Directing sequence-specific proteolysis to new targets. The influence of loop size and target sequence on selective proteolysis by tissue-type plasminogen activator and urokinase-type plasminogen activator

Gary S. Coombs, Robert C. Bergstrom, Edwin L. Madison, David R. Corey

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

We have previously used substrate phage display to identify peptide sequences that are efficiently and selectively cleaved by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). We demonstrate that this information can be used to direct selective proteolysis to new protein targets. Sequences that were labile to selective cleavage by t-PA or u-PA when in the context of a peptide were introduced into the 43-52 (or Ω) loop of staphyloooccal nuclease. Both t-PA and u-PA hydrolyze the engineered proteins at the inserted target sequences, and K(m) values for protein cleavage were reduced up to 200-fold relative to values for cleavage of analogous sequences within 15 residue peptides. Variation of loop size surrounding a target sequence affects the efficiency of t-PA approximately 5-fold more strongly than that of trypsin, suggesting that cleavage by t-PA is more dependent on target site mobility. Cleavage of proteins by t-PA and u-PA is sequence selective. u-PA is 47-fold more active than t-PA for cleavage of a sequence known to be u-PA selective within small peptide substrates, whereas t-PA is 230-fold more active toward a t-PA- selective sequence.

Original languageEnglish (US)
Pages (from-to)4323-4328
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number8
DOIs
StatePublished - Feb 20 1998

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Proteolysis
Plasminogen Activators
Urokinase-Type Plasminogen Activator
Tissue Plasminogen Activator
Peptides
Proteins
Bacteriophages
Substrates
Trypsin

ASJC Scopus subject areas

  • Biochemistry

Cite this

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abstract = "We have previously used substrate phage display to identify peptide sequences that are efficiently and selectively cleaved by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). We demonstrate that this information can be used to direct selective proteolysis to new protein targets. Sequences that were labile to selective cleavage by t-PA or u-PA when in the context of a peptide were introduced into the 43-52 (or Ω) loop of staphyloooccal nuclease. Both t-PA and u-PA hydrolyze the engineered proteins at the inserted target sequences, and K(m) values for protein cleavage were reduced up to 200-fold relative to values for cleavage of analogous sequences within 15 residue peptides. Variation of loop size surrounding a target sequence affects the efficiency of t-PA approximately 5-fold more strongly than that of trypsin, suggesting that cleavage by t-PA is more dependent on target site mobility. Cleavage of proteins by t-PA and u-PA is sequence selective. u-PA is 47-fold more active than t-PA for cleavage of a sequence known to be u-PA selective within small peptide substrates, whereas t-PA is 230-fold more active toward a t-PA- selective sequence.",
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AU - Madison, Edwin L.

AU - Corey, David R.

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