Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain.
To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.
Burck
P J
PJ
Department of Molecular Genetics Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
Berg
D H
DH
Warrick
M W
MW
Berg
D T
DT
Walls
J D
JD
Jaskunas
S R
SR
Crisel
R M
RM
Weigel
B
B
Vlahos
C J
CJ
McClure
D B
DB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D005091:Exons; D005784:Gene Amplification; D006801:Humans; D007438:Introns; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011487:Protein Conformation; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D010959:Tissue Plasminogen Activator