A synthetic DNA encoding a modified human urokinase resistant to inhibition by serum plasminogen activator inhibitor.
Using DNA synthesis technology we constructed two synthetic DNAs, designated syn-uPA-DNA and mut-uPA-DNA. Syn-uPA-DNA contains the complete coding sequence of human presecretion form of single-chain u-PA. Mut-uPA-DNA was derived from syn-uPA-DNA by deleting 18 base pairs coding for amino acids Arg179-Ser184. Each synthetic DNA was inserted into a bovine papilloma viral genome-based expression vector to obtain expression in mouse cells. The results indicate that both syn-uPA and mut-uPA proteins are secreted predominantly in single-chain form. The single-chain form of both enzymes can be completely converted to two-chain form by treatment with plasmin. Both enzymes are as active as natural urokinase (std-uPA) isolated from urine. Analysis of enzymatic activity indicates that under conditions where syn-uPA and std-uPA are completely inhibited by endothelial-type plasminogen activator inhibitor (PAI-1), mut-uPA retains 90% activity. In identical experiments with placental-type PAI (PAI-2), mut-uPA retains 80% activity. Syn-uPA is capable of forming a approximately 100-kDa complex with PAI, whereas mut-uPA can not. PAI-treated mut-uPA has kinetic properties similar to untreated syn-uPA or std-uPA. Overall, the data indicate that amino acids Arg179-Ser184 function at least in part as a binding site for PAI. Resistance to PAI inhibition may increase the potency of mut-uPA as a thrombolytic agent.
Adams
D S
DS
Transgenic Sciences, Inc., Worcester, Massachusetts 01608.
Griffin
L A
LA
Nachajko
W R
WR
Reddy
V B
VB
Wei
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000138:chemical synthesis*; D004789:Enzyme Activation; D005091:Exons; D005341:Fibrinolysin / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D015849:Plasminogen Inactivators / Q000494:pharmacology*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism
D015849:Plasminogen Inactivators; D004247:DNA; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator