Screening for the Preferred Substrate Sequence of Transglutaminase Using a Phage-displayed Peptide Library

Abstract

Mammalian transglutaminase (TGase) catalyzes covalent crosslinking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPD(P), QxP, and QxxDP; Factor XIIIa, QxxxWP (where x and represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.Transglutaminase (TGase, 2 2.3.2.13) is an enzyme that catalyzes the formation of isopeptide cross-links between glutamine and lysine residues in a variety of proteins and also attaches other primary amines to peptide-bound glutamines (1-5). To date, eight human TGase isozymes (Factor XIII, TGases 1-7) have been identified, comprising a large protein family with unique tissue distributions and physiological roles. Among the isozymes, TGase 2 and Factor XIII are two major members, although their locations and regulation differ. TGase 2 is expressed ubiquitously and has been implicated in many biological processes, including apoptosis, stabilization of the extracellular matrix, and regulation of growth factors (6, 7). Plasma Factor XIII is synthesized as a zymogen that comprises two A and two B subunits (8). The A subunit contains the catalytic domain and is converted by thrombin-dependent proteolysis into its active form during clot formation. Activated Factor XIII (Factor XIIIa) is involved in fibrin stabilization and wound healing (9).Generally, TGase reactions involve a Ca 2ϩ -dependent acyl transfer via a double displacement mechanism. In the first step, a glutamine ␥-carboxyamide group in the substrate binds to a cysteine at the active site, resulting in formation of a ␥-glutamylthioester bond and release of ammonia. Formation of the covalent acyl enzyme intermediate is the rate-limiting step. The -amino group of a peptide-bound lysine, as a nucleophilic substrate, binds to the acyl enzyme intermediate and then attacks the thioester bond, thereby generating an intermolecular isopeptide -(␥-glutamyl)lysine cross-link. Primary amines can replace lysine in transamidation reactions and become incorp...

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.…”

“…Each of them contained several glutamine residues showing high reactivities as GST-fusion proteins. Although the precise reactive glutamine residue(s) have not been identified, there are possible primary sequences: glutamine residue ''island'' (QQQ) or Q-x-P-w (x and w are any and hydrophobic amino acids, respectively) that was proposed as preferred consensus sequence for TGase 2 (Sollid 2002;Sugimura et al 2006). Interestingly, the same sequence (WQAPV) is observed in RNG157 and hCG2024782, suggesting the possibility of a novel primary sequence as favored substrate.…”

“…In the expressed protein, hexahistidine was attached to the N-terminus to GST (glutathione-S-transferase), in which all glutamine residues were changed to asparagine prior to construction of the vector (Sugimura et al 2006). Bacterial strain BL21(DE3)LysS was transformed with the expression vectors and then used for inducible expression by IPTG according to the established procedure.…”

“…Phage clones that were covalently bound to bio-Cd were selected by avidin affinity purification. The selected phages were amplified and then subjected to four additional cycles, as carried out in the screening using M13 phage-displayed random peptide library (Sugimura et al 2006). During the procedure, in each cycle, DNA from the amplified phage clones was prepared and then used as a template to amplify their encoding cDNA containing region by PCR, which were then analyzed by polyacrylamide electrophoresis (Fig.…”

“…To know physiological roles of TGases in these processes, investigation of substrates is essential research. So far, several approaches have revealed substrates for TGases by proteomics analyses of cross-linked/polymerized products, affinity purification using labeled primary amines, and screening from peptide display (Orru et al 2003;Lee et al 1992;Ikura et al 1998;Ichikawa et al 2008;Sugimura et al 2006;Keresztessy et al 2006). Based on the results of the studies described above, database for substrates and their sequences are available on the web site and summarized as several publications (Csosz et al 2009;Esposito and Caputo 2005;Facchiano and Facchiano 2009).…”