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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.36, #4)

An overview of the first 50 years of transglutaminase research by Simone Beninati; Carlo M. Bergamini; Mauro Piacentini (pp. 591-598).

Transglutaminases and their substrates in biology and human diseases: 50 years of growing by Angelo Facchiano; Francesco Facchiano (pp. 599-614).

Transglutaminase is an enzyme able to play more than one enzymatic action, acting on a variety of different substrates. The growth of knowledge about the members of the enzyme transglutaminase’s family and its substrates since the last 50 years indicates a large interest and curiosity about this protein, whose function and structure was, but still is, an important object of research. On the other hand, the involvement in a number of human diseases together with the lack of knowledge about the biological functions played by some of the most studied members of this family, make this enzyme a fascinating field of study. The history of this enzyme and its substrates, whose cross-linking action was reported for the first time 50 years ago, suggests that an effort to increase knowledge and communication among researchers is required. To achieve this important result, 10 years ago an internet web page called worldwide happening around transglutaminase (WHAT) was created. Driven by these experiences, novel points-of-view to look at Transglutaminase and its substrates may be identified.

Keywords: TGase family; Enzyme substrate; G-protein; Crosslink reaction

Transdab wiki: the interactive transglutaminase substrate database on web 2.0 surface by Éva Csősz; Bertalan Meskó; László Fésüs (pp. 615-617).

TRANSDAB wiki is a database of transglutaminase substrate proteins. This wiki is designed to provide quality content of all the details (including synonyms, structures, references) about transglutaminase substrate proteins and interaction partners. Currently TRANSDAB contains 243 articles about substrate proteins for 6 transglutaminase types in a user-friendly, editable format. Our aim was to collect structural information about substrate proteins and this information is provided in form of images, videos and links. The scientific community is invited to edit the database and besides providing up-to-date information, this wiki should serve as a platform for valuable discussions.

Keywords: TG2; Transglutaminase; Kinase; Deamidase; Interaction partners; Wiki

Preferred substrate sequences for transglutaminase 2: screening using a phage-displayed peptide library by Kiyotaka Hitomi; Miyako Kitamura; Yoshiaki Sugimura (pp. 619-624).

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.

Keywords: Transglutaminase; Substrate; Phage-displayed peptide library; Immobilization

Some lessons from the tissue transglutaminase knockout mouse by Z. Sarang; B. Tóth; Z. Balajthy; K. Köröskényi; É. Garabuczi; L. Fésüs; Z. Szondy (pp. 625-631).

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca²⁺-dependent protein modifications. It acts as a G protein in transmembrane signaling and as a cell surface adhesion mediator, this distinguishes it from other members of the transglutaminase family. The sequence motifs and domains revealed in the TG2 structure, can each be assigned distinct cellular functions, including the regulation of cytoskeleton, cell adhesion, and cell death. Though many biological functions of the enzyme have already been described or proposed previously, studies of TG2 null mice by our laboratory during the past years revealed several novel in vivo roles of the protein. In this review we will discuss these novel roles in their biological context.

Keywords: Tissue transglutaminase; Knockout mice; Macrophage; Liver; Heart; Neutrophils

Unfolding studies of tissue transglutaminase by Carlo Cervellati; Lorella Franzoni; Monica Squerzanti; Carlo M. Bergamini; Francesco Spinozzi; Paolo Mariani; Vincenzo Lanzara; Alberto Spisni (pp. 633-641).

Activation of tissue transglutaminase by calcium involves a conformational change which allows exposition of the active site to the substrate via movements of domains 3 and 4 that lead to an increase of the inter-domain distance. The inhibitor GTP counteracts these changes. Here we investigate the possible existence of non-native conformational states still compatible with the enzyme activity produced by chemical and thermal perturbations. The results indicate that chemical denaturation is reversible at low guanidine concentrations but irreversible at high concentrations of guanidine. Indeed, at low guanidine concentrations tissue TG-ase exists in a non-native state which is still affected by the ligands as in the native form. In contrast, thermal unfolding is always irreversible, with aggregation and protein self-crosslinkage in the presence of calcium. DSC thermograms of the native protein in the absence of ligands consist of two partly overlapped transitions, which weaken in the presence of calcium and merge together and strengthen in the presence of GTP. Overall, the present work shows, for the first time, the reversible denaturation of a TG-ase isoenzyme and suggests the possibility that also in in vivo, the enzyme may acquire non-native conformations relevant to its patho-physiological functions.

Keywords: Tissue transglutaminase; Denaturation; Guanidine hydrochloride; Thermal unfolding; Differential scanning calorimetry; Ligands

Plant and animal transglutaminases: do similar functions imply similar structures? by Donatella Serafini-Fracassini; Massimiliano Della Mea; Gianluca Tasco; Rita Casadio; Stefano Del Duca (pp. 643-657).

In plants the post-translational modification of proteins by polyamines catalysed by transglutaminases has been studied since 1987; it was identified by the production of glutamyl-polyamine derivatives, biochemical features, recognition by animal antibodies and modification of typical animal substrates. Transglutaminases are widespread in all plant organs and cell compartments studied until now, chloroplast being the most studied. Substrates are: photosynthetic complexes and Rubisco in chloroplasts, cytoskeleton and cell wall proteins. Roles either specific of plants or in common with animals are related to photosynthesis, fertilisation, stresses, senescence and programmed cell death, showing that the catalytic function is conserved across the kingdoms. AtPng1p, the first plant transglutaminase sequenced shows undetectable sequence homology to the animal enzymes, except for the catalytic triad. It is, however, endowed with a calcium-dependent activity that allowed us to build a three-dimensional model adopting as a template the animal tranglutaminase 2.

Keywords: Transglutaminases; Polyamines; Chloroplast; Cytoskeleton; Programmed cell death; Protein modelling

Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications by R. J. Collighan; M. Griffin (pp. 659-670).

This review summarises the functions of the enzyme tissue transglutaminase (TG2) in the extracellular matrix (ECM) both as a matrix stabiliser through its protein cross-linking activity and as an important cell adhesion protein involved in cell survival. The contribution of extracellular TG2 to the pathology of important diseases such as cancer and fibrosis are discussed with a view to the potential importance of TG2 as a therapeutic target. The medical applications of TG2 are further expanded by detailing the use of transglutaminase cross-linking in the development of novel biocompatible biomaterials for use in soft and hard tissue repair.

Keywords: Tissue transglutaminase; TG2; Biomaterials; Extracellular matrix; Cross-linking

Novel interactions of TG2 with heparan sulfate proteoglycans: reflection on physiological implications by E. A. M. Verderio; A. Scarpellini; T. S. Johnson (pp. 671-677).

This mini-review brings together information from publications and recent conference proceedings that have shed light on the biological interaction between transglutaminase-2 and heparan sulphate proteoglycans. We subsequently draw hypotheses of possible implications in the wound healing process. There is a substantial overlap in the action of transglutaminase-2 and the heparan sulphate proteoglycan syndecan-4 in normal and abnormal wound repair. Our latest findings have identified syndecan-4 as a possible binding and signalling partner of fibronectin-bound TG2 and support the idea that transglutaminase-2 and syndecan-4 act in synergy.

Keywords: Transglutaminase-2; Heparan sulphate proteoglycans; Syndecan-4; Wound healing; Fibrosis

FXIIIA and TGF-β over-expression produces normal musculo-skeletal phenotype in TG2-/- mice by U. Tarantino; F. Oliva; G. Taurisano; A. Orlandi; V. Pietroni; E. Candi; G. Melino; N. Maffulli (pp. 679-684).

Transglutaminase (TGs) enzymes and proteins crosslinking have for long time been implicated in the formation of hard tissue development, matrix maturation and mineralization. Among the TGs family members, in the context of connective tissue formation, TG2 and Factor XIII are expressed in cartilage by hypertrophic chondrocytes. Here, we analyse the morphological consequences of TG2 deficiency, during the development of skeletal elements. When TG2 is absent, there are not gross abnormalities in the development of the skeletal system, probably from compensatory mechanisms resulting in increased expression of FXIIIA and TGF-ß 1. In vivo other TGs may be involved in promoting chondrocytes and osteoblast differentiation and matrix mineralisation

Keywords: Endochondral ossification; Transglutaminase

A role for anti-transglutaminase 2 autoantibodies in the pathogenesis of coeliac disease? by K. Lindfors; K. Kaukinen; M. Mäki (pp. 685-691).

Coeliac disease is an autoimmune-mediated disorder with both innate and adaptive immune components. The disease is triggered by dietary gluten, which provokes the development of a massive immune reaction leading to the destruction of the small-intestinal mucosal morphology and intestinal dysfunction. Besides the typical small-bowel symptoms extraintestinal manifestations may also arise in a subset of coeliac disease patients. In addition, gluten evokes the production of antibodies mainly targeting deamidated gluten peptides or transglutaminase 2. Although coeliac disease has traditionally been regarded as a T cell-mediated disorder, this review discusses the role of the gluten-induced disease-specific anti-transglutaminase 2-autoantibodies in the pathogenesis of the disease.

Keywords: Coeliac disease; Transglutaminase 2; Autoantibodies; Pathogenesis

Tissue transglutaminase in celiac disease: role of autoantibodies by Ivana Caputo; Maria Vittoria Barone; Stefania Martucciello; Marilena Lepretti; Carla Esposito (pp. 693-699).

In celiac disease (CD), gluten, the disease-inducing toxic component in wheat, induces the secretion of IgA-class autoantibodies which target tissue transglutaminase (tTG). These autoantibodies are produced in the small-intestinal mucosa, and, during gluten consumption, they can also be detected in patients’ serum but disappear slowly from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The finding of IgA deposits on extracellular tTG in the liver, kidney, lymph nodes and muscles of patients with CD indicates that tTG is accessible to the gut-derived autoantibodies. Although the specific autoantibody response directed against tTG is very characteristic in celiac patients, their role in the immunopathology of the celiac mucosal lesion is a matter of debate. Here we report a brief summary of anti-tTG antibody effects demonstrating that these antibodies are functional and not mere bystanders in the disease pathogenesis. In fact, they inhibit intestinal epithelial cell differentiation, induce intestinal epithelial cell proliferation, increase epithelial permeability and activate monocytes and disturb angiogenesis.

Keywords: Celiac disease; Tissue transglutaminase; Autoantibodies; Autoimmunity

Protein–polyamine conjugates by transglutaminase 2 as potential markers for antineoplastic screening of natural compounds by A. Lentini; B. Provenzano; C. Tabolacci; S. Beninati (pp. 701-708).

The role of post-translational modification of cell proteins with polyamines, a reaction catalyzed by a tissue tranglutaminase (TG, EC 2.3.2.13), in the induction of cell differentiation, represents an intriguing strategy to control cell proliferation and metastatic ability of different tumor cell lines. In this review, we focus our attention on the metabolic aspects of some natural compounds (methylxantines, retinoids and flavonoids) responsible of their antitumor effects exerted through the induction of TG activity in cancer cells.

Keywords: Polyamines; Transglutaminase; Melanoma; Differentiation

Biological and therapeutic significance of tissue transglutaminase in pancreatic cancer by K. Mehta (pp. 709-716).

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest cancers world-wide with an estimated annual incidence and mortality rates of approximately 6,500 cases in the UK, over 40,000 cases in Europe, 19,000 cases in Japan and over 30,000 cases in the United States. Difficulty to diagnose the disease at an early stage, rapid progression and intrinsic resistance to currently available therapies are major factors that contribute to poor disease outcome in these patients (overall 5 years survival, <3%). Identification of cancer cell-encoded genes that contribute to the development of intrinsic resistance and metastatic spread of the PDA tumors, may yield immediate clinical benefits in terms of revealing new therapeutic targets for effective treatment of the disease. This article discusses the significance of tissue-type transglutaminase (TG2) whose expression is elevated in the majority of PDA tumors and cell lines. Based on the published data and the results discussed in this review, TG2 appears to be a promising target for containment and treatment of this formidable disease.

Keywords: Chemoresistance; Metastasis; Invasion; Autophagy; NF-κB; FAK; PTEN

In vivo evaluation of type 2 transglutaminase contribution to the metastasis formation in melanoma by G. Di Giacomo; A. Lentini; S. Beninati; M. Piacentini; C. Rodolfo (pp. 717-724).

One of the most relevant problems in tumour treatment resides on the ability of the tumour to form metastasis and disseminate among the organism. The formation of metastases is a complex process, which requires the action of various effectors, not yet completely identified. The analysis of various types of tumours revealed a complex picture about the relationship between type 2 transglutaminase (TG2) expression and outcome and/or metastatic potential of the tumour itself. In some tumours, the transition to a highly invasive state is paralleled by an up-regulation of TG2 expression and/or activity while in some other a down-regulation has been reported. In addition, host tissues seem to react to tumour invasion by up-regulating TG2 expression. In order to analyse whether TG2 might be involved in the metastatic process in melanoma, we studied the metastases formation and development by means of the B16-F10 murine melanoma cell line and with TG2^−/− mice as experimental model. Our results indicate that TG2 absence in the host is a favouring condition for the formation and development of the metastasis, while the presence of TG2 in the tumour’s cell might be requested for the development of the metastasis.

Keywords: Transglutaminase 2; Melanoma; B16-F10; Metastasis

Homocysteine-induced toxicity increases TG2 expression in Neuro2a cells by M. Currò; S. Condello; D. Caccamo; N. Ferlazzo; G. Parisi; R. Ientile (pp. 725-730).

High levels of homocysteine promote cell damage mainly through induction of oxidative stress, endoplasmic reticulum (ER) stress, and activation of pro-inflammatory factors. The effects of homocysteine were here examined in the continuously dividing neuroblastoma cell line Neuro2a. Cell treatment with homocysteine (100–500 μM) for 4 h increased ROS production while reducing cell viability in a dose-dependent manner. Cell exposure to 250 μM homocysteine was able to induce transglutaminase 2 up-regulation and increased in situ transglutaminase activity. These effects were prevented by the incubation with the transglutaminase activity inhibitor cystamine. Homocysteine also induced NF-κB activation that seemed associated with transglutaminase 2 up-regulation since the specific NF-κB inhibition by SN50 was able to reduce transglutaminase expression and activity levels. In the light of these observations, it may be postulated that TG2 up-regulation is involved in cell response to homocysteine-induced stress, in which NF-κB activation plays also a pivotal role.

Keywords: Homocysteine; Transglutaminases; NF-κB; Neuro2a cells; ROS

Role of transglutaminase 2 in quercetin-induced differentiation of B16-F10 murine melanoma cells by C. Forni; R. Braglia; A. Lentini; M. Nuccetelli; B. Provenzano; C. Tabolacci; S. Beninati (pp. 731-738).

Flavonoids belong to the class of plant polyphenolic compounds with over 6,000 individual structures known. These phytochemicals have attracted the interest of the scientists, because they possess a remarkable spectrum of biological activities, such as antiallergic, antiinflammatory, antioxidant, antimutagenic and anticarcinogenic. In this work, we compared the anticancer potential of two flavonoids, quercetin and pelargonidin, on highly metastatic B16-F10 melanoma murine cells. We have evaluated different parameters related to cell proliferation and differentiation, such as cell number, toxicity, intracellular content of polyamines and transglutaminase (TG, EC 2.3.2.13) activity. The higher inhibition of tumor cell growth, with respect to control, was obtained with quercetin cell treatment, i.e. 32% reduction after 48 h and 39% reduction after 72 h of incubation (P < 0.001). In parallel, quercetin-treated cells showed a similar decrease in polyamine content. TG activity was fourfold increased, with respect to control, after 48 h and twofold increased after 72 h (P < 0.001). Pelargonidin treatment did not show significant antiproliferative effects and any increase in TG activity. Proteomic approach was used to investigate changes in protein expression profiles in tumor cells following quercetin treatment. Changes in expression of 60 proteins were detected, i.e. 8 proteins were down-regulated, 35 up-regulated, 11 “de novo” synthetized proteins and 6 suppressed proteins were present in treated cells. A 80 kDa spot, identified as TG type 2 by Western blot analysis, presented a fourfold increase in intensity, confirming the key role played by TG in the induction of cancer cell differentiation.

Keywords: Melanoma; Quercetin; Pelargonidin; Transglutaminase; Polyamines; Proteomics

TIG3: a regulator of type I transglutaminase activity in epidermis by Richard L. Eckert; Michael T. Sturniolo; Ralph Jans; Catherine A. Kraft; Haibing Jiang; Ellen A. Rorke (pp. 739-746).

Keratinocytes undergo a process of terminal cell differentiation that results in the construction of a multilayered epithelium designed to produce a structure that functions to protect the body from dehydration, abrasion and infection. These protective properties are due to the production of a crosslinked layer of protein called the cornified envelope. Type I transglutaminase (TG1), an enzyme that catalyzes the formation of ε-(γ-glutamyl)lysine bonds, is the key protein responsible for generation of the crosslinks. The mechanisms that lead to activation of transglutaminase during terminal differentiation are not well understood. We have identified a protein that interacts with TG1 and regulates its activity. This protein, tazarotene-induced gene 3 (TIG3), is expressed in the differentiated layers of the epidermis and its expression is associated with transglutaminase activation and cornified envelope formation. We describe a novel mechanism whereby TIG3 regulates TG1 activity.

Keywords: Transglutaminase; TIG3; Keratinocyte differentiation; Calcium; RIG1; H-rev 107 tumor suppressor

Enhanced osteoblast adhesion on transglutaminase 2-crosslinked fibronectin by J. Forsprecher; Z. Wang; V. Nelea; M. T. Kaartinen (pp. 747-753).

Fibronectin (FN) is a cell adhesion protein that binds integrins in a process also involving the protein-crosslinking enzyme transglutaminase 2 (TG2) as a co-receptor. The cell-adhesive property of TG2 has been linked to a complex formation with FN and to its ability to crosslink and polymerize FN on the cell surface. We tested here the effects of extracellular FN, before and after in vitro crosslinking and polymerization by TG2, on MC3T3-E1 osteoblast adhesion. We show that TG2-mediated crosslinking creates large, compacted chain-like protein clusters that include both TG2 and FN molecules as analyzed by Western blotting and atomic force microscopy. Crosslinking of FN significantly promotes osteoblast adhesion as measured by crystal violet staining, and enhances β₁-integrin clustering on the cell surface as visualized by immunofluorescence microscopy. We hypothesize that TG2-mediated crosslinking enhances the cell-adhesive properties of FN by increasing the molecular rigidity of FN in the extracellular matrix.

Keywords: Transglutaminase 2; Protein crosslinking; Fibronectin; Osteoblast adhesion; Integrin clustering

Transglutaminase-2 differently regulates cartilage destruction and osteophyte formation in a surgical model of osteoarthritis by A. Orlandi; F. Oliva; G. Taurisano; E. Candi; A. Di Lascio; G. Melino; L. G. Spagnoli; U. Tarantino (pp. 755-763).

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodeling. Transglutaminases catalyze a calcium-dependent transamidation reaction that produces covalent cross-linking of available substrate glutamine residues and modifies the extracellular matrix. Increased transglutaminases-mediated activity is reported in osteoarthritis, but the relative contribution of transglutaminases-2 (TG2) is uncertain. We describe TG2 expression in human femoral osteoarthritis and in wild-type and homozygous TG2 knockout mice after surgically-induced knee joint instability. Increased TG2 levels were observed in human and wild-type murine osteoarthritic cartilage compared to the respective controls. Histomorphometrical but not X-ray investigation documented in osteoarthritic TG2 knockout mice reduced cartilage destruction and an increased osteophyte formation compared to wild-type mice. These differences were associated with increased TGFβ-1 expression. In addition to confirming its important role in osteoarthritis development, our results demonstrated that TG2 expression differently influences cartilage destruction and bone remodeling, suggesting new targeted TG2-related therapeutic strategies.

Keywords: Osteoarthritis; Transglutaminase-2; Cartilage; Osteophyte; TGF-β1

Molecular farming of human tissue transglutaminase in tobacco plants by Angela Sorrentino; Stefan Schillberg; Rainer Fischer; Raffaele Porta; Loredana Mariniello (pp. 765-772).

In this study we have utilized Nicotiana tabacum with a molecular farming purpose in attempt of producing transgenic plants expressing the human tissue transglutaminase (htTG). Three plant expression constructs were used enabling targeting and accumulation of the recombinant protein into the plant cell cytosol (cyto), the chloroplasts (chl) and the apoplastic space (apo). Analysis of transgenic T₀ plants revealed that recombinant htTG was detectable in all three transgenic lines and the accumulation levels were in a range of 18–75 μg/g of leaf material. In the T₁ generation, the recombinant htTG was still expressed at high level and a significant catalytic activity was detected into the leaf protein extracts. Southern blot analyses revealed that apo and chl plants of T₁ generation possess a high copy number of the recombinant htTG in their genome, while the cyto plants carry a single copy.

Keywords: Tissue transglutaminase; Bioreactor; Molecular farming; Nicotiana tabacum

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.36, #4)