Identification of preferred substrate sequences for transglutaminase 1--development of a novel peptide that can efficiently detect cross-linking enzyme activity in the skin.
Transglutaminase 1 (TGase 1) is an essential enzyme for cornified envelope formation in stratified squamous epithelia. This enzyme catalyzes the cross-linking of glutamine and lysine residues in structural proteins in differentiating keratinocytes. To gain insight into the preferred substrate structure of TGase 1, we used a phage-displayed random peptide library to screen primary amino acid sequences that are preferentially selected by human TGase 1. The peptides selected as glutamine donor substrate exhibited a marked tendency in primary structure, conforming to the sequence: QxK/RpsixxxWP (where x and psi represent non-conserved and hydrophobic amino acids, respectively). Using glutathione S-transferase (GST) fusion proteins of the selected peptides, we identified several sequences as preferred substrates and confirmed that they were isozyme-specific. We generated GST-fused alanine mutants of the most reactive sequence (K5) to determine the residues that were critical for reactivity. Even in peptide form, K5 appeared to have high and specific reactivity as substrate. In situ analysis of mouse skin sections using fluorescence-conjugated K5 peptide resulted in detection of TGase 1 activity with high sensitivity, but no signal was detected in a TGase 1-null mouse. In conclusion, we were successful in generating a novel substrate peptide for sensitive detection of endogenous TGase 1 activity in the skin.
Sugimura
Yoshiaki
Y
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
Hosono
Masayo
M
Kitamura
Miyako
M
Tsuda
Tatsuya
T
Yamanishi
Kiyofumi
K
Maki
Masatoshi
M
Hitomi
Kiyotaka
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D004798:Enzymes; D007527:Isoenzymes; D051379:Mice; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000032:analysis*
D004798:Enzymes; D007527:Isoenzymes; D019151:Peptide Library; D010455:Peptides; D011503:Transglutaminases; C105804:transglutaminase 1