Characterization of Highly Reactive Sequences for Transglutaminase 2 and Factor XIHa

摘要

Transglutaminases (TGase) are enzymes that catalyze the Ca~(2+) dependent cross-linking reaction between a gamma-carboxyamide group of glutamine and an s-amino group of lysine or other primary amine. Among these isozymes, TGases 2 and Factor XIII aremajor isozymes, which have been investigated since they are involved in various physiological functions. We developed a screening system to identify highly reactive sequences for TGase substrate using Ml3 phage-displayed random peptide library. Using themethod, we identified several amino acid sequences that were preferred as glutamine-donor substrates on the catalytic reaction of TGase 2 and Factor XIHa (active form of Factor XIII). In these sequences, there was a tendency of the sequence around the reactive glutamine residues. Furthermore, the highly reactive substrate sequences were quite different between TGase 2 and Factor XIII. We further confirmed the reactivity of the sequences by producing fusion proteins with glutathione-S-transferase (GST)as histidine-tagged peptide-GST. Most of the fusion proteins exhibited a significant increase in incorporation of fluorescent primary amines over that of GST protein alone. Moreover, among the obtained sequences, we selected the peptide sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reaction by TGase 2 and Factor XIHa.