Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain.
The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.
Gething
M J
MJ
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235.
Adler
B
B
Boose
J A
JA
Gerard
R D
RD
Madison
E L
EL
McGookey
D
D
Meidell
R S
RS
Roman
L M
LM
Sambrook
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004815:Epidermal Growth Factor / Q000235:genetics; D005091:Exons; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics*; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000235:genetics*
D010446:Peptide Fragments; D021382:Protein Sorting Signals; D004815:Epidermal Growth Factor; D004247:DNA; D010959:Tissue Plasminogen Activator