Tissue-type plasminogen activator variants with domain duplications and rearrangements.
The interactions between tPA domains that are important for catalysis are poorly understood. We have probed the function of interdomain interactions by generating tPA variants in which domains are duplicated or rearranged. The proteins were expressed in a transient mammalian expression system and tested in vitro for their ability to activate plasminogen, induce fibrinolysis and bind to a forming fibrin clot. Duplication of the heavy chain domains of tPA produced enzymatically active tPA variants, many of which demonstrated similar in vitro amidolytic and fibrinolytic activity and similar fibrin affinity to the parent molecule. Zymographic analysis of the domain duplication tPA variants showed one major active species for each variant. Selection of the residues duplicated and the interdomain spacing were found to be critical considerations in the design of tPA variants with duplicated domains. We also rearranged the domains of tPA such that kringle 1 replaced the second kringle domain and vice versa. An analysis of these variants indicates that the first kringle domain can confer fibrin affinity to a tPA variant and function in place of kringle 2. Therefore, in wild-type tPA, the functions of kringle 1 and kringle 2 must be dependent partially on their orientation within the heavy chain of the protein. The functional autonomy of the heavy and light chains of tPA is demonstrated by the activity of a tPA variant in which the order of the heavy and light chains was reversed.
Markland
W
W
Protein Engineering Laboratory, Integrated Genetics Inc., Framingham, MA 01701.
Pollock
D
D
Livingston
D J
DJ
D001483:Base Sequence; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D004262:DNA Restriction Enzymes; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000235:genetics
D021382:Protein Sorting Signals; D004247:DNA; D004262:DNA Restriction Enzymes; D010959:Tissue Plasminogen Activator