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Transglutaminase 2: an enigmatic enzyme with diverse functions Laszlo Fesus and Mauro Piacentini Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca2+-dependent protein modifications. It acts as a G protein in transmembrane signalling 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 recent TG2 structure, can each be assigned distinct cellular functions, including the regulation of cytoskeleton, cell adhesion and cell death. Ablation of TG2 in mice results in impaired wound healing, autoimmunity and diabetes, reflecting the number and variety of TG2 functions. An important role for the enzyme in the pathogenesis of coeliac disease, fibrosis and neurodegenerative disorders has also been demonstrated, making TG2 an important therapeutic target.

signalling, turned out to be TG2 [5]. TG2 binds and hydrolyzes GTP with an affinity and catalytic rate similar to the α subunits of large heterotrimeric G proteins and small Ras-type G proteins. Gh/TG2 couples α1b- and α1d- adrenoreceptors, thromboxane and oxytocin receptors to phospholipase C (PLCδ1), mediating inositol phosphate production in response to agonist activation. The GDP/GTP-bound form cannot act as a transglutaminase. This inhibition is suspended by Ca2+, which serves as a switch between the two distinct functions [6].

Published online: 12 September 2002

Structural explanation of biochemical functions

The first transglutaminase was identified by Heinrich Waelsch more than 40 years ago as a liver enzyme incorporating amines into proteins [1,2]. This enzyme, transglutaminase 2 (TG2), was used to determine the catalytic mechanism of the transglutaminases, which involves an active site cysteine, the formation of an acyl-enzyme thioester intermediate between this cysteine and a polypeptide-bound glutamine and the reaction of the thioester intermediate with a suitable nucleophile [3]. Because of their similarities in the catalytic triad and reaction mechanism, transglutaminases (EC 2.3.2.13), papain (EC 3.4.22.2) and papain-like cysteine proteases are classified within the same superfamily in the Structural Classification of Proteins database (SCOP, http://scop.mrc-lmb.cam. ac.uk/scop/). In invertebrates, only a single transglutaminase gene has been found, whereas nine evolutionary related genes (encoding blood coagulation FXIIIa, TG1–7 and the inactive epb42), clustered on five different chromosomes, have evolved in vertebrates by successive duplications [4]. Human TG2 is a 76-kD protein, consisting of 686 amino acids. Laszlo Fesus* Dept of Biochemistry and Molecular Biology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, H-4012 Hungary. *e-mail: fesus@ indi.dote.hu Mauro Piacentini Dept of Biology, University of Rome ‘Tor Vergata’ and INMI-IRCCS ‘Lazzaro Spallanzani’, Rome, Italy.

TG2 is a multifunctional protein

TG2 ‘moonlights’ between several distinct biochemical functions at various cellular locations (for details see Fig. 1.) In addition to crosslinking, TG2 can modify proteins by amine incorporation and deamidation, and by acting as an isopeptidase in a Ca2+-dependent manner (Fig. 1). Furthermore, TG2 is externalized from cells, where it mediates the interaction of integrins with fibronectin and crosslinks proteins of the extracellular matrix (ECM). In 1994, a novel G protein (Gh), observed in rat liver plasma membrane as a mediator of transmembrane http://tibs.trends.com

The structure of TG2, crystallized in a dimer form in complex with GDP, has been reported recently [7]. Similar to another transglutaminase, FXIIIa [8], TG2 has four distinct domains (Fig. 2): an N-terminal β-sandwich (with fibronectin and integrin binding site), catalytic core (containing the catalytic triad for the acyl-transfer reaction and a conserved Trp essential for this catalytic activity [9]) and two C-terminal β-barrel domains (the second contains a phospholipase C binding sequence [10]). A unique guanidine nucleotide-binding site, which has not been found in any other protein, is located in a cleft between the catalytic core and the first β-barrel [7]; this sequence is coded by exon 10 of the TG2 gene, which has very poor sequence homology with the same exons in other TGs. Some GDP/GTP-interacting residues and those essential for GTP hydrolysis are situated in other domains (Fig. 2), as predicted by site-directed mutagenesis [11]. In the GDP-bound form of TG2, access to the transamidation active site is blocked by two loops, and the active site cysteine is hydrogen-bonded to a Tyr residue [7]. The structure of the Ca2+-bound form of TG2 is unresolved. A putative Ca2+-binding site, homologous to one demonstrated in FXIIIa [12], is distorted in the TG2 structure by the bound nucleotide [7]. Ca2+-binding at this site, or others [13], could weaken nucleotide binding, and consequent conformational changes – a process which involves substrate binding and related displacement of the hydrogen-bonded Tyr [14] – might make the active site accessible [15]. The recently solved three dimensional structure of TG3 has revealed that it has the same domain structure as TG2, and after binding Ca2+ a channel opens to expose two critical Trp residues that control access of substrates to the active site [16]; similar structural changes might occur in TG2. It has been suggested that the two

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O NH2-R

Gln

Glu

C, N, E

ar

m ya

P

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Pr o -Cys

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-b

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Incorporation of amines into proteins

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Lys

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Lys

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H

H2O

Gln

H2O

Glu

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NH3

H2 O

TG2

O Glu

C

N

H2O

Lys

Glu

Lys

Isopeptidase activity

H

Ca2+

Ca2+

Appearance on the cell surface

Fibronectin Promotion of cell–matrix interactions

TG2

TG2 GDP C

Integrins

GTP E Receptor stimulation

Oxytocin receptor, TPα thromboxane A2 receptor, α1B- and α1D adrenoreceptors

GDP VI VII I

III II

V IV

Transmembrane signaling PIP2

DAG

PLC-δ1 IP3

TG2 M Fig. 1. Biochemical activities of transglutaminase 2 (TG2). TG2 catalyzes Ca2+-dependent acyl-transfer reaction [3] between γ-carboxamide group of a specific protein-bound glutamine and either the ε-amino group of a distinct protein-bound lysine residue (covalent protein crosslinking; the principal in vivo activity) or primary amines such as polyamines and histamine. Water can replace amine donor substrates, leading to deamidation of the recognized glutamines. TG2, similar to factor XIIIa, has Ca2+-dependent isopeptidase activity and, at least under test tube conditions, can hydrolyse γ:ε isopeptides [55]. TG2 can be exposed on the external leaflet of the plasma membrane ([40,42] and references therein). The presence of TG2 outside the cell has been proposed to depend on its interaction with fibronectin and integrins [39,56,57]. TG2 binds and thereby activates phospholipase C following stimulation of several kinds of cell surface receptors; its endogenous GTPase activity ensures proper regulation of transmembrane signalling through these receptors ([11] and references therein). Functions of TG2 are performed in the cytosol (C), the nucleus (N), at the cell membrane (M) and in the extracellular space (E). Except for its isopeptidase activity, all other functions have been shown to occur in intact cells and/or tissues.

non-proline cis peptide bonds (one close to the active site cysteine) present in FXIIIa [17], TG2 [7] and TG3 [16] might be involved in the activation process [17]. http://tibs.trends.com

GTP Ti BS

Additional motifs of TG2 (Fig. 2) might be used to allow externalization without a signal peptide, interaction with integrins and fibronectin [18], and translocation to the nucleus [19] or perhaps other organelles. The function of TG2 in cells

How are the diverse biochemical activities of TG2 related to cellular functions? The in vivo expression pattern of the enzyme, its subcellular locations and identified substrates (Table 1) suggest multiple roles. There are cell types (e.g. endothelial and smooth muscle cells) which constitutively express TG2 at high levels [20], whereas in other cell types it is induced by distinct signalling pathways, targeting specific response elements in the regulatory region of the gene. Retinoic acid (RA), TGFβ, NF-κB and AP responsive sites and regions have been functionally identified – all are related to induction of cellular defence mechanisms and cellular maturation ([21] and references therein).

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W241 C277 H335 D358 N398 E447 R476 Y516 D400 E452 R478 273KY274 C336 387KY388 V479 M483

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R580 Y583

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Fig. 2. Functional elements and three-dimensional structure of the human tissue-type transglutaminase (TG2) [7]. (a) The four structural domains are indicated by arrows with amino acid positions (top). Exon boundaries of the gene encoding TG2 are indicated by arrowheads with numbers corresponding to the last amino acids of each exon-encoded region. Exon 10, which contains residues forming the GDP-binding site, is marked. Functional regions and amino acid positions indicated are as follows. FN, fibronectin binding site [18], 2AEELVLE7 (cyan); integrin-binding region, N-terminal 28-kDa fragment; BH3, BH3 motif of the Bcl-2 protein family, 200PKFLKNAGRDCSRR214 [34] (green); NLS1 and NLS2, nuclear localization signals predicted on the basis of homology to the NLS of the NS1 non-structural protein of influenza virus [19], 259DILRR263 and 597PKQKRK602 (grey); catalytic triad, Cys277-His335-Asp358 (blue) and active site residues W241, C336 and Y516 of transamidating activity (purple ball and stick representation) involved in transition state stabilization, forming an inhibitory H-bond with Cys277 and potential inhibition of activity by disulfide bonding of Cys277, respectively [7,9,14]; Ca2+, Ca2+-binding site predicted from the FXIIIa structure [12] (red); non-proline cis peptide bonds 273KY274, 387KY388 (yellow); GTP, GDP-binding and GTPase catalytic site residues (orange); PLCδ1, interaction site for phospholipase Cδ1 657LHMGLHKLVVNFESDK LKAVK677 (magenta). Putative sites are labelled by an asterisk. (b) Three-dimensional structure of TG2 in complex with GDP shown from directions facing the transamidating (left) and the GTPase (right) active sites based on the reported crystal structure [7]. Functional regions indicated in (a) are coloured correspondingly. N- and C-terminals are designated by N and C, respectively. The fibronectin binding site is not indicated because the revealed structure lacks the coordinates for the N-terminal amino acids, M1–L14. The catalytic triad and the surrounding residues of the transamidating active site, as well as amino acids contributing to the GTPase site, are shown in ball and stick representation. The bound GDP molecule is colored green. Clusters of Glu/Gln and Asp/Asn residues forming putative Ca2+-binding sites predicted on the basis of surface potentional analysis [13] are coloured red.

At membrane locations, the role of TG2 in transmitting signals from seven-transmembrane helix receptors to phospholipase C is clearly established [11]. Phospholipase C itself becomes active when its inhibition by GDP–TG2 is suspended after TG2 binds GTP [22]. Interaction of TG2 with specific molecules (e.g. with sphingosylphosphocholine [23]) might reduce the Ca2+ requirement for the http://tibs.trends.com

transglutaminase activity [24]. This activity is strongly influenced by nitric oxide: up to 15 of the 18 cysteine residues can be nitrosylated and denitrosylated in a Ca2+-dependent manner, inhibiting and activating the enzyme, respectively [25]. TG2 activated by Ca2+ interacts with and modifies major components of the cytoskeleton (Table 1). In response to RA treatment, TG2-dependent transamidation of RhoA results in the increased binding of RhoA GTPase to ROCK-2 protein kinase, autophosphorylation of ROCK-2 and phosphorylation of vimentin [26] which can lead to the formation of stress fibers and increased cell adhesion. These events are prevented by TG2 inhibition. TG2 can interact with β-tubulin and with microtubule-binding proteins [27] including tau, which can be crosslinked by the enzyme [28]. An intriguing aspect of TG2 function is its translocation to the nucleus under certain conditions [24] – presumably with the help of importin-α3 [19] – where it can function either as a G protein [29] or as a transamidase activated by nuclear Ca2+-signals to crosslink histones [30], retinoblastoma (Rb) [31] and SP1 (S. Kojima, pers. commun.) proteins (Table 1). This suggests that TG2 could have a direct role in chromatin modifications and/or gene expression regulation. TG2 is induced in cells undergoing apoptosis in vivo [32]. Its overexpression primes cells for suicide and inhibition of its expression by antisense strategy results in decreased cell death [33]. It has been reported recently that TG2 sensitizes cells for apoptosis by interacting with mitochondria [34], shifting them to a higher polarized state and altered redox status. This might provoke activation of transglutaminase crosslinking activity [24]. During the late stage of apoptosis, the massive increase of cytosolic Ca2+ determines the switch of TG2 to its crosslinking configuration in all subcellular compartments leading to extensive polymerization of intracellular proteins (including actin [35] and Rb [31]) and formation of detergent-insoluble structures [36]. These protein scaffolds stabilize the structure of the dying cell before its clearance by phagocytosis, limiting the release of harmful intracellular components and consequently inflammatory or autoimmune responses [37]. Under pathological conditions the death of cells expressing high amounts of TG2 can occur as a result of a ‘mummification’ event caused by extensive crosslinking of cytosolic proteins without signs of either apoptosis or necrosis [38]. TG2 on the cell surface and in the extracellular matrix

Integrin-bound TG2 on the cell surface provides a binding site for fibronectin, which TG2 binds with high affinity (Fig. 1). The TG2 binding site on integrins probably involves sequences outside the integrin ligand-binding pocket. It is estimated that, on the surface of different cell types, 5–40% of β1 integrins could be complexed with TG2 and that all

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Table 1. Intracellular localization of TG2 and examples of its protein a partners Subcellular compartment Cytosol Plasma membrane

Interacting proteins

Substrates

Receptors, PLCδ1 [11] Integrins [39, 40]

RhoA [26], DLK [58] LTGF-β [59] b Ankyrin , LC1 [60]

Cytoskeleton Microfilaments

Intermediate filaments Microtubules

Tubulin-β [27]

Nuclear

Importin α3 [19]

Actin [35], Myosin [61] b Spectrin , Thymosin β [62] b Troponin T , b b Keratin , Vimentin Neurofilaments [63] S100A7, S100A10, S100A11 [64]; Tau [28] b Histones pRB [31]

a

Abbreviations: DLK, dual leucine zipper-bearing kinase; LTGF, latent tumor growth factor; LC1, lipocortin; S100As, family of 10–14 kDa EF hand containing calcium-binding proteins; pRB, retinoblastoma protein; PLC, phospholipase. b For references see [65].

2

TG2 on the cell surface is present as 1:1 complexes with integrins [39]. The interaction of TG2 with integrins occurs primarily at the extracellular domains of integrin β subunits, does not require crosslinking activity [40] and facilitates adhesion, spreading and motility of cells [39,40]. The significance of TG2 function in the extracellular space goes beyond promotion of cell adhesion and spreading; it is directly involved in wound healing and angiogenesis [41]. TG2 is also involved in the assembly, remodelling and stabilization of the ECM in various tissues [42]. This is done by crosslinking fibronectin, fibrinogen/fibrin, von Willebrand factor, vitronectin, lipoprotein ‘a’, dermatane sulfate proteoglycans, collagen V, osteonectin, laminin, nidogen and osteopontin ([42] and references therein). Furthermore, the secreted enzyme contributes to the covalent modification and activation of several growth factors [43] including TGFβ [44], which promotes transcriptional regulation of ECM genes and of TG2 itself [45]. Consequences of TG2 deletion

Knowing the multifunctionality and unique cellular biochemistry of TG2, it came as a surprise to learn that homozygous deletion of TG2 does not result in an embryonic lethal phenotype [46,47]. The homozygous null animals are viable, of normal size and weight, and born with mendelian frequency. No obvious alterations have been observed in apoptosis, the structure of the ECM or heart function (in which the G protein activity of TG2 thought to be important). The most probable explanation for the lack of severe phenotypes is that other transglutaminases in mammalian tissues can compensate for the loss of TG2. However, the other mammalian transglutaminases do not bind GDP/GTP and, with the exception of FXIIIa, they have not been found on the cell surface. Therefore, alterations are expected http://tibs.trends.com

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in TG2–/– mice, especially under certain stresses and pathological conditions. In fact, decreased adherence of primary fibroblasts [47] and impaired wound healing related to altered cytoskeletal dynamics of fibroblasts [R. Graham, pers. commun.] have been observed in these mice, consistent with the suggested extra- and intra-cellular functions of TG2. The participation of TG2 in apoptosis could explain the findings that on increasing the frequency of cell death in the knock out mice, clearance of apoptotic cells by phagocytosis is defective in the thymus and the liver and inflammatory as well as autoimmune reactions develop (Z. Szondy, pers. commun.). TG2-deficient mice also show glucose intolerance and hyperglycaemia because of reduced insulin secretion, a phenomenon similar to a subtype of diabetes called MODY (for maturity-onset diabetes of the young)[48]. In humans, no definitive TG2 deficiency has been observed so far, and therefore the above findings clearly point to possible areas for future clinical investigation. Pathogenic role of the enzyme in disease

Coeliac disease is a malabsorbtion syndrome characterized by almost total atrophy of villi in the jejunum on exposure to dietary glutens. TG2 is involved in generating T cell stimulatory gluten peptides through deamidation of specific glutamines [49,50]. In HLA-DQ2 or HLA-DQ8 settings, the TG2-formed disease-triggering epitopes provoke a pathological immune response that destroys the jejunal epithelium. In parallel, a T-cell-mediated autoimmune response is initiated, producing IgA-type autoantibodies against TG2 [51], the detection of which has become a widely used diagnostic marker of coeliac disease. Dysregulation of the suggested functions of TG2 in various pathological settings might significantly contribute to the development of fibrosis in susceptible organs such as the lung, liver and kidney ([52] and references therein). Huntington disease (HD) is a neurodegenerative diseases caused by the expansion of CAG trinucleotide repeats in the gene encoding huntingtin (htt). This results in a large number of contiguous glutamine residues in the htt protein. Accumulations of ubiquitinated htt aggregate in the nucleus and progressive loss of neuronal cells is observed. One of the proposed mechanisms of htt aggregation is based on the action of TG2 because expanded polyglutamine repeats are excellent glutaminyl-donor substrates for TG2-catalyzed cross-linking ([24] and references therein). By crossing HD R6/1 transgenic mice with TG2–/– mice, a reduction in cell death was observed in R6/1/TG2–/– compared with TG2–/– mice, together with the potentiation of the formation of htt aggregates and significant improvement in both motor performance and survival [53], suggesting that the involvement of TG2 in the loss of neurons in HD is not related to the formation of htt aggregates.

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Acknowledgements This work was partially supported by grants (‘Apoptosis Mechanisms’ and ‘Apoclear’) from the European Community, by funds from the Hungarian National Research Fund (OTKA) and Hungarian Ministry of Health (ETT), from AIRC and Ricerca Corrente and Finalizzata from the Italian Ministry of Health. We thank Zoltan Nemes and Zsolt Keresztessy (University of Debrecen) for their helpful suggestions.

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Intron–exon swapping of TG2 mRNA in the brains of patients with Alzheimer disease results in the appearance of an alternatively spliced short form of TG2, which lacks GDP/GTP binding residues and thereby has increased crosslinking activity [54]. The latter has been connected to the formation of neurofibrillary tangles and death of neuronal cells [24]. Conclusion

TG2 is emerging as a well-characterized, multifunctional molecular player in various cellular processes, ranging from intracellular signalling to apoptosis and pathological conditions such as autoimmune and Huntington diseases. The evidence accumulated in recent years points to the enzyme having a general protective and stabilizing role in cells and tissues; in fact, in the absence of the enzyme mice show impaired wound healing, autoimmunity and diabetes. However, under pathological condition, the uncontrolled activation of TG2 can turn its

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protective function into a pathological one. In fibrogenesis, coeliac and Huntington disease, the enzyme contributes to the formation of fibrotic tissues with scars, creates stimulatory epitopes for the immune system and determines excessive cell death, respectively. Despite these major advance, basic issues remain to be elucidated. These include the intracellular regulation of TG2, both in its crosslinking and signalling configuration, the physiological significance of its nuclear function(s), and the mechanism of externalization. Another question is the extent to which its functions can be replaced by other transglutaminase(s). Considering the emerging possibility of TG2-based therapies – potentially made available by the detailed structural and functional characterization of the enzyme in recent years – for fibrosis, Huntington and coeliac diseases, it is easy to predict that tissue transglutaminase will make further headlines in the future.

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