Identification of preferred substrate sequences for transglutaminase 1 – development of a novel peptide that can efficiently detect cross‐linking enzyme activity in the skin

Abstract

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/RψxxxWP (where x and ψ 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.

In Situ Detection of Active Transglutaminases for Keratinocyte Type (TGase 1) and Tissue Type (TGase 2) Using Fluorescence-Labeled Highly Reactive Substrate Peptides

“…1A; Sugimura et al 2008). No apparent signal was observed in the section from the TGase 1-deficient mouse or in the reaction using FITC-pepK5QN.…”

“…Although many in vitro assay systems have been established to measure the TGase activity, there is no method for monitoring isozymespecific activity in situ. We recently obtained highly reactive substrate peptides that act as efficient tools for detecting enzymatic activity of several TGases (Sugimura et al 2006;Sugimura et al 2008). More recently, using a fluorescence-labeled peptide (pepK5) specific for TGase 1, we have successfully completed in situ detection of active TGases both in human and mouse skin (Sugimura et al 2008;Akiyama et al 2010).…”

“…Recently, using a random peptide library, we have characterized the preferred glutamine-donor substrate sequences for TGases that shows a unique reaction tendency to each isozyme (Sugimura et al 2006). In these studies, several 12-mer substrate peptide sequences for major TGases have been identified: factor XIII, TGase 1, and TGase 2 (Sugimura et al 2008;Hitomi, Kitamura, and Sugimura 2009). The most favorable sequence for each isozyme appeared to act as a substrate with high reactivities and isozyme specificities, even in the peptide form.…”

Distinct protein expression and activity of transglutaminases found in different epidermal tumors

“…In situ activity assays specific for TG1 or TG2 in BCC To examine whether TG2 is functional in BCC, we performed in situ activity assays using FITC-labelled peptides K5 and T26, which are specific substrates for TG1 and TG2, respectively (22,23). In BCC, TG1 activity was seen in overlying epidermis, but not in BCC tumor cells (Fig.…”

Screening of substrate peptide sequences for tissue-type transglutaminase (TGase 2) using T7 phage cDNA library

“…Therefore, in order to clarify substrate specificity, the possible sequence around the reactive glutamine residues should be investigated. In recent years, we have established a screening system to identify the reactive substrate sequence around the glutamine residue using M13 phage-displayed random peptide library (Sugimura et al 2006(Sugimura et al , 2008aHitomi et al 2009). Regarding several isozymes including TGase 2, the preferred 12-mer amino acid sequences specific for each isozyme have been identified.…”