Signal and propeptide processing of human tissue plasminogen activator: activity of a pro-tPA derivative.
We have explored the heterogeneity in the proteolytic processing of the N-terminus of human tissue plasminogen activator. We demonstrate that normal propeptide processing occurs following Arg-4, preceding the sequence Gly-Ala-Arg-Ser+1. Generation of the previously designated Ser+1 occurs via secondary proteolysis following secretion. By site-directed mutagenesis, we have eliminated this cleavage site resulting in a derivative containing the propeptide sequence. N-terminal sequence analysis of this form indicated that signal peptide cleavage occurs following Ser-13. The pro-tPA derivative had near normal serine protease and plasminogen activating activities, and could be stimulated by fibrin. An additional derivative, containing the tribasic sequence from the human protein C propeptide preceding Ser+1, was secreted with full processing of the propeptide. Our data have defined the cleavages for the signal peptide and propeptide and demonstrate that a tribasic sequence can be used to eliminate N-terminal heterogeneity in this molecule. In addition, we demonstrate that, unlike several other serine proteases, a propeptide sequence does not alter the activity of this enzyme.
Cardiovascular Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002461:Cell Line, Transformed; D002846:Chromatography, Affinity; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics*; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004247:DNA; D010959:Tissue Plasminogen Activator