Artificial exon shuffling between tissue-type plasminogen activator (t-PA) and urokinase (u-PA): a comparative study on the fibrinolytic properties of t-PA/u-PA hybrid proteins.
We constructed two human tissue-type plasminogen activator/urokinase (t-PA/u-PA) hybrid cDNAs which were expressed by transfection of mouse Ltk- cells. The properties of the secreted proteins were compared with those of recombinant t-PA (rt-PA) and high molecular weight (HMW) u-PA. The hybrid proteins each contain the amino-terminal fibrin-binding chain of t-PA fused to the carboxy-terminal serine protease moiety of u-PA but differ by a stretch of 13 amino acid residues between kringle 2 of t-PA and the plasmin cleavage site of u-PA. Hybrid protein rt-PA/u-PA I contains amino acids 1-262 of t-PA connected with amino acids 147-411 of u-PA, whereas hybrid protein rt-PA/u-PA II consists of the same t-PA segment and residues 134-411 of u-PA. We demonstrated fibrin binding for rt-PA, whereas the hybrid proteins bind to a lesser extent and HMW u-PA has no affinity for fibrin. Plasminogen activation by either one of the hybrid proteins in the absence of a fibrin substitute was similar to that by HMW u-PA, while rt-PA was much less active. The catalytic efficiency, in the presence of a fibrin substitute, increases more than 2000-fold for rt-PA, about 250-fold for hybrid proteins I and II, and 12-fold for HMW u-PA, respectively. Under these conditions the hybrid proteins are more efficient plasminogen activators than the parental ones. The hybrid molecules form a 1:1 molar complex with the human endothelial plasminogen activator inhibitor (PAI-1), analogous to that formed by rt-PA and HMW u-PA. The relative affinity of rt-PA for PAI-1 is 4.6-fold higher than that of HMW u-PA.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003062:Codon; D004247:DNA / Q000235:genetics; D005091:Exons; D005342:Fibrinolysis; D006801:Humans; D007700:Kinetics; D007739:L Cells / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D013937:Thymidine Kinase / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism
D003062:Codon; D004247:DNA; D013937:Thymidine Kinase; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator