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

Abstract

Transglutaminase (TGase) is a family of enzymes that catalyzes cross-linking reaction between glutamine-and lysine residue of substrate proteins in several mammalian biological events. Substrate proteins for TGase and their physiological relevance have been still in research, continuously expanding. In this study, we have established a novel screening system that enables identification of cDNA sequence encoding favorable primary structure as a substrate for tissue-type transglutaminase (TGase 2), a multifunctional and ubiquitously expressing isozyme. By the screening, we identified several T7 phage clones that displayed substrate peptides for TGase 2 as a translated product from human brain cDNA library. Among the selected clones, the C-terminal region of IKAP, IkappaB kinase complex associated protein, appeared as a highly reactive substrate sequence for TGase 2. This system will open possibility of rapid identification of substrate sequences for transglutaminases at a genetic level.

Transglutaminse 2 and EGGL, the Protein Cross-Link Formed by Transglutaminse 2, As Therapeutic Targets for Disabilities of Old Age

“…Extensive studies of the sequence specificity of TG2 show that it has some restriction on the sequence around glutamine, although the amino acids immediately flanking the Gln residue have little effect on the TG2 reaction rate. [35][36][37][38] TG2 has almost no sequence preference for the sequence around lysine. (I note, however, that surface lysines in ECM proteins are twice as likely to be flanked by hydrophobic amino acids as expected by chance from the distribution of all exposed surface amino acids, and so we might expect some limited selectivity for a flanking hydrophobic environment in TG2.…”

Recent advances in the development of tissue transglutaminase (TG2) inhibitors

“…The majority target the active site CYS residue of the mammalian TGs, and incorporate in their structure an essential electrophilic warhead able to covalently react with the highly nucleophilic sulphur atom from the catalytic site CYS residue, therefore totally inactivating the enzyme. Importantly, using the information gathered from the active sites of crystal structures of TG3, Factor XIII and TG2 and from information gathered from the preferred peptide amino acid sequences acting as TGase substrates (Hitomi et al 2009;Sugimura et al 2011) they allow greater specificity to be achieved against the target TG.…”