Expression of an anchored urokinase in the apical endothelial cell membrane. Preservation of enzymatic activity and enhancement of cell surface plasminogen activation.
A mutant single chain urokinase plasminogen activator (scu-PA) was constructed by the addition of an apical membrane targeting signal from decay accelerating factor to the scu-PA carboxyl terminus. Bovine aortic endothelial cells (EC) were transduced with the mutant scu-PA. Metabolic labeling, immunoprecipitation, and gel electrophoresis revealed that the mutant scu-PA was present in a single-chain form at the EC surface. Immunohistochemistry and enzyme-linked immunosorbent assay before and after treatment of EC with phosphotidylinositol-specific phospholipase C confirmed that scu-PA was attached to the EC surface by a glycosyl-phosphotidylinositol anchor. Approximately 10(6) anchored scu-PA molecules/cell were present; however, anchoring was not 100% efficient, with scu-PA released into the medium as well. Selective biotinylation of the apical and basolateral surfaces revealed that anchored scu-PA was polarized to the apical surface. Apically anchored scu-PA could be converted by plasmin to two-chain urokinase, with a normal specific activity (140,000 IU/mg) as measured with the chromogenic substrate S-2444. Expression of anchored scu-PA resulted in an increase in EC surface plasminogen activator activity, as compared with the activity of either untransduced EC or EC transduced with a wild type scu-PA. These experiments demonstrate: 1) apical membrane targeting can be accomplished in EC; 2) scu-PA can be anchored to the EC surface with preservation of enzymatic activity; 3) EC surface plasminogen activator activity is significantly increased by the presence of anchored scu-PA. Cell surface targeted plasminogen activators may eventually be useful in the prevention and treatment of intravascular thrombosis.
Lee
S W
SW
Molecular Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Kahn
M L
ML
Dichek
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D004247:DNA; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D006017:Glycolipids / Q000378:metabolism; D017261:Glycosylphosphatidylinositols; D007124:Immunoenzyme Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism*; D011233:Precipitin Tests; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014161:Transduction, Genetic; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D010958:Plasminogen; D004247:DNA; D014568:Urokinase-Type Plasminogen Activator