Structure-function analysis of tissue-type plasminogen activator by linker-insertion, point and deletion mutagenesis

We undertook a structure–function analysis of human tissueplasminogen activator (tPA) using linker-scanning and deletionmutagenesis. Synthetic oligonucleotide linkers were introducedinto the tPA cDNA at pre-existing restriction enzyme sites.This generated a series of tPA variants which contained smallprimary sequence alterations consisting of point mutations,deletions or insertions. The majority of the linker-insertionvariants demonstrate a significant reduction in amidolytic andfibrinolytic activity in comparison to wild-type tPA. The exceptionsare 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 (proteasedomain) of tPA are the lowest in enzymatic activity. Particularlysensitive to mutation are highly conserved amino acids. Heavychain deletion variants were constructed from point mutantsat the domain boundaries of tPA. Deletion of the kringle domainslowers the fibrinolytic activity to a greater extent than deletionof the finger or growth factor domains. We conclude that alterationsin any domain of the tPA molecule, and particularly in the highlyconserved residues within these domains, can affect fibrinolyticactivity.