Structure-function analysis of tissue-type plasminogen activator by linker-insertion, point and deletion mutagenesis.
We undertook a structure--function analysis of human tissue plasminogen activator (tPA) using linker-scanning and deletion mutagenesis. Synthetic oligonucleotide linkers were introduced into the tPA cDNA at pre-existing restriction enzyme sites. This generated a series of tPA variants which contained small primary sequence alterations consisting of point mutations, deletions or insertions. The majority of the linker-insertion variants demonstrate a significant reduction in amidolytic and fibrinolytic activity in comparison to wild-type tPA. The exceptions are the variants with linker-inserts placed at the BglII(115) and StyI(277) sites of the tPA cDNA (4SLEG5 and 57LEA58 respectively), which encode insertions at the boundaries of the finger domain. The variants with linker-inserts in the light chain (protease domain) of tPA are the lowest in enzymatic activity. Particularly sensitive to mutation are highly conserved amino acids. Heavy chain deletion variants were constructed from point mutants at the domain boundaries of tPA. Deletion of the kringle domains lowers the fibrinolytic activity to a greater extent than deletion of the finger or growth factor domains. We conclude that alterations in any domain of the tPA molecule, and particularly in the highly conserved residues within these domains, can affect fibrinolytic activity.
Markland
W
W
Protein Engineering Laboratory, Integrated Genetics Inc., Framingham, MA 01701.
Pollock
D
D
Livingston
D J
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000235:genetics; D014162:Transfection
D004247:DNA; D010959:Tissue Plasminogen Activator