Abstract
Differential scanning calorimetry was used to study the domain structure and intramolecular interactions of tPA/uPA chimeras. A high temperature transition centered near 90 degrees C was observed upon melting of the tPA/uPA chimera (amino acids 1-274 of tPA and 138-411 of uPA) and its variant lacking the finger and epidermal growth factor-like modules (residues 1-3 and 87-274 of tPA and 138-411 of uPA). Since neither of the two parent plasminogen activators display such a stable structure, one may suggest that a new stabilizing intramolecular interaction occurs in the chimeras. We found that occupation of the lysine binding site of tPA by a lysine or arginine side chain from the urokinase moiety is responsible for the high temperature transition as well as for the failure of the chimeras to exhibit the expected fibrin binding properties. All uPA species, single- and two-chain high molecular weight uPA (Pro-Uk and HMW-Uk) and two-chain low molecular weight uPA (LMW-Uk), interact intermolecularly with tPA and its kringle-containing derivatives. This intermolecular interaction was strongly inhibited by epsilon-aminocaproic acid indicating that the lysine binding site of tPA is involved. The binding of uPA with the fluorescein-labeled A-chain of tPA, registered by changes in fluorescence anisotropy, was estimated to have a Kd range of 1-7 microM. The interaction of tPA with uPA determined by solid-phase assays appeared to be tighter, with a Kd range of 50-300 nM. Two synthetic peptides, with and without carboxyl-terminal lysine, corresponding to urokinase residues 144-158 and 144-157, were approximately 100-fold more potent than epsilon-aminocaproic acid with respect to inhibition of the tPA-uPA interaction, indicating that the tPA binding site on urokinase is located within this sequence, close to the activation site Lys158-Ile159. The discovered intermolecular interaction may be related to the reported synergistic effect of simultaneous administration of these two plasminogen activators.
Highlights
From the Walland Laboratory, American Red Cross, Rockville, Maryland 20855 and the §Center for Molecular and Vascular Biology, KU Leuuen, B-3000 Leuuen, Belgium
We found that occupation of the lysine binding site of type Plasminogen Activator (tPA) by a lysine or arginine side chain from the urokinase moiety is responsible for the high temperature transition as well as for the failure of the chimeras to exhibit the expected fibrin binding properties
All Urokinasetype Plasminogen Activator (uPA) species, single- and twochain high molecular weight uPA (Pro-Uk and HMW-Uk) and two-chain low molecular weight uPA (LMW-Uk), interact intermolecularly with tPA and its kringle-containing derivatives. This intermolecular interaction was strongly inhibited by e-aminocaproic acid indicating that the lysine binding site of tPA is involved
Summary
From the Walland Laboratory, American Red Cross, Rockville, Maryland 20855 and the §Center for Molecular and Vascular Biology, KU Leuuen, B-3000 Leuuen, Belgium. All uPA species, single- and twochain high molecular weight uPA (Pro-Uk and HMW-Uk) and two-chain low molecular weight uPA (LMW-Uk), interact intermolecularly with tPA and its kringle-containing derivatives. This intermolecular interaction was strongly inhibited by e-aminocaproic acid indicating that the lysine binding site of tPA is involved. TPA and uPA are mosaic proteins consisting of similar homologous modules that are found in other plasma proteins These include an epidermal growth factor-like (E) module, a kringle module (K), and a serine protease module (SP) which are common for the two plasminogen activators. The E module of uPA is responsible for the interaction with the uPA receptor [13]
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