibrinolysin / Q000494:pharmacology; D005342:Fibrinolysis; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase