tailieunhanh - Báo cáo khoa học: 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 enve-lope 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. | ỊFEBS Journal Identification of preferred substrate sequences for transglutaminase 1 - development of a novel peptide that can efficiently detect cross-linking enzyme activity in the skin Yoshiaki Sugimura1 z Masayo Hosono1 Miyako Kitamura1 Tatsuya Tsuda2 Kiyofumi Yamanishi2 Masatoshi Maki1 and Kiyotaka Hitomi1 1 Department of Applied Molecular Biosciences Graduate Schoolof BioagriculturalSciences Nagoya University Japan 2 Department of Dermatology Hyogo College of Medicine Nishinomiya Japan Keywords epidermis keratinocyte phage display skin transglutaminase Correspondence K. Hitomi Department of Applied Molecular Biosciences Graduate School of Bioagricultural Sciences Nagoya University Chikusa 464 8601 Nagoya Japan Fax 81 52 789 5542 Tel 81 52 789 5541 E-mail hitomi@ These authors contributed equally to this work Received 1 August 2008 revised 10 September 2008 accepted 18 September 2008 doi 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 RWxxxWP where x and w 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 .