Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGASE 2 and Factor XIIIA.
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.
Sugimura
Yoshiaki
Y
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Hosono
Masayo
M
Wada
Fumitaka
F
Yoshimura
Tohru
T
Maki
Masatoshi
M
Hitomi
Kiyotaka
K
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D003001:Cloning, Molecular; D026122:Factor XIIIa / Q000235:genetics* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005820:Genetic Testing / Q000379:methods*; D005973:Glutamine / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013379:Substrate Specificity; D011503:Transglutaminases / Q000235:genetics* / Q000378:metabolism*
D002364:Caseins; D019151:Peptide Library; D005973:Glutamine; D026122:Factor XIIIa; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins