Screening for the Preferred Substrate Sequence of Transglutaminase Using a Phage-Displayed Peptide Library: Identification of Peptide Substrates for TGASE 2 and Factor XIIIA

J Biol Chem. 2006 Jun 30;281(26):17699-706. doi: 10.1074/jbc.M513538200. Epub 2006 Apr 24.


Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPphiD(P), QxPphi, and QxxphiDP; Factor XIIIa, QxxphixWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Caseins / metabolism
  • Cloning, Molecular
  • Factor XIIIa / genetics*
  • Factor XIIIa / metabolism*
  • GTP-Binding Proteins / genetics*
  • GTP-Binding Proteins / metabolism*
  • Genetic Testing / methods*
  • Glutamine / metabolism
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism
  • Guinea Pigs
  • Humans
  • Molecular Sequence Data
  • Peptide Library*
  • Substrate Specificity
  • Transglutaminases / genetics*
  • Transglutaminases / metabolism*


  • Caseins
  • Peptide Library
  • Glutamine
  • transglutaminase 2
  • Factor XIIIa
  • Transglutaminases
  • Glutathione Transferase
  • GTP-Binding Proteins