The construction and expression of chimeric urokinase-type plasminogen activator genes containing kringle domains of human plasminogen.
A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA-->K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA-->K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA-->K2HPgK3HPg]uPA), and K4HPgK5HPg (r-[KuPA-->K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37 degrees C) of 120 s-1 mM-1. This value ranges from 92 s-1 mM-1 (tcr-[KuPA-->K1HPg]uPA) to 166 s-1 mM-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 x 10(7) M-1s-1 (pH 7.4, 10 degrees C), ranges from 0.29 x 10(7) M-1s-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) to 1.08 x 10(7) M-1s-1 (tcr-[KuPA-->K4HPgK5HPg]uPA), for the tcr-chimeric variants. Neither wtr-uPA nor any of its chimeric r-variants interacted macroscopically with a fibrin clot under conditions that allowed binding of 74% of single-chain r-tissue-type plasminogen activator. However, the tcr-chimeric uPA variants provided HPg-enriched clot lysis times between 0.2 (r-[KuPA-->K1HPgK4HPg]uPA) and 2.4 (r-[KuPA-->K2HPgK3HPg]uPA) relative to that of wtr-uPA.(ABSTRACT TRUNCATED AT 400 WORDS)
Boutaud
A
A
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D015119:Aminocaproic Acid / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003488:Cyanogen Bromide; D004789:Enzyme Activation / Q000187:drug effects; D005337:Fibrin / Q000378:metabolism / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C028190:5-oxo-prolyl-glycyl-arginine-4-nitroanilide; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator; D003488:Cyanogen Bromide; D015119:Aminocaproic Acid