Domain-domain interactions in hybrids of tissue-type plasminogen activator and urokinase-type plasminogen activator.
Fibrin-dependent plasminogen activation by tissue-type plasminogen activator (t-PA) is in part associated with the presence of the kringle 2 domain in t-PA. Within this kringle 2 domain a lysyl-binding site has been described. The plasminogen to plasmin conversion by urokinase-type plasminogen activator (u-PA), in contrast to that of t-PA, is not enhanced in the presence of fibrin. Within the u-PA kringle domain no lysyl-binding site is found. To study whether introduction of a lysyl-binding site in the u-PA kringle domain will make u-PA a fibrin-dependent plasminogen activator, three stretches of amino acid residues of the u-PA kringle domain (A28-Q33, D55-N57 and G67-V72) were substituted by three stretches of amino acids from the corresponding positions of the kringle 2 domain of t-PA (M28-K33, D55-D57 and N67-W72). These changes resulted in the creation of the lysyl-binding site consensus of the kringle 2 domain (K33, D55, D57, W62 and W72) in the u-PA kringle. However, the resulting u-PA mutant did not interact with lysyl-Sepharose, nor did it display fibrin-enhanced plasminogen activation in the presence of soluble fibrin mimic. When the kringle domain of u-PA was replaced by the kringle 2 domain of t-PA, similar results were obtained. The hybrid protein hardly interacted with lysyl-Sepharose and the plasminogen activation was not enhanced in the presence of fibrin mimic. However, the N-terminal fragment isolated from this hybrid molecule (consisting of growth factor domain and kringle 2 domain) did interact with lysyl-Sepharose, suggesting that in the hybrid molecule a functional lysyl-binding site is present but not operational. Indeed, lysine analogue (epsilon-amino-caproic acid) sensitive binding of isolated t-PA kringle 2 domain to u-PA could be observed. The modified u-PA kringle, the wild type u-PA kringle and the kringle 2 of the u-PA hybrid were also placed N-terminal of the protease domain of t-PA. As expected, the t-PA mutant consisting of the kringle 2 domain and the protease domain bound to lysyl-Sepharose and showed fibrin-dependent plasminogen activation. Further, the hybrid molecule consisting of the u-PA kringle placed N-terminal of the t-PA protease domain did not display these features. Introduction of the modified u-PA kringle N-terminal of the t-PA protease domain resulted in a very weak interaction with lysyl-Sepharose. Despite the high overall similarity in primary structure of the modified u-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogen activation of this hybrid molecule was observed. The above-mentioned results question the concept that the structural auto-nomous domains within hybrid plasminogen activators t-PA and u-PA function as autonomous domains and suggest that interactions between the kringle and the protease domain in hybrid molecules strongly influences their functional features.
Bakker
A H
AH
Gaubius Laboratory TNO-PG, Leiden, Netherlands.
Nieuwenbroek
N M
NM
Verheijen
J H
JH
D000595:Amino Acid Sequence; D015119:Aminocaproic Acid / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D016384:Consensus Sequence; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism / Q000494:pharmacology; D018082:Kringles / Q000235:genetics*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D005337:Fibrin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine; D015119:Aminocaproic Acid