Transglutaminases Research Articles

Patients with congenital ichthyosis and TGM1 mutations overexpress other ARCI genes in the skin: Part of a barrier repair response?

Autosomal recessive congenital ichthyosis (ARCI) is a group of monogenic skin disorders caused by mutations in any of at least 12 different genes, many of which are involved in the epidermal synthesis of ω‐O‐acylceramides (acylCer). AcylCer are essential precursors of the corneocyte lipid envelope crosslinked by transglutaminase‐1 (TGm‐1), or a yet unidentified enzyme, for normal skin barrier formation. We hypothesized that inactivating TGM1 mutations will lead to a compensatory overexpression of the transcripts involved in skin barrier repair, including many other ARCI‐causing genes. Using microarray, we examined the global mRNA expression profile in skin biopsies from five ARCI patients with TGM1 mutations and four healthy controls. There were a total of 599 significantly differentially expressed genes (adjusted P < 0.05), out of which 272 showed more than 1.5 log2fold‐change (FC) up‐ or down‐regulation. Functional classification of the latter group of transcripts showed enrichment of mRNA encoding proteins mainly associated with biological pathways involved in keratinocyte differentiation and immune response. Moreover, the expression of seven out of twelve ARCI‐causing genes was significantly increased (FC = 0.98‐2.05). Also, many of the genes involved in keratinocyte differentiation (cornified envelope formation) and immune response (antimicrobial peptides and proinflammatory cytokines) were upregulated. The results from the microarray analysis were also verified for selected genes at the mRNA level by qPCR and at the protein level by semi‐quantitative immunofluorescence. The upregulation of these genes might reflect a compensatory induction of acylCer biosynthesis as a part of a global barrier repair response in the patient′s epidermis.

Transglutaminases in Monocytes and Macrophages.

Macrophages are key players in various inflammatory disorders and pathological conditions via phagocytosis and orchestrating immune responses. They are highly heterogeneous in terms of their phenotypes and functions by adaptation to different organs and tissue environments. Upon damage or infection, monocytes are rapidly recruited to tissues and differentiate into macrophages. Transglutaminases (TGs) are a family of structurally and functionally related enzymes with Ca2+-dependent transamidation and deamidation activity. Numerous studies have shown that TGs, particularly TG2 and Factor XIII-A, are extensively involved in monocyte- and macrophage-mediated physiological and pathological processes. In the present review, we outline the current knowledge of the role of TGs in the adhesion and extravasation of monocytes, the expression of TGs during macrophage differentiation, and the regulation of TG2 expression by various pro- and anti-inflammatory mediators in macrophages. Furthermore, we summarize the role of TGs in macrophage phagocytosis and the understanding of the mechanisms involved. Finally, we review the roles of TGs in tissue-specific macrophages, including monocytes/macrophages in vasculature, alveolar and interstitial macrophages in lung, microglia and infiltrated monocytes/macrophages in central nervous system, and osteoclasts in bone. Based on the studies in this review, we conclude that monocyte- and macrophage-derived TGs are involved in inflammatory processes in these organs. However, more in vivo studies and clinical studies during different stages of these processes are required to determine the accurate roles of TGs, their substrates, and the mechanisms-of-action.

Biocatalysis by Transglutaminases: A Review of Biotechnological Applications.

The biocatalytic activity of transglutaminases (TGs) leads to the synthesis of new covalent isopeptide bonds (crosslinks) between peptide-bound glutamine and lysine residues, but also the transamidation of primary amines to glutamine residues, which ultimately can result into protein polymerisation. Operating with a cysteine/histidine/aspartic acid (Cys/His/Asp) catalytic triad, TGs induce the post-translational modification of proteins at both physiological and pathological conditions (e.g., accumulation of matrices in tissue fibrosis). Because of the disparate biotechnological applications, this large family of protein-remodelling enzymes have stimulated an escalation of interest. In the past 50 years, both mammalian and microbial TGs polymerising activity has been exploited in the food industry for the improvement of aliments’ quality, texture, and nutritive value, other than to enhance the food appearance and increased marketability. At the same time, the ability of TGs to crosslink extracellular matrix proteins, like collagen, as well as synthetic biopolymers, has led to multiple applications in biomedicine, such as the production of biocompatible scaffolds and hydrogels for tissue engineering and drug delivery, or DNA-protein bio-conjugation and antibody functionalisation. Here, we summarise the most recent advances in the field, focusing on the utilisation of TGs-mediated protein multimerisation in biotechnological and bioengineering applications.

Transglutaminase activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics.

Osteoclasts, bone resorbing cells, derive from monocyte/macrophage cell lineage. Increased osteoclast activity is responsible for bone destruction in diseases such as osteoporosis, periodontitis and rheumatoid arthritis. Transglutaminases (TGs), protein crosslinking enzymes, were recently found involved in osteoclastogenesis in vivo, however their mechanisms of action have remained unknown. In this study, we have investigated the role of TG activity in osteoclastogenesis in vitro using four TG inhibitors, NC9, Z006, T101, and monodansyl cadaverine. Our results showed that all TG inhibitors were capable of blocking the entire osteoclastogenesis process. The most potent of the inhibitors, NC9 when added to cultures at different phases of osteoclastogenesis, inhibited differentiation, migration, and fusion of pre-osteoclasts as well as resorption activity of mature osteoclasts. Further investigation into the mechanisms revealed that NC9 increased RhoA levels and blocked podosome belt formation suggesting that TG activity regulates actin dynamics in pre-osteoclasts. The inhibitory effect of NC9 on osteoclastogenesis as well as podosome belt formation was completely reversed with a Rho-family inhibitor Exoenzyme C3. Microtubule architecture, acetylation, and detyrosination of α-tubulin were not affected. Finally, we demonstrated that macrophages and osteoclasts expressed mRNA of three TGs:TG1, TG2, and Factor XIII-A which were all differentially regulated in these cells during differentiation. Immunofluoresence microscopic analysis showed that all three enzymes co-localized to podosomes in osteoclasts. Taken together, our data suggests that TG activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics and that this may involve contribution from all three TG enzymes.

Improvement of skin barrier dysfunction by Scutellaria baicalensis GEOGI extracts through lactic acid fermentation

The development of an alternative medicine to treat atopic dermatitis (AD) from natural sources is necessary. To improve skin barrier dysfunction by enhancing the differentiation of human keratinocytes with the fermented Scutellaria baicalensis. Scutellaria baicalensis was fermented with Lactobacillus plantarum and extracted with 70% ethanol (FE). Antioxidant activities and the regulation of the gene expression related to keratinocyte differentiation were measured as well as its proliferation. This work first proved that the FE had multiple activities, both increasing keratinocyte differentiation and proliferation: The FE greatly up-regulated expression of the genes of keratinocyte differentiation such as involucrin, keratin 10, and transglutaminase-1 (TG-1) up to 4.06-fold, which was 3 times higher than the 2 other extracts. The effect of baicalein on keratinocyte differentiation was also first found; however, its efficacy was lower than that of the fermented extract. The FE proved to effectively accelerate keratinocyte differentiation, rather than to initiate the differentiation, and also showed an ability of stimulating keratinocyte proliferation up to 2.8 × 106 viable cells/mL as well as 70.24 ng/mL of collagen production in fibroblasts. High efficacy of the FE was confirmed by synergistic effects of large amounts of various bioactive substances in the extracts as baicalein alone did not show remarkable effects and even positive controls had not much better activities than the FE. The fermented extract was able to improve skin barrier dysfunction, and the ointment with 1%-5% (v/v) of the extract be directly used for skin clinical trials to treat AD.

Transglutaminases in autoimmune and inherited skin diseases: The phenomena of epitope spreading and functional compensation.

Transglutaminases (TGs) are structurally and functionally related enzymes that modify the post-translational structure and activity of proteins or peptides, and thus are able to turn on or switch off their function. Depending on location and activities, TGs are able to modify the signalling, the function and the fate of cells and extracellular connective tissues. Besides mouse models, human diseases enable us to appreciate the function of various TGs. In this study, skin diseases induced by genetic damages or autoimmune targeting of these enzymes will be discussed. TG1, TG3 and TG5 contribute to the cutaneous barrier and thus to the integrity and function of epidermis. TGM1 mutations related to autosomal recessive ichthyosis subtypes, TGM5 mutations to a mild epidermolysis bullosa phenotype and as novelty TGM3 mutation to uncombable hair syndrome will be discussed. Autoimmunity to TG2, TG3 and TG6 may develop in a few of those genetically determined individuals who lost tolerance to gluten, and manifest as coeliac disease, dermatitis herpetiformis or gluten-dependent neurological symptoms, respectively. These gluten responder diseases commonly occur in combination. In autoimmune diseases, the epitope spreading is remarkable, while in some inherited pathologies, a unique compensation of the lost enzyme function is noted.

Transglutaminase in Receptor and Neurotransmitter-Regulated Functions.

Transglutaminases (TGs) and especially TG2 play important roles in neurotransmitter and receptor signaling pathways. Three different mechanisms by which TG2 interacts with neurotransmitter and receptor signaling systems will be discussed in this review. The first way in which TG2 interacts with receptor signaling is via its function as a guanine nucleotide binding protein (G-protein) coupling to G-protein coupled receptors (GPCRs) to activate down-stream signaling pathways. TG2 can exist in a least two conformations, a closed GTP-bound conformation and an open calcium-bound conformation. In the closed GTP-bound conformation, TG2 is capable of functioning as a G-protein for GPCRs. In the open calcium-bound conformation, TG2 catalyzes a transamidation reaction cross-linking proteins or catalyzing the covalent binding of a mono- or polyamine to a protein. The second mechanism is regulation of the transamidation reaction catalyzed by TG2 via receptor stimulation which can increase local calcium concentrations and thereby increase transamidation reactions. The third way in which TG2 plays a role in neurotransmitter and receptor signaling systems is via its use of monoamine neurotransmitters as a substrate. Monoamine neurotransmitters including serotonin can be substrates for transamidation to a protein often a small G-protein (also known as a small GTPase) resulting in activation of the small G-protein. The transamidation of a monoamine neurotransmitter or serotonin has been designated as monoaminylation or more specifically serotonylation, respectively. Other proteins are also targets for monoaminylation such as fibronectin and cytoskeletal proteins. These receptor and neurotransmitter-regulated reactions by TG2 play roles in physiological and key pathophysiological processes.

Phenotypic diversity of the recurrent p.Val379Leu missense mutation of the TGM1 gene

Abstract Autosomal recessive congenital ichthyosis type 1 (ARCI1), a clinically heterogeneous group of keratinization disorders, develops due to mutations in the transglutaminase 1 (TGM1) gene. Here we report a Hungarian pedigree affected by the lamellar ichthyosis clinical form of the ARCI1 phenotype. Direct sequencing revealed two recurrent heterozygous mutations: a splice site (c.877-2A > G) and a missense (c.1135G > C, p.Val379Leu) mutation. This splice site mutation is the most frequently observed in ARCI1 worldwide. The missense mutation is relatively rare and has been reported in only 13 Scandinavian patients. Comparison of the clinical phenotypes of our Hungarian patients and the Scandinavian patients demonstrates great phenotypic diversity associated with the p.Val379Leu genotype.

Relationship between the serum level of transglutaminase 2-specific IgE and disease condition in patients with atopic dermatitis

Objective To detect the serum level of transglutaminase 2 (TG2) -specific IgE (sIgE) in patients with atopic dermatitis (AD) , and to analyze its correlation with the disease condition. Methods A total of 77 patients with AD were enrolled into this study, including 44 patients aged ≥ 12 years and 33 patients aged < 12 years. Of the 77 patients, 20 were diagnosed with intrinsic AD, which was characterized by the absence of sIgE and total serum IgE values < 150 kU/L, and 49 with extrinsic AD characterized by positive (++) or even strongly positive sIgE for more than 1 kind of exogenous allergens, or total serum IgE values ≥ 150 kU/L. Immunocapture-biotinylated detector enzyme immunoassay was performed to detect the serum level of TG2-sIgE in 77 patients with AD, 40 adult patients with psoriasis vulgaris (PV) and 30 healthy adult controls. Clinical data on the AD patients were recorded, including age, disease duration, SCORAD score, blood eosinophil count, levels of total IgE and TG2-sIgE. Results The serum levels of TG2-sIgE in AD patients aged ≥ 12 years, AD patients aged 0.05) . Conclusion The serum level of TG2-sIgE obviously increases in AD patients, so TG2 may be one kind of autoantigen in AD patients, but there is no significant correlation between the TG2-sIgE level and AD severity. Key words: Dermatitis, atopic; Transglutaminases; Immunoglobulin E; Autoantigens; Severity of illness index

Al mena: a comprehensive resource of human genetic variants integrating genomes and exomes from Arab, Middle Eastern and North African populations.

Middle East and North Africa (MENA) encompass very unique populations, with a rich history and encompasses characteristic ethnic, linguistic and genetic diversity. The genetic diversity of MENA region has been largely unknown. The recent availability of whole-exome and whole-genome sequences from the region has made it possible to collect population-specific allele frequencies. The integration of data sets from this region would provide insights into the landscape of genetic variants in this region. We integrated genetic variants from multiple data sets systematically, available from this region to create a compendium of over 26 million genetic variations. The variants were systematically annotated and their allele frequencies in the data sets were computed and available as a web interface which enables quick query. As a proof of principle for application of the compendium for genetic epidemiology, we analyzed the allele frequencies for variants in transglutaminase 1 (TGM1) gene, associated with autosomal recessive lamellar ichthyosis. Our analysis revealed that the carrier frequency of selected variants differed widely with significant interethnic differences. To the best of our knowledge, al mena is the first and most comprehensive repertoire of genetic variations from the Arab, Middle Eastern and North African region. We hope al mena would accelerate Precision Medicine in the region.

Calcium‐dependent activation of transglutaminase 2 by nanosecond pulsed electric fields

Exposure of cultured human cells to nanosecond pulsed electric fields (nsPEFs) elicits various cellular events, including Ca2+ influx and cell death. Recently, nsPEFs have been regarded as a novel physical treatment useful for biology and medicine, but the underlying mechanism of action remains to be fully elucidated. In this study, we investigated the effect of nsPEFs on transglutaminases (TGs), enzymes that catalyze covalent protein modifications such as protein–protein crosslinking. Cellular TG activity was monitored by conjugation of cellular proteins with biotin‐cadaverine, a cell‐permeable pseudosubstrate for TGs. We applied nsPEFs to HeLa S3 cells and found that overall catalytic activity of cellular TGs was greatly increased in a Ca2+‐dependent manner. The Ca2+ ionophore ionomycin significantly augmented nsPEF‐induced TG activation, further supporting the importance of Ca2+. Among human TG family members, TG2 is known to be the most ubiquitously expressed, and its catalytic activity requires elevated intracellular Ca2+. Given the requirement of Ca2+ for TG activation by nsPEFs, we performed depletion of TG2 by RNA interference (RNAi). We observed that TG2 RNAi suppressed the nsPEF‐induced TG activation and partially alleviated the cytotoxic effects of nsPEFs. These findings demonstrate that TG2 activation is a Ca2+‐dependent event in nsPEF‐exposed cells and exerts negative effects on cell physiology.

Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca2+ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes.

Interferon Regulatory Factor 6 Promotes Keratinocyte Differentiation in Response to Porphyromonas gingivalis.

We recently demonstrated that the expression of the interferon regulatory factor 6 (IRF6) transcription factor in oral keratinocytes was stimulated by the periodontal pathogen Porphyromonas gingivalis Here, we have established that IRF6 promotes the differentiation of oral keratinocytes in response to P. gingivalis This was evidenced by the IRF6-dependent upregulation of specific markers of keratinocyte terminal differentiation (e.g., involucrin [IVL] and keratin 13 [KRT13]), together with additional transcriptional regulators of keratinocyte differentiation, including Grainyhead-like 3 (GRHL3) and Ovo-like zinc finger 1 (OVOL1). We have previously established that the transactivator function of IRF6 is activated by receptor-interacting protein kinase 4 (RIPK4). Consistently, the silencing of RIPK4 inhibited the stimulation of IVL, KRT13, GRHL3, and OVOL1 gene expression. IRF6 was shown to also regulate the stimulation of transglutaminase-1 (TGM1) gene expression by P. gingivalis, as well as that of small proline-rich proteins (e.g., SPRR1), which are covalently cross-linked by TGM1 to other proteins, including IVL, during cornification. The expression of the tight junction protein occludin (OCLN) was found to also be upregulated in an IRF6-dependent manner. IRF6 was demonstrated to be important for the barrier function of oral keratinocytes; specifically, silencing of IRF6 increased P. gingivalis-induced intercellular permeability and cell invasion. Taken together, our findings potentially position IRF6 as an important mediator of barrier defense against P. gingivalis.

Genomic variants reveal differential evolutionary constraints on human transglutaminases and point towards unrecognized significance of transglutaminase 2.

Transglutaminases (TGMs) catalyze Ca2+-dependent transamidation of proteins with specified roles in blood clotting (F13a) and in cornification (TGM1, TGM3). The ubiquitous TGM2 has well described enzymatic and non-enzymatic functions but in-spite of numerous studies its physiological function in humans has not been defined. We compared data on non-synonymous single nucleotide variations (nsSNVs) and loss-of-function variants on TGM1-7 and F13a from the Exome aggregation consortium dataset, and used computational and biochemical analysis to reveal the roles of damaging nsSNVs of TGM2. TGM2 and F13a display rarer damaging nsSNV sites than other TGMs and sequence of TGM2, F13a and TGM1 are evolutionary constrained. TGM2 nsSNVs are predicted to destabilize protein structure, influence Ca2+ and GTP regulation, and non-enzymatic interactions, but none coincide with conserved functional sites. We have experimentally characterized six TGM2 allelic variants detected so far in homozygous form, out of which only one, p.Arg222Gln, has decreased activities. Published exome sequencing data from various populations have not uncovered individuals with homozygous loss-of-function variants for TGM2, TGM3 and TGM7. Thus it can be concluded that human transglutaminases differ in harboring damaging variants and TGM2 is under purifying selection suggesting that it may have so far not revealed physiological functions.

Microglial endocytosis and transglutaminases

Microglial endocytosis and transglutaminases

ADAM17/EGFR axis promotes transglutaminase-dependent skin barrier formation through phospholipase C γ1 and protein kinase C pathways.

The vitally important skin barrier is formed by extensive cross-linking activity of transglutaminases (TGs) during terminal epidermal differentiation. We have previously shown that epidermal deficiency of a disintegrin and metalloproteinase 17 (ADAM17), the principal EGFR ligand sheddase, results in postnatal skin barrier defects in mice due to impeded TG activity. However, the mechanism by which ADAM17/EGFR signalling maintains TG activity during epidermal differentiation remains elusive. Here we demonstrate that ADAM17-dependent EGFR signalling promotes TG activity in keratinocytes committed to terminal differentiation by direct induction of TG1 expression. Restored TG1 expression of EGF-stimulated differentiated Adam17−/− keratinocytes was strongly repressed by inhibitors for PLCγ1 or protein kinase C (PKC) pathways, while treatment with the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate restored TG activity in the epidermis of keratinocyte-specific Adam17−/− (AD17ΔKC) mice. Further investigations emphasized the expression of PKCη, a mediator of TGM1 transcription, to be sensitive to EGFR activation. In agreement, topical skin application of cholesterol sulfate, an activator of PKCη, significantly improved TG activity in epidermis of AD17ΔKC mice. Our results suggest ADAM17/EGFR-driven PLCγ1 and PKC pathways as important promoters of TG1 expression during terminal keratinocyte differentiation. These findings may help to identify new therapeutic targets for inflammatory skin diseases related to epidermal barrier defects.

Characterization of TG2 and TG1-TG2 double knock-out mouse epidermis.

Transglutaminases (TGs) are a family of enzymes that catalyse the formation of isopeptide bonds between the γ-carboxamide groups of glutamine residues and the ε-amino groups of lysine residues leading to cross-linking reactions among proteins. Four members, TG1, TG2, TG3, and TG5, of the nine mammalian enzymes are expressed in the skin. TG1, TG3 and TG5 crosslinking properties are fundamental for cornified envelope assembly. In contrast, the role of TG2 in keratinization has never been studied at biochemical level in vivo. In this study, taking advantage of the TG2 knock-out (KO) and TG1 heterozygous mice, we generated and characterized the epidermis of TG1-TG2 double knock-out (DKO) mice. We performed morphological analysis of the epidermis and evaluation of the expression of differentiation markers. In addition, we performed analysis of the amino acid composition from isolated corneocytes. We found a significant change in amino acid composition in TG1KO cornified cell envelopes (CEs) while TG2KO amino acid composition was similar to wild-type CEs. Our results confirm a key role of TG1 in skin differentiation and CE assembly and demonstrate that TG2 is not essential for CE assembly and skin formation.

Tissue transglutaminase-1 promotes stemness and chemoresistance in gastric cancer cells by regulating Wnt/β-catenin signaling.

Gastric cancer is a common malignancy, and is one of the most frequent causes of cancer deaths worldwide. Recently, members of the transglutaminases (TGM) family, especially TGM2, have been implicated in the progression and drug resistance of cancers, but the function of TGM1 in cancer development has been largely overlooked. In this study, we demonstrate the roles of TGM1 in development of gastric cancer. We found that expression levels of TGM1 were upregulated in both gastric cancer tissues and cultured gastric cancer cells, and that TGM1 expression levels were correlated with patient survival. In cultured gastric cancer cells, loss of TGM1 expression inhibited cell proliferation and promoted apoptosis, as well increased gastric cancer cell sensitivity to chemotherapeutic drugs and reducing stemness. These results strongly supported the participation of TGM1 in the regulation of gastric cancer development. In addition, we found evidence that the mechanism of action of TGM1 in regulating gastric cancer cell might involve the Wnt signaling pathway, as loss of TGM1 expression in gastric cancer cells led to a significant suppression of Wnt signaling activities.

Expression of Transglutaminase in Foreskin of Children with Balanitis Xerotica Obliterans.

Balanitis xerotica obliterans (BXO) is a chronic inflammatory skin disorder of unclear etiology. The etiology and the exact molecular mechanisms underlying the disease are still unknown. The human transglutaminase (TG) family consists of several proteins with catalytic activity essential for biological processes. In the present research we investigated the transcript levels of three TGs in patients operated on for congenital phimosis without or with histologically confirmed BXO; Thirty children with acquired phimosis were enrolled. The removed foreskins were sent both for histological diagnosis and for quantitative real-time PCR to evaluate the transcript levels of keratinocyte (TG1), tissue (TG2), and epidermal (TG3) transglutaminase; We observed a decrease in TG1 and TG3 transcripts by about 70% (p < 0.001) in foreskins from patients with BXO (n = 15) in comparison with patients without BXO (n = 15) and an increase in TG2 mRNA levels by 2.9 folds (p < 0.001); Reduced expression of both TG1 and TG3 was associated with the altered structure of the foreskin in BXO and can be a consequence of damage to keratinocytes. Increased expression of TG2 can be the result of chronic inflammation. TG2 overexpression can play a pivotal role in triggering and maintaining the inflammatory response in BXO patients.

FRET-based detection of isozyme-specific activities of transglutaminases.

Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Expression studies on its three major members, FXIII, TG1, and TG2, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. We previously identified a highly reactive substrate peptide, including glutamine, for each isozyme from a phage display library and developed a method for detecting isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. Here, we describe genetically encoded Förster resonance energy transfer (FRET)-based probes composed of each fluorescence protein (Cerulean and EVenus) fused with substrate peptides. The probe pairs, designated as Trac-MTG (His-CerΔ11-LQ/EV-K-His) containing linker and substrate peptide sequence for microbial TG (MTG), increased the EVenus:Cerulean fluorescence intensity ratio by more than 1.5-fold. Furthermore, we demonstrated that Trac-TG1 (His-CerΔ11-K5) and Trac-TG2 (His-CerΔ11-T26) containing substrate peptide sequence for mammalian TGs successfully detected the isozyme-specific activity of TG1 and TG2, respectively. In this study, we developed a rapid and convenient experimental system for measuring the isozyme-specific activity of TGs. The application of these probes for analyses in cells and tissues will be helpful for elucidating the physiological and pathological functions of TGs.