Autonomous functions of structural domains on human tissue-type plasminogen activator.
Transfected mouse Ltk- cells were employed for transient expression of recombinant human tissue-type plasminogen activator (t-PA; EC 3.4.21.31) or of recombinant-t-PA deletion proteins, encoded by SV40-pBR322-derived t-PA cDNA plasmids. The t-PA cDNA deletion mutants have two features in common, i.e., cDNA programming the signal peptide and the coding region for the light chain. Consequently, recombinant t-PA mutant proteins are efficiently secreted and display plasminogen activator activity. The gene encoding the amino-terminal heavy chain [an array of structural domains homologous to other plasma proteins (finger, epidermal growth factor, and kringle domains)] was mutated using restriction endonucleases to delete one or more structural domains. The stimulatory effect of fibrinogen fragments on the plasminogen activator activity of t-PA was demonstrated to be mediated by the kringle K2 domain and to a lesser extent by the finger/epidermal growth factor region but not by the kringle K1 domain. These data correlate well with the fibrin-binding properties of the recombinant t-PA deletion proteins, indicating that the stimulation of the activity by fibrinogen fragments is based on aligning the substrate plasminogen and t-PA on the fibrin matrix. Our results support the evolutionary concept of exon shuffling, arranging structural domains that constitute autonomous functions of the protein.
D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D010958:Plasminogen / Q000378:metabolism; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000378:metabolism; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000502:physiology*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D021382:Protein Sorting Signals; D005337:Fibrin; D010958:Plasminogen; D004247:DNA; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator