Yoshiaki Sugimura

Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library : Identification of peptide substrates for tgase 2 and factor XIIIA

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking 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, QxPϕD(P), QxPϕ, and QxxϕDP; Factor XIIIa, QxxϕxWP (where x and ϕ 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.

Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library

Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.

Preferred substrate sequences for transglutaminase 2: screening using a phage-displayed peptide library

A large number of substrate proteins for tissue transglutaminase (TGase 2) have been identified in vivo and in vitro. Preference in primary sequence or secondary structure around the reactive glutamine residues in the substrate governs the reactivity for TGase 2. We established a screening system to identify preferable sequence as a glutamine-donor substrate using a phage-displayed peptide library. The results showed that several peptide sequences have higher reactivity and specificity to TGase 2 than those of preferable sequences previously reported. By analysis of the most reactive 12-amino acid sequence, T26 (HQSYVDPWMLDH), residues crucial to the enzymatic reaction were investigated. The following review summarizes the screening system and also the preference in substrate sequences that were obtained by this method and those previously reported.

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 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.

Identification of a preferred substrate peptide for transglutaminase 3 and detection of in situ activity in skin and hair follicles

Transglutaminases (TGases) are a family of enzymes that catalyze cross‐linking reactions between proteins. During epidermal differentiation, these enzymatic reactions are essential for formation of the cornified envelope, which consists of cross‐linked structural proteins. Two main transglutaminases isoforms, epidermal‐type (TGase 3) and keratinocyte‐type (TGase 1), are cooperatively involved in this process of differentiating keratinocytes. Information regarding their substrate preference is of great importance to determine the functional role of these isozymes and clarify their possible co‐operative action. Thus far, we have identified highly reactive peptide sequences specifically recognized by TGases isozymes such as TGase 1, TGase 2 (tissue‐type isozyme) and the blood coagulation isozyme, Factor XIII. In this study, several substrate peptide sequences for human TGase 3 were screened from a phage‐displayed peptide library. The preferred substrate sequences for TGase 3 were selected and evaluated as fusion proteins with mutated glutathione S‐transferase. From these studies, a highly reactive and isozyme‐specific sequence (E51) was identified. Furthermore, this sequence was found to be a prominent substrate in the peptide form and was suitable for detection of in situ TGase 3 activity in the mouse epidermis. TGase 3 enzymatic activity was detected in the layers of differentiating keratinocytes and hair follicles with patterns distinct from those of TGase 1. Our findings provide new information on the specific distribution of TGase 3 and constitute a useful tool to clarify its functional role in the epidermis.

Transglutaminase 2 activity promotes membrane resealing after mechanical damage in the lung cancer cell line A549

Transglutaminase is a Ca2+‐dependent enzyme catalyzing protein cross‐linking reactions. We investigated the contribution of this enzyme to the resealing of the injured plasma membrane in animal cells, using a lung cancer‐derived cell line, A549. After mechanical injury by razor‐scratching, the level of membrane resealing was estimated by differential incorporation of dextrans labeled with two distinct fluorescent dyes. The recovery level was decreased in the presence of excess primary amine, as a competitive inhibitor of transglutaminase. We established a cell line that stably expresses shRNA (short hairpin RNA) to specifically inhibit the expression of TGase 2 (tissue‐type isozyme of transglutaminase) and confirmed the suppressed resealing level in the cell. Furthermore, additional expression of TGase 2 rescued the ability for membrane resealing. These results show that, after mechanical damage, this enzyme appeared to contribute to membrane resealing.

Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damage.

Transglutaminases are Ca2+‐dependent enzymes that post‐translationally modify proteins by crosslinking or polyamination at specific polypeptide‐bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin‐labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity‐purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca2+‐binding protein with four EF‐hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS/PAGE. Of the three proteins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transglutimase reactions function in the response to mechanical injury.

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

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.

Characterization of Highly Reactive Sequences for Transglutaminase 2 and Factor XIIIa

Transglutaminases (TGase) are enzymes that catalyze the Ca2+ dependent cross-linking reaction between a γ-carboxyamide group of glutamine and an e-amino group of lysine or other primary amine. Among these isozymes, TGases 2 and Factor XIII are major isozymes, which have been investigated since they are involved in various physiological functions.

Immunological analysis of epidermal-type transglutaminase (TGase 3) in epithelium

Transglutaminases (TGase) are enzymes that catalyze the Ca 2+ dependent cross-linking reaction between a γ-carboxyamide group of glutamine and an e-amino group of lysine or other primary amine. Among these isozymes, TGases 1 and 3 are involved in the formation of the cornified envelope, where they function to cross-link structural proteins during epidermal terminal differentiation. We developed monoclonal antibodies against human recombinant TGase 3. Using these antibodies, we analyzed TGase 3 expression in epidermis as well as cultured epidermal keratinocytes as a function of differentiation. In skin epidermis, TGase 3 was expressed in the cells of granular and cornified layers consistent with its role in cornified envelop formation. In the primary cultured keratinocytes, TGase 3 was expressed entirely in the cytoplasm of differentiating cells. TGase 3 activation during keratinocyte differentiation involves cleavage of a 77 kDa zymogen by limited proteolysis to release 47 and 30 kDa fragments. Although the dispase (bacterial protease) was only known enzyme for limited proteolysis in vitro, the responsible protease in vivo had not been clear. Recently, cathepsins S and L appeared to proteolyze the recombinant TGase 3. We have confirmed the enzymatic activation of TGase 3 zymogen by cathepsins and then determined the cleavage sites by these proteases.