Transglutaminases (TG) belong to a family of structurally and functionally related enzymes that catalyze Ca2+-dependent posttranslational modifications of proteins by introducing protein-protein cross-links, amine incorporation and site-specific deamidation (Lorand L et al. 2003).
In humans, nine members of the TG family (TG1-TG9) have been identified, eight of which are catalytically active. TG2 is the best-studied multifunctional member of the transglutaminase family and is unique among them in that it possesses enzymatic GTPase, protein disulfide isomerase and protein kinase activities in addition to being a transglutaminase (Fesus L et al. 2002). TG2 is expressed in almost all cell compartments such as the cytoplasm, mitochondria, recycling endosomes and the nucleus. It is also present on the cell surface and is secreted into the extracellular matrix via non-classical mechanisms.
The structure of TG2 contains four domains: an N-terminal β-sandwich domain, a catalytic core domain and two C-terminal β-barrel 1 and β-barrel 2 domains. The protein can exist in both a closed (in the presence of GTP) and an open active conformation when Ca2+ is bound to the enzyme (Pinkas DM et al. 2007). TG2 has a conserved 3D structure and catalytic triad shared by other members of the family, but also other unique protein sequences, very often intrinsically disordered regions and short linear motifs that make the protein an ideal protein-protein interaction partner.
These and many other non-enzymatic interactions have physiological functions that are carried out in different protein networks. This functional diversity also explains why TG2 acts as a mediator in so many human diseases.
Exome sequencing data from different populations failed to reveal individuals with homozygous loss-of-function variants for TG2, suggesting that TG2 is subject to purifying selection that does not even allow the emergence of heterozygous common variants (Thangaraju K et al. (2017).
Transglutaminase 2 in fibroproliferative diseases: Fibroproliferative diseases (e.g. pulmonary fibrosis, systemic sclerosis, liver cirrhosis and cardiovascular disease) are partly due to activation of fibroblast activity by TGM2 (Wynn TA 2007). Under healthy conditions, a regeneration program is initiated after injury involving activated T lymphocytes that produce profibrotic cytokines such as transforming growth factor (TGF)-β and interleukin (IL)-13, as well as activated B lymphocytes that produce IL-6. These cytokines activate both macrophages and fibroblasts. As a result, activated fibroblasts transform into alpha-SMA-expressing, collagen-producing myofibroblasts (Phillips RJ et al. 2004; Varga J et al. 2007).
TG2 can promote tissue fibrosis in several ways. First of all, TG2 and the production of active TGF-β are closely linked. TGF-β is secreted in latent form and is non-covalently linked to its cleaved propeptide, which is disulfide-linked to proteins of the TGF-β latent binding protein (LTBP) family that aid in its folding, secretion and localization and enable mechanical activation of the cytokine (Troilo H et al. 2016). The N-terminus of LTBPs has been shown to be a substrate for TG2. In addition, TG2 has been found to contribute to macrophage TGF-β activation and promote TGF-β1 transcription (Yen JHet al. (2015).
TG2 is not only associated with active TGF-β formation, but also has a profibrotic effect as it can cross-link various matrix proteins, making them more resistant to protein degradation (Colligham RJ et al. 2014; Sarang Z et al. 2005). TGF-β itself in turn promotes TG2 transcription, thus TGF-β drives TG2 expression, while TG2 contributes to TGF-β transcription, secretion and activation, leading to the formation of an additional layer of the self-amplification loop in the pathogenesis of fibrosis. Comment: It is suggested that inhibition of extracellular TG2 activity may be beneficial in the treatment of fibrotic diseases.
Transglutaminase 2 and neoplasia: Another group of diseases in which inhibition of TG2 may be beneficial is cancer. Versch. Studies suggest that cancer cells have elevated TG2 levels, and that elevated TG2 levels are associated with an aggressive cancer phenotype and drug resistance in most of these tumors (Huang L et al. 2015). Thus, an association between elevated TG2 levels and cancer aggressiveness has been found in colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, esophageal cancer, glioblastoma, malignant melanoma (Fok JYet al. 2006), among others. Although several mechanisms by which TG2 promotes cancer survival, tumor progression and invasion are known, many of these effects are attributed to extracellularly localized TG2.
As a protein cross-linking enzyme, TG2 can alter the structure and stability of the extracellular matrix (ECM) to support integrin-dependent ECM binding and migration of cancer cells. Extracellular TG2 can cross-link S100A4 and thus promote metastasis. TG2 also functions as an integrin coreceptor for the β1, β3, β4 and β5 integrins and facilitates integrin-mediated signaling pathways, some of which promote growth factor signaling, thereby promoting cell growth (Multhaupt HA et al. 2016). It appears that chronic inflammation, which is a strong predisposing factor for carcinogenesis, and hypoxia, which is characteristic of rapidly growing cancer cells, are the two main drivers leading to TG2 overexpression in cancer cells, as TG2 expression is directly regulated by pro-inflammatory cytokines activating NF-κB, TGF-β and hypoxia-activated HIFs.
Transglutaminase 2 in cardiovascular disease: Under normal circumstances, TG2 is widely expressed in macrophages, smooth muscle cells and endothelial cells. It has been reported to accumulate in atherosclerotic plaques (Haroon ZA et al. 2001) and to interact with atherosclerotic processes in various ways. For example, TG2 has been shown to activate the NFκB signaling pathway and promote inflammation by cross-linking the NFκB inhibitor IKB-α, leading to the expression of TNF-α and nitric oxide synthase. The promoter of TG2 contains an NFκB- and cytokine-responsive element that contributes to the formation of an activation loop in inflammatory macrophages. In this way, TG2 may facilitate the initial damage of endothelial cells by promoting the inflammatory response in macrophages.
TG2 and platelets: TG2 can also be detected in platelets (Lorand Let al. 1987). During platelet activation, platelets release the contents of their α-granules and dense bodies to promote blood clotting. Activated platelets bind several proteins released from the α-granules, including fibrinogen, von Willebrand factor, thrombospondin and fibronectin, all of which are substrates for TG2. Both the release of the contents of the α-granules and the binding of these procoagulants occur through TG2-mediated covalent binding of serotonin to proteins, known as serotonylation (Walther DJ et al. (2003).
Transglutaminase 2 in celiac disease and other gastroenterological diseases: In celiac disease, TG2 is a specific target of an autoimmune mechanism triggered by exogenous cereal peptides (glutens). In genetically predisposed individuals, the consumption of wheat, rye and barley leads to atrophy of the small intestinal villi, malabsorption and the formation of antibodies against TG2. The gluten peptides derived from these cereals are rich in glutamine and proline residues (especially those from the alcohol-soluble gliadin fraction of gluten) and are good substrates for the transamidating enzyme reaction catalyzed by TG2 (Qiao SWet al. 2009). Deamidation according to the pattern characteristic of TG2 (Q-X-P motifs) makes gliadin peptides more immunogenic, as they fit better into the HLA-DQ groove of antigen-presenting cells (Sollid LM et al. 2011). In this context, it is important to note that only HLA-DQ2.5, 2.2 and DQ8 can present gliadin peptides to T cells. In this respect, coeliac disease only occurs in people with these genetic alleles. This is the reason why coeliac disease is widespread in the Caucasian population, in the Arab population and in India. HLA-DQ2 and DQ8 molecules are rare in other African and Asian countries, where celiac disease occurs only in exceptional cases (Korponay-Szabô IR et al. 2015). HLA-DQ2 and DQ8 molecules require acidic residues at specific positions in order for peptides to dock and for effective binding of gliadin-specific T cells to occur. Due to the high content of proline residues, gliadin peptides are resistant to proteolytic enzymes of the gastrointestinal tract, so that longer peptides are transported through the absorbent epithelial layer and taken up by T cells (Shan L et al. 2002).
TG2 antibodies: Originally, celiac disease antibodies were detected using the indirect immunofluorescence method by incubating patient serum on normal tissue sections. The resulting binding patterns (referred to in the 20th century as endomysial [EMA], reticulin [ARA] or anti-jejunal antibodies [JEA]) are exclusively TG2-dependent and thus EMA, ARA and JEA represent celiac disease-specific TG2 autoantibodies against extracellular TG2 epitopes (Dahlbom I et al. 2010) .
TG2-specific antibodies are produced in all celiac disease patients (Simon-Vecsei Zet al. 2013). Although TG2 antibodies cannot be detected in the serum of up to 10 % of patients, TG2 autoantibodies bound in the tissue are also found in these patients and cause an inflammatory reaction. Extraintestinal manifestations of coeliac disease affect almost all organs, including the liver, heart, kidney, pancreas, skin, brain and placenta (Mogyorosy G et al. 2014). In this respect, celiac disease is now considered a systemic autoimmune disease (Husby S et al. 2012) and not just a malabsorptive bowel disease. In addition to TG2, some celiac disease antibodies can also be directed against TG3 (epidermal transglutaminase) or TG6 (in the brain) (Zone JJ et al. 2011; Hadjivassiliou M et al. 2006; Giersiepen K et al. 2012).
Gliadin-specific T cells are responsible as helpers for the activation of specific groups of B cells that produce antibodies against gliadin peptides and also against transglutaminase 2 (TG2). There may also be some molecular mimicry between gliadin peptides and TG2. In any case, the autoimmune response to TG2 only occurs in the presence of gliadin peptides and ceases when the patient is placed on a gluten-free diet. A gluten-free diet also leads to regression of all disease manifestations (if they are still reversible) and is an effective treatment for celiac disease and dermatitis herpetiformis.
A high concentration of circulating anti-TG2 antibodies (above 10 times the upper limit of detection in ELISA), which is also confirmed by a positive serum EMA result in patients with malabsorptive symptoms and HLA-DQ2 or DQ8 background, reliably predicts villous atrophy and can therefore be used as a substitute for histologic evaluation according to the new European diagnostic guidelines. A gluten-free diet leads to a decrease in antibodies. If they remain positive for more than 1-2 years under control, this indicates dietary errors, so that anti-TG2 measurements are also successfully used in daily practice as follow-up tests (Dahlbom I et al. 2010).
Transglutaminase 2 and other inflammatory diseases: TG2 antibodies can also be produced in other autoimmune or inflammatory diseases, but the use of epitopes and IgG subclasses differs from celiac disease. The clinical significance of these antibodies is uncertain.
TG2 is expressed by both apoptotic cells and macrophages. As part of the apoptosis-phagocytosis program, TG2 has an anti-inflammatory effect as it promotes apoptosis in apoptotic cells. The release of pro-inflammatory cell contents is prevented by TG2 cross-linking the proteins of the apoptotic cells (Piredda L et al. 1997). Furthermore, TG2 accelerates the appearance of phosphatidylserine on the cell surface, the most important recognition signal for macrophages. It has also been shown that TG2 expression is upregulated in synovial tissue and synovial fluid mononuclear cells of patients with gouty arthritis (Yen JH et al. 2015).
Psoriasis: Increased TG2 expression was also found in skin biopsies from patients with psoriasis, although no correlation was found between TG2 expression level and disease duration, disease stage and subtype of psoriasis (Su CC et al. (2017). TG2 from mast cells has been reported to be involved in the pathogenesis of chronic urticaria.
Multiple sclerosis: TG2 has also been implicated in the pathogenesis of experimental multiple sclerosis, rheumatoid arthritis.
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TG2 therapeutic approaches: TG2 is a potential therapeutic target. There are several strategies to downregulate TG2 activity. The classical approach is to inhibit the catalytic activity of the enzyme.
Based on their mechanism of action, TG2 inhibitors can be divided into three main groups:
- competitive amines
- reversible
and
The first TG2 inhibitors were amines, e.g. cadaverine derivatives and putrescine, which compete with biogenic amine or lysine donor protein substrates and prevent the formation of naturally occurring isopeptide cross-linking. Superficially, this group contains cystamine, a specific disulfide diamine, with multiple inhibitory mechanisms and side effects such as inhibition of caspase-3.
One group of reversible inhibitors are non-hydrolysable GTP analogues and mimics that stabilize the inactive, closed conformation of the enzyme. Small molecules have been described that target the GTP-binding pocket of TG2 and block its function (Keillor JWet al. 2016). To date, there is only one TG2 inhibitor in clinical trials (phase IIa). The positive results of this study are available. Zedira (Germany) has developed ZED1227 (Dr. Falk Pharma), a small pyridinone derivative, for the treatment of celiac disease that blocks TG2-mediated deamidation of gliadin peptides (Schuppan Det al. 2021).