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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #1)

Transglutaminases: future perspectives by S. Beninati; F. Facchiano; M. Piacentini (pp. 1-9).

This is the third special issue focused on “Transglutaminases” that is now available on this journal and dedicated to one of the pioneers of these enzymes, John Edward Folk, who died December 2010 [see in this issue Beninati et al. 2012a]. The first edition, “Polyamines and Transglutaminases” was published in Amino Acids, vol 26, no. 4, 2004, with the contribution of two prestigious Guest Editors as Alberto Abbruzzese and Mauro Piacentini. This editorial initiative was followed by the second special issue published in occasion of the 50th years of the discovery of transglutaminase. Indeed, “Transglutaminase 2: 50th Anniversary of the Discovery” Amino Acids, vol 36, no. 4, 2009, was published with the valuable collaboration of Carlo Maria Bergamini and Mauro Piacentini (Beninati et al. 2009). To continue with this editorial tradition, on this occasion, an outstanding board of Guest Editors composed by Francesco Facchiano and Mauro Piacentini has also been invited to promote this initiative and recruit a selected panel of Authors, many of who participated in the first and second edition of the Gordon Conference on Transglutaminases: “Transglutaminases in Human Diseases Processes” chaired by Rickard L Eckert and Kapil Mehta on July 18–23, 2010, and by Kapil Mehta and Mauro Piacentini on July 15–20, 2012, held at Davidson College, NC, USA. In this Amino Acids special issue, the manuscripts were selected to reflect the progress and the future perspectives of transglutaminases.

In memoriam: John E. Folk (1925–2010) by Simone Beninati; Myung Hee Park; Edith Wolff; László Fésüs; Alberto Abbruzzese; Soo II Chung; Franco Carmassi; Enzo Cocuzzi; Mary Lynn Trawick; Mauro Piacentini (pp. 11-18).

Transglutaminases and receptor tyrosine kinases by Manaswini Sivaramakrishnan; Gary K. Shooter; Zee Upton; Tristan I. Croll (pp. 19-24).

Transglutaminases are confounding enzymes which are known to play key roles in various cellular processes. In this paper, we aim to bring together several pieces of evidence from published research and literature that suggest a potentially vital role for transglutaminases in receptor tyrosine kinases (RTK) signalling. We cite literature that confirms and suggests the formation of integrin:RTK:transglutaminase complexes and explores the occurrence and functionality of these complexes in a large fraction of the RTK family.

Keywords: Transglutaminases; Integrins; Extracellular matrix; Receptor tyrosine kinases; Tissue transglutaminase; Fibronectin

Transglutaminases: key regulators of cancer metastasis by Alessandro Lentini; Alberto Abbruzzese; Bruno Provenzano; Claudio Tabolacci; Simone Beninati (pp. 25-32).

The ability to metastasize represents the most important characteristic of malignant tumors. The biological details of the metastatic process remain somewhat unknown, due to difficulties in studying tumor cell behaviour with high spatial and temporal resolution in vivo. Several lines of evidence involve transglutaminases (TGs) in the key stages of tumor progression cascade, even though the molecular mechanisms remain controversial. TG expression and activity display a different role in the primary tumor or in metastatic cells. In fact, TG expression is low in the primary tumor mass, but augmented when cells acquire the metastatic phenotype. Nevertheless, in other cases, the use of inducers of TG transamidating activity seems to contrast tumor cell plasticity, migration and invasion. In the following review, the function of TGs in cancer cell migration into the extracellular matrix, adhesion to the capillary endothelium and its basement membrane, invasion and angiogenesis is discussed.

Keywords: Polyamines; Transglutaminase; Invasion; Extracellular matrix

Expression of transglutaminase-2 isoforms in normal human tissues and cancer cell lines: dysregulation of alternative splicing in cancer by V. M. Phatak; S. M. Croft; S. G. Rameshaiah Setty; A. Scarpellini; D. C. Hughes; R. Rees; S. McArdle; E. A. M. Verderio (pp. 33-44).

The multiple enzymatic activities and functions of transglutaminase type 2 (TG2) may be attributed to alternative TG2 molecules produced by differential splicing of TG2 mRNA. Different RNA transcripts of the human TG2 gene (TGM2) have been identified, but the expression of TG2 multiple transcripts has never been systematically addressed. We have confirmed and rationalized the main TG2 variants and developed a screening assay for the detection of alternative splicing of TG2, based on real-time reverse-transcription PCR. We have quantified the multiple TG2 transcripts in a wide range of normal tissues and in cancer cell lines from four different sites of origin. Our data show a significant correlation in the expression of canonical and alternative TG2 isoforms in normal human tissue, but differences in alternative splicing of TG2 in cancer cell lines, suggesting that in cancer cells the alternative splicing of TG2 is a more active process.

Keywords: Transglutaminase-2; Alternative splicing; Splicing dysregulation; Normal tissue; Cancer; Cell lines

γ-Tocopherol inhibits human prostate cancer cell proliferation by up-regulation of transglutaminase 2 and down-regulation of cyclins by P. Torricelli; M. Caraglia; A. Abbruzzese; S. Beninati (pp. 45-51).

To establish a system to study differentiation therapy drugs, we used the androgen-independent human prostate PC-3 tumor cell line as a target and α- and γ-tocopherol as inducers. Effects of α- and γ-tocopherol on the cell cycle, proliferation and differentiation, were examined. A more significant growth inhibition activity for γ- than for α-tocopherol was observed. Flow cytometry analysis of α- and γ-tocopherol-treated prostate carcinoma PC3 cells showed decreased progression into the S-phase. This effect, particularly evident for γ-tocopherol, was associated with an up-regulation and increased activity of transglutaminase 2 (TG2), a reduced DNA synthesis and a remarkable decreased levels of cyclin D1 and cyclin E. Activation of TG2 suggests that γ-tocopherol has an evident differentiative capacity on PC3 cells, leading to an increased expression of TG2, and reduced cyclin D1 and cyclin E levels, affecting cell cycle progression. It is feasible that up-regulation and activation of TG2, associated with a reduced proliferation, are parts of a large-scale reprogramming that can attenuate the malignant phenotype of PC3 cells in vitro. These data suggest further investigation on the potential use of this γ-form of vitamin E as a differentiative agent, in combination with the common cytotoxic treatments for prostate cancer therapy.

Keywords: Human prostate cancer; Transglutaminase; Cyclins; Vitamin E

Tissue transglutaminase activity protects from cutaneous melanoma metastatic dissemination: an in vivo study by Francesco Facchiano; Daniela D’Arcangelo; Alessandro Lentini; Stefania Rossi; Cinzia Senatore; Tania Pannellini; Claudio Tabolacci; Angelo M. Facchiano; Antonio Facchiano; Simone Beninati (pp. 53-61).

The role of tissue transglutaminase (TG-2, TGase-2) in cancer development is still a fascinating field of research. The available reports do not elucidate fully its mechanism of action, due to the limitations of in vitro approaches. Therefore, to understand TG-2 role in cancer, we carried out an in vivo study with a more direct approach. TG-2 was in vivo overexpressed in a murine model of melanoma (intravenous injection of B16 melanoma cells in C57BL/6N mice) by means of a plasmid carrying the TG-2 cDNA. The evaluation of the frequency and size of the metastases indicated that the number of melanoma lung foci was more markedly reduced by TG-2 overexpression than the metastatic size. Then, TG-2 overexpressing mice showed a prolonged survival with respect to control mice. Further analyses were carried by means of proteomic analysis of melanoma cell lysates and meta-analysis of published transcriptomic datasets. Proteomic analysis of cell lysates from a human melanoma cell line compared to human keratinocytes showed significant differences in the expression of TG-2 substrates known to be involved in proliferation/differentiation and cancer progression. Taken together, these findings indicate a protective role of TG-2 enzymatic activity in melanoma progression in vivo.

Keywords: Cutaneous melanoma; Transglutaminase type 2; Microenvironment; Cell proteome; Bioinformatics

Tissue transglutaminase: a new target to reverse cancer drug resistance by Alfredo Budillon; Carmine Carbone; Elena Di Gennaro (pp. 63-72).

Cancer resistance mechanisms, which result from intrinsic genetic alterations of tumor cells or acquired genetic and epigenetic changes, limit the long-lasting benefits of anti-cancer treatments. Tissue transglutaminase (TG2) has emerged as a putative gene involved in tumor cell drug resistance and evasion of apoptosis. Although some reports have indicated that TG2 can suppress tumor growth and enhance the growth inhibitory effects of anti-tumor agents, several studies have presented both pro-survival and anti-apoptotic roles for TG2 in malignant cells. Increased TG2 expression has been found in several tumors, where it was considered a potential negative prognostic marker, and it is often associated with advanced stages of disease, metastatic spread and drug resistance. TG2 mediates drug resistance through the activation of survival pathways and the inhibition of apoptosis, but also by regulating extracellular matrix (ECM) formation, the epithelial-to-mesenchymal transition (EMT) or autophagy. Because TG2 knockdown or inhibition of TG2 enzymatic activity may reverse drug resistance and sensitize cancer cells to drug-induced apoptosis, many small molecules capable of blocking TG2 have recently been developed. Additional insight into the multifunctional nature of TG2 as well as translational studies concerning the correlation between TG2 expression, function or location and cancer behavior will aid in translating these findings into new therapeutic approaches for cancer patients.

Keywords: Tissue transglutaminase; Cancer; Chemotherapy; Resistance

Depletion of cathepsin D by transglutaminase 2 through protein cross-linking promotes cell survival by Se-Jin Kim; Kyung-Hee Kim; Eu-Ree Ahn; Byong Chul Yoo; Soo-Youl Kim (pp. 73-80).

Transglutaminase 2 (TGase 2) promotes nuclear factor-κB (NF-κB) activity through depletion of the inhibitory subunit of NF-κB (I-κBα) via protein cross-linking, leading to resolution of inflammation. Increased expression of TGase 2 contributes to inflammatory disease pathogenesis via constitutive NF-κB activation. Conversely, TGase 2 inhibition often reverses inflammation in animal models. The role of TGase 2 in apoptosis remains less clear, as both pro- and anti-apoptotic functions of TGase 2 have been demonstrated under different experimental conditions. Apoptosis is intact in a TGase 2 knock out mouse (TGase2^−/−), which is phenotypically normal. However, upon exposure to tumor necrosis factor (TNF)-α-induced apoptotic stress, mouse embryonic fibroblasts (MEFs) from TGase2^−/− mice were more sensitive to cell death than MEFs from wild-type (TGase 2^+/+) mice. In the current study, to explore the role of TGase 2 in apoptosis, TGase 2-binding proteins were identified by LC/MS. TGase 2 was found to associate with cathepsin D (CTSD). Binding of TGase 2 to CTSD resulted in the depletion of CTSD via cross-linking in vitro as well as in MEFs, leading to decreased levels of apoptosis. Furthermore, cytoplasmic CTSD levels were higher in MEFs from TGase 2^−/− mice than in those from TGase 2^+/+ mice, as were caspase 3 activation and poly (ADP-ribose) polymerase (PARP) processes. These results suggest that TGase 2, while not previously implicated as a major regulatory factor in apoptosis, may regulate the balance between cell survival and cell death through the modulation of CTSD levels.

Keywords: Transglutaminase 2; Apoptosis; Cathepsin D

Tissue transglutaminase, inflammation, and cancer: how intimate is the relationship? by Santosh Kumar; Kapil Mehta (pp. 81-88).

Despite significant advances in surgery and biology, cancer remains a major health problem. It is now well accepted that metastasis and cancer cells’ acquired or inherent resistance to conventional therapies are major roadblocks to successful treatment. Chronic inflammation is an important driving force that provides a favorable platform for cancer’s progression and development and suggests a link between inflammation and metastatic transformation. However, how chronic inflammation contributes to metastatic cell transformation is not well understood. According to the current theory of cancer progression, a small subpopulation of cancer stem cells (CSCs) in tumors is responsible for their metastasis, resistance, and sustenance. Whether CSCs originate from normal stem cells or from dedifferentiation of terminally differentiated cells remains unknown. Recent evidence indicates that stem cells are not unique; malignant or nonmalignant cells can reprogram and de-differentiate to acquire a stemness phenotype. Thus, phenotypic plasticity may exist between stem cells and non-stem cells, and a dynamic equilibrium may exist between the two phenotypes. Moreover, this equilibrium may shift in one direction or another on the basis of contextual signals in the microenvironment that influence the interconversion between stem and non-stem cell compartments. Whether the inflammatory microenvironment influences this interconversion and shifts the dynamic equilibrium towards stem cell compartments remains unknown. We recently found that aberrant tissue transglutaminase (TG2) expression induces the mesenchymal transition (EMT) and stem cell characteristics in epithelial cells. This finding, in conjunction with the observation that inflammatory signals (e.g., TGFβ, TNFα, and NF-κB) which induce EMT, also induce TG2 expression, suggests a possible link between TG2, inflammation, and cancer progression. In this review, we summarize TG2-driven processes in inflammation and their implications in cancer progression.

Keywords: Chemoresistance; Metastasis; Cancer stem cells; EMT; Inflammation; Transglutaminase 2

The role of TG2 in ECV304-related vasculogenic mimicry by Richard A. Jones; Zhuo Wang; Shakthi Dookie; Martin Griffin (pp. 89-101).

Tumour vasculogenesis can occur by a process referred to as vasculogenic mimicry, whereby the vascular structures are derived from the tumour itself. These tumours are highly aggressive and do not respond well to anti-angiogenic therapy. Here, we use the well characterised ECV304 cell line, now known as the bladder cancer epithelial cell line T24/83 which shows both epithelial and endothelial characteristics, as a model of in vitro vasculogenic mimicry. Using optimised ratios of co-cultures of ECV304 and C378 human fibroblasts, tubular structures were identifiable after 8 days. The tubular structures showed high levels of TG2 antigen and TG in situ activity. Tubular structures and in situ activity could be blocked either by site-directed irreversible inhibitors of TG2 or by silencing the ECV304 TG2 by antisense transfection. In situ activity for TG2 showed co-localisation with both fibronectin and collagen IV. Deposition of these proteins into the extracellular matrix could be reduced by inclusion of non-cell penetrating TG inhibitors when analysed by Western blotting suggesting that the contribution of TG2 to tube formation is extracellular. Incubation of ECV304 cells with these same irreversible inhibitors reduced cell migration which paralleled a loss in focal adhesion assembly, actin cytoskeleton formation and fibronectin deposition. TG2 appears essential for ECV304 tube formation, thus representing a potential novel therapeutic target in the inhibition of vasculogenic mimicry.

Keywords: Tissue transglutaminase; ECV304; Co-culture; Vasculogenic mimicry; Inhibitors

eIF5A isoforms and cancer: two brothers for two functions? by M. Caraglia; M. H. Park; E. C. Wolff; M. Marra; A. Abbruzzese (pp. 103-109).

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [N ^ε-(4-amino-2-hydroxybutyl)lysine]. The role of hypusine formation in the eIF5A protein in the regulation of cell proliferation and apoptosis is addressed in the present review. Moreover, vertebrates carry two genes that encode two eIF5A isoforms, eIF5A-1 and eIF5A-2, which, in humans, are 84% identical. However, the biological functions of these two isoforms may be significantly different. In fact, eIF5A-1 is demonstrable in most cells of different histogenesis, whereas eIF5A-2 protein is detectable only in certain human cancer cells or tissues, suggesting its role as a potential oncogene. In this review we focus our attention on the involvement of eIF5A-1 in the triggering of an apoptotic program and in the regulation of cell proliferation. In addition, the potential oncogenic role and prognostic significance of eIF5A-2 in the prediction of the survival of cancer patients is described. eIF5A-1 and/or the eIF5A-2 isoform may serve as a new molecular diagnostic or prognostic marker or as a molecular target for anti-cancer therapy.

Keywords: eIF5A isoform; Hypusine; Tissue transglutaminase; Cancer; Apoptosis; Prognostic markers; Hypusine synthesis inhibitors

Possible involvement of transglutaminase-catalyzed reactions in the physiopathology of neurodegenerative diseases by Antonio Martin; Alessandro Giuliano; Domenico Collaro; Giulia De Vivo; Carla Sedia; Enrica Serretiello; Vittorio Gentile (pp. 111-118).

Transglutaminases are ubiquitous enzymes, which catalyze post-translational modifications of proteins. Recently, transglutaminases and tranglutaminase-catalyzed post-translational modification of proteins have been shown to be involved in the molecular mechanisms responsible for several human diseases. Transglutaminase activity has been hypothesized to be involved also in the pathogenetic mechanisms responsible for human neurodegenerative diseases. Neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, supranuclear palsy, Huntington’s disease and other polyglutamine diseases, are characterized in part by aberrant cerebral transglutaminase activity and by increased cross-linked proteins in affected brains. In this review, we focus on the possible molecular mechanisms by which transglutaminase activity could be involved in the pathogenesis of neurodegenerative diseases, and on the possible therapeutic effects of selective transglutaminase inhibitors for the cure of patients with diseases characterized by aberrant transglutaminase activity.

Keywords: Transglutaminases; Post-translational modifications of proteins; Nervous System; Neurodegenerative diseases

Recent advances in the development of tissue transglutaminase (TG2) inhibitors by E. Badarau; R. J. Collighan; M. Griffin (pp. 119-127).

Tissue transglutaminase (TG2) is a Ca²⁺-dependent enzyme and probably the most ubiquitously expressed member of the mammalian transglutaminase family. TG2 plays a number of important roles in a variety of biological processes. Via its transamidating function, it is responsible for the cross-linking of proteins by forming isopeptide bonds between glutamine and lysine residues. Intracellularly, Ca²⁺ activation of the enzyme is normally tightly regulated by the binding of GTP. However, upregulated levels of TG2 are associated with many disease states like celiac sprue, certain types of cancer, fibrosis, cystic fibrosis, multiple sclerosis, Alzheimer’s, Huntington’s and Parkinson’s disease. Selective inhibitors for TG2 both cell penetrating and non-cell penetrating would therefore serve as novel therapeutic tools for the treatment of these disease states. Moreover, they would provide useful tools to fully elucidate the cellular mechanisms TG2 is involved in and help comprehend how the enzyme is regulated at the cellular level. The current paper is intended to give an update on the recently discovered classes of TG2 inhibitors along with their structure–activity relationships. The biological properties of these derivatives, in terms of both activity and selectivity, will also be reported in order to translate their potential for future therapeutic developments.

Keywords: Tissue transglutaminase; TG2; Inhibitors; Structure–activity relationships (SAR)

γ-Glutamylamines and neurodegenerative diseases by Thomas M. Jeitner; Kevin Battaile; Arthur J. L. Cooper (pp. 129-142).

Transglutaminases catalyze the formation of γ-glutamylamines utilizing glutamyl residues and amine-bearing compounds such as lysyl residues and polyamines. These γ-glutamylamines can be released from proteins by proteases in an intact form. The free γ-glutamylamines can be catabolized to 5-oxo-L-proline and the free amine by γ-glutamylamine cyclotransferase. Free γ-glutamylamines, however, accumulate in the CSF and affected areas of Huntington Disease brain. This observation suggests transglutaminase-derived γ-glutamylamines may play a more significant role in neurodegeneration than previously thought. The following monograph reviews the metabolism of γ-glutamylamines and examines the possibility that these species contribute to neurodegeneration.

Keywords: γ-Glutamylamine; Transglutaminase; Neurodegeneration; γ-Glutamylamine cyclotransferase

Probing the specificity of gamma-glutamylamine cyclotransferase: an enzyme involved in the metabolism of transglutaminase-catalyzed protein crosslinks by Todd E. Bowser; Mary Lynn Trawick (pp. 143-150).

γ-Glutamylamine cyclotransferase (gGACT) catalyzes the intramolecular cyclization of a variety of l-γ-glutamylamines producing 5-oxo-l-proline and free amines. Its substrate specificity implicates it in the downstream metabolism of transglutaminase products, and is distinct from that of γ-glutamyl cyclotransferase which acts on l-γ-glutamyl amino acids. To elucidate the mechanism by which gGACT distinguishes between l-γ-glutamylamine and amino acid substrates, the specificity of the rabbit kidney enzyme for the amide region of substrates was probed through the kinetic analysis of a series of l-γ-glutamylamines. The isodipeptide N ^ɛ-(l-γ-glutamyl)-l-lysine 1 was used as a reference. The kinetic constants of the l-γ-glutamyl derivative of n-butylamine 7, were nearly identical to those of 1. Introduction of a methyl or carboxylate group on the carbon adjacent to the side-chain amide nitrogen in l-γ-glutamylamine substrates resulted in a dramatic decrease in substrate properties for gGACT thus providing an explanation of why gGACT does not act on l-γ-glutamyl amino acids except for l-γ-glutamylglycine. Placement of substituents on carbons further removed from the side-chain amide nitrogen in l-γ-glutamylamines restored activity for gGACT, and l-γ-glutamylneohexylamine 19 had a higher specificity constant (k _cat /K _m) than 1. gGACT did not exhibit any stereospecificity in the amide region of l-γ-glutamylamine substrates. In addition, analogues (26–30) with heteroatom substitutions for the γ methylene position of the l-γ-glutamyl moiety were examined. Several thiocarbamoyl derivatives of l-cysteine (28–30) were excellent substrates for gGACT.

Keywords: Gamma-glutamylamine cyclotransferase; Gamma-glutamyl cyclotransferase; Transglutaminase; l-γ-glutamylamines; S-(n-butylcarbamyl)-l-cysteine; O-(n-butylcarbamyl)-l-serine

Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress by Daniela Caccamo; Salvatore Condello; Nadia Ferlazzo; Monica Currò; Martin Griffin; Riccardo Ientile (pp. 151-159).

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role.

Keywords: Transglutaminase 2; Excitotoxicity; Neuronal-like SH-SY5Y cells; Small heat shock proteins; Transglutaminase inhibitor R283

Transglutaminase 6: a protein associated with central nervous system development and motor function by Helen Thomas; Konrad Beck; Magdalena Adamczyk; Pascale Aeschlimann; Martin Langley; Radu C. Oita; Lars Thiebach; Martin Hils; Daniel Aeschlimann (pp. 161-177).

Transglutaminases (TG) form a family of enzymes that catalyse various post-translational modifications of glutamine residues in proteins and peptides including intra- and intermolecular isopeptide bond formation, esterification and deamidation. We have characterized a novel member of the mammalian TG family, TG6, which is expressed in a human carcinoma cell line with neuronal characteristics and in mouse brain. Besides full-length protein, alternative splicing results in a short variant lacking the second β-barrel domain in man and a variant with truncated β-sandwich domain in mouse. Biochemical data show that TG6 is allosterically regulated by Ca²⁺ and guanine nucleotides. Molecular modelling indicates that TG6 could have Ca²⁺ and GDP-binding sites related to those of TG3 and TG2, respectively. Localization of mRNA and protein in the mouse identified abundant expression of TG6 in the central nervous system. Analysis of its temporal and spatial pattern of induction in mouse development indicates an association with neurogenesis. Neuronal expression of TG6 was confirmed by double-labelling of mouse forebrain cells with cell type-specific markers. Induction of differentiation in mouse Neuro 2a cells with NGF or dibutyryl cAMP is associated with an upregulation of TG6 expression. Familial ataxia has recently been linked to mutations in the TGM6 gene. Autoantibodies to TG6 were identified in immune-mediated ataxia in patients with gluten sensitivity. These findings suggest a critical role for TG6 in cortical and cerebellar neurons.

Keywords: Transglutaminase 6; Sequence; Structural model; Regulation; Central nervous system; Ataxia

Tissue-specific responses to loss of transglutaminase 2 by Stephanie Deasey; Shobana Shanmugasundaram; Maria Nurminskaya (pp. 179-187).

Of the eight catalytic transglutaminases (TGs), transglutaminase 2 (TG2) has been the most comprehensively studied due to its ubiquitous expression in multiple cell types. Despite the observed critical role for this enzyme in multiple biological processes in vitro, TG2 knockout mouse models have shown no severe developmental phenotypes, suggesting compensation by other TGs. To begin characterization of the compensating mechanisms, we analyzed total transamidating activity and expression patterns of all catalytically active TGs in seven different tissues/organs from wild-type and TG2 knockout mice. Inhibitory analysis with TG2-specific inhibitor KCC-009 suggests that relative contribution of TG2 in total transamidating activity differs in various tissues. Accordingly, our data indicate tissue-specific mechanisms of compensation for the loss of TG2, including transcriptional compensation in heart and liver versus functional compensation in aorta, kidney and skeletal/cartiagenous tissues. On the contrary, no compensation has been detected in skeletal muscle, suggesting a limited role for the TG2-mediated transamidation in normal development of this tissue.

Keywords: Transglutaminase; Inhibitors; Cartilage; Heart; Aorta; Liver; Kidney; Muscle; Compensation

Divergent results induced by different types of septic shock in transglutaminase 2 knockout mice by Hyun Yoo; Eu-Ree Ahn; Se-Jin Kim; Seon-Hyeong Lee; Seung Hyun Oh; Soo-Youl Kim (pp. 189-197).

Acute sepsis can be induced by cytokines such as TNF-α and biological products such as LPS. All of these agents cause systemic inflammation, which is characterized by hemodynamic shock and liver toxicity. However, the outcomes of different septic shock models were totally opposite in transglutaminase 2 knockout (TGase 2^−/−) mice. The aim of our study was to clarify the role of TGase 2 in liver injury. Therefore, we explored the role of TGase 2 in liver damage using two different stress models: LPS-induced endotoxic shock and TNF-α/actinomycin D (ActD)-induced sepsis. TNF-α-dependent septic shock resulted in increased liver damage in TGase 2^−/− mice compared with wild-type (WT) mice, and was accompanied by increased levels of caspase 3 and cathepsin D (CTSD) in the damaged liver. Conversely, LPS-induced septic shock resulted in ablation of inflammatory endotoxic shock in TGase 2^−/− mice and decreased liver injury. We found that TGase 2 protected liver tissue from TNF-α-dependent septic shock by reducing the expression of caspase 3 and CTSD. However, TGase 2 differently participated in increased the hemodynamic shock in LPS-induced septic shock through macrophage activation rather than protecting direct liver damage. Therefore, these findings demonstrate that septic shock caused by different agents may induce different results in TGase 2^−/− mice depending on the primary target organs affected.

Keywords: Transglutaminase; Acute liver failure; TNF-α; Cathepsin D; Caspase 3

Transglutaminase 2 as a biomarker of osteoarthritis: an update by Umberto Tarantino; Amedeo Ferlosio; Gaetano Arcuri; Luigi Giusto Spagnoli; Augusto Orlandi (pp. 199-207).

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodelling. Under physiologic conditions, articular cartilage displays a stable chondrocyte phenotype, whereas in osteoarthritis a chondrocyte hypertrophy develops near the sites of cartilage surface damage and associates to the pathologic expression of type X collagen. Transglutaminases (TGs) include a family of Ca²⁺-dependent enzymes that catalyze the formation of γ-glutamyl cross-links. Their substrates include a variety of intracellular and extracellular macromolecular components. TGs are ubiquitously and abundantly expressed and implicated in a variety of physiopathological processes. TGs activity is modulated by inflammatory cytokines. TG2 (also known as tissue transglutaminase) mediates the hypertrophic differentiation of joint chondrocytes and interleukin-1-induced calcification. Histomorphometrical and biomolecular investigations document increased TG2 expression in human and experimental osteoarthritis. Consequently, the level of TG2 expression may represent an adjuvant additional marker to monitor tissue remodelling occurring in osteoarthritic joint tissue. Experimental induction of osteoarthritis in TG2 knockout mice is followed from reduced cartilage destruction and increased osteophyte formation compared to wild-type mice, suggesting a different influence on joint bone and cartilage remodelling. The capacity of transamidation by TG2 to regulate activation of latent TGF-β seems to have a potential impact on the regulation of inflammatory response in osteoarthritic tissues. Additional studies are needed to define TG2-regulated pathways that are differently modulated in osteoblasts and chondrocytes during osteoarthritis.

Keywords: Osteoarthritis; Transglutaminase 2; Joint tissue; Transforming growth factor-beta

Transglutaminase 2 and Factor XIII catalyze distinct substrates in differentiating osteoblastic cell line: utility of highly reactive substrate peptides by Kazuya Watanabe; Kanako Tsunoda; Miho Itoh; Mina Fukui; Hitoshi Mori; Kiyotaka Hitomi (pp. 209-214).

Differentiated osteoblastic cell line, MC3T3-E1 expresses transglutaminase 2 (TG2) and Factor XIII (FXIII). In previous studies, we identified isozyme-specific and highly reactive glutamine-donor substrate peptides (pepF11KA and pepT26) for each isozyme. Using these peptides, we compared the reaction products with lysine-donor substrates for each isozyme in differentiating MC3T3-E1 cells. By this analysis, distinct substrates for the activated TG2 and FXIII were detected in cultured cellular extract. Possible substrates that incorporated biotin-labeled peptides were further purified using streptavidin-affinity chromatography. Several isozyme-specific substrates were identified by mass spectrometry analysis of the purified fractions. These analyses also indicate the benefit of the substrate peptides for obtaining distinct substrates in a reaction mixture where two isozymes co-exist.

Keywords: Transglutaminase; Osteoblast; TG2; Factor XIII

Polymorphism of transglutaminase 2: unusually low frequency of genomic variants with deficient functions by Róbert Király; Endre Barta; László Fésüs (pp. 215-225).

Transglutaminase 2 (TG2) is a multifunctional member of an enzyme family: it modifies glutamine residues by cross-linking proteins and incorporating primary amines into them, has protein disulphide isomerase and protein kinase activities, mediates trans-membrane signal transduction and interactions between cell surface proteins and the extracellular matrix. These unusual multiple roles encoded into one polypeptide chain suggest that genomic variations in the TGM2 gene should be limited. Indeed, the available information in databases shows that unlike in the case of most other transglutaminases there are no common single nucleotide polymorphisms in exons of human TGM2. We collected data on and produced some of the rare genetic variants of TGM2 by site directed mutagenesis and found that some were less stable than the most abundant (wild type) enzyme variant and the majority had deficient transamidating activity. Further studies are required to clarify the pathologic significance of these rare TGM2 alleles in the human population.

Keywords: Human transglutaminase 2; Wild type sequence; SNPs; No common variants; Rare variants; Deficient activities

The side chain of glutamine 13 is the acyl-donor amino acid modified by type 2 transglutaminase in subunit T of the native rabbit skeletal muscle troponin complex by Monica Squerzanti; Carlo Cervellati; Blendi Ura; Carlo Mischiati; Piero Pucci; Stefano Annunziata; Carla Iannone; Rita Casadio; Carlo M. Bergamini; Carla Esposito (pp. 227-234).

Subunit T of the native muscle troponin complex is a recognised substrate of transglutaminase both in vitro and in situ with formation of isopeptide bonds. Using a proteomic approach, we have now determined the precise site of in vitro labelling of the protein. A preparation of troponin purified from ether powder from mixed rabbit skeletal muscles was employed as transglutaminase substrate. The only isoform TnT2F present in our preparation was recognised as acyl-substrate by human type 2 transglutaminase which specifically modified glutamine 13 in the N-terminal region. During the reaction, the troponin protein complex was polymerized. Results are discussed in relation to the structure of the troponin T subunit, in the light of the role of troponins in skeletal and cardiac muscle diseases, and to the rules governing glutamine side chain selection by tissue transglutaminase.

Keywords: Transglutaminase; Skeletal troponin T; Protein post-translational modification

Retinoids produced by macrophages engulfing apoptotic cells contribute to the appearance of transglutaminase 2 in apoptotic thymocytes by Éva Garabuczi; Beáta Kiss; Szabolcs Felszeghy; Gregory J. Tsay; László Fésüs; Zsuzsa Szondy (pp. 235-244).

Transglutaminase 2 (TG2) has been known for a long time to be associated with the in vivo apoptosis program of various cell types including T cells. Though the expression of the enzyme was strongly induced in mouse thymocytes following apoptosis induction in vivo, no significant induction of TG2 could be detected, when thymocytes were induced to die by the same stimuli in vitro indicating that signals arriving from the tissue environment are required for the in vivo induction of the enzyme in apoptotic thymocytes. Previous studies have shown that one of these signals is transforming growth factor-β (TGF-β) which is released by macrophages engulfing apoptotic cells. Besides TGF-β, the TG2 promoter contains retinoic acid response elements as well. Here we show that in vitro retinoic acids, or TGF-β and retinoic acids together can significantly enhance the TG2 mRNA expression in dying thymocytes, and the apoptotic signal contributes to the TG2 induction. Inhibition of retinoic acid synthesis either by alcohol or retinaldehyde dehydrogenases significantly attenuates the in vivo induction of TG2 following apoptosis induction indicating that retinoids indeed might contribute in vivo to the apoptosis-related TG2 expression. What is more, the in vivo apoptosis induction in the thymus is accompanied by an enhanced retinoid-dependent transcriptional activity due to the enhanced retinoid synthesis by macrophages engulfing apoptotic cells. Our data reveal a new crosstalk between macrophages and apoptotic cells, in which apoptotic cell uptake-induced retinoid synthesis in macrophages enhances TG2 expression in the dying thymocytes.

Keywords: Tissue transglutaminase; Macrophage; Thymocytes; Retinoid; Apoptosis

Identification of human salivary transglutaminases by Mileidys Perez Alea; Vincent Thomas; Guillaume Martin; Saïd El Alaoui (pp. 245-250).

Transglutaminases (TGs) expression and enzymatic activities in human saliva were investigated. Specific antibodies showed the co-existence of TG1, TG2, TG3 and TG4. TG2 and TG3 were found in native and multiple proteolytic forms. Our data indicate that TG1 and TG2 isoenzymes are highly active with the major activity attributed to TG1. These findings pave the way for future studies on the physiological role of TG in the oral cavity and the potential impact of their deregulation in TG-associated oral diseases.

Keywords: Transglutaminase; Saliva fluid; Transamidase activity; TG-specific assay

Anti-tissue transglutaminase antibodies activate intracellular tissue transglutaminase by modulating cytosolic Ca²⁺ homeostasis by Ivana Caputo; Marilena Lepretti; Agnese Secondo; Stefania Martucciello; Gaetana Paolella; Daniele Sblattero; Maria Vittoria Barone; Carla Esposito (pp. 251-260).

Anti-tissue transglutaminase (tTG) antibodies are specifically produced in the small-intestinal mucosa of celiac disease (CD) patients. It is now recognized that these antibodies, acting on cell-surface tTG, may play an active role in CD pathogenesis triggering an intracellular response via the activation of different signal transduction pathways. In this study, we report that anti-tTG antibodies, both commercial and from a CD patient, induce a rapid Ca²⁺ mobilization from intracellular stores in Caco-2 cells. We characterized the mechanism of Ca²⁺ release using thapsigargin and carbonylcyanide-p-trifluoromethoxyphenylhydrazone, which are able to deplete specifically endoplasmic reticulum and mitochondria of Ca²⁺, respectively. Our data highlight that both pathways of calcium release were involved, thus indicating that the spectrum of cellular responses downstream can be very wide. In addition, we demonstrate that the increased Ca²⁺ level in the cells evoked by anti-tTG antibodies was sufficient to activate tTG, which is normally present as a latent protein due to the presence of low Ca²⁺ and to the inhibitory effect of GTP/GDP. Herein, we discuss the importance of intracellular tTG activation as central in the context of CD pathogenesis.

Keywords: Transglutaminase; Celiac disease; Autoantibodies; Ca²⁺ homeostasis; Intracellular Ca²⁺ stores; Endoplasmic reticulum

Nitric oxide regulates tissue transglutaminase localization and function in the vasculature by Simran K. Jandu; Alanah K. Webb; Alina Pak; Baris Sevinc; Daniel Nyhan; Alexey M. Belkin; Nicholas A. Flavahan; Dan E. Berkowitz; Lakshmi Santhanam (pp. 261-269).

The multifunctional enzyme tissue transglutaminase (TG2) contributes to the development and progression of several cardiovascular diseases. Extracellular rather than intracellular TG2 is enzymatically active, however, the mechanism by which it is exported out of the cell remains unknown. Nitric oxide (NO) is shown to constrain TG2 externalization in endothelial and fibroblast cells. Here, we examined the role of both exogenous and endogenous (endothelial cell-derived) NO in regulating TG2 localization in vascular cells and tissue. NO synthase inhibition in endothelial cells (ECs) using N-nitro l-arginine methyl ester (l-NAME) led to a time-dependent decrease in S-nitrosation and increase in externalization of TG2. Laminar shear stress led to decreased extracellular TG2 in ECs. S-nitrosoglutathione treatment led to decreased activity and externalization of TG2 in human aortic smooth muscle and fibroblast (IMR90) cells. Co-culture of these cells with ECs resulted in increased S-nitrosation and decreased externalization and activity of TG2, which was reversed by l-NAME. Aged Fischer 344 rats had higher tissue scaffold-associated TG2 compared to young. NO regulates intracellular versus extracellular TG2 localization in vascular cells and tissue, likely via S-nitrosation. This in part, explains increased TG2 externalization and activity in aging aorta.

Keywords: Aging; Tissue transglutaminase; Nitric oxide; S-nitrosation

Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices by Simone Beninati; Rosa Anna Iorio; Gianluca Tasco; Donatella Serafini-Fracassini; Rita Casadio; Stefano Del Duca (pp. 271-283).

Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca²⁺-dependent transglutaminase (TG, EC 2.3.2.13) activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally.

Keywords: Plant transglutaminases; (γ-Glutamyl)-polyamines; Spermidine; Post-translational modification; Helianthus tuberosus ; Meristems

Transglutaminase-mediated modification of ovomucoid: effects on its trypsin inhibitory activity and antigenic properties by Raffaele Porta; Concetta Valeria L. Giosafatto; Prospero di Pierro; Angela Sorrentino; Loredana Mariniello (pp. 285-292).

Hen egg can cause food hypersensitivity in infants and young children, and ovomucoid is the most allergenic factor among proteins contained in egg white. Since proteinase treatment, a well-recognized strategy in reducing food allergenicity, is ineffective when applied to ovomucoid because of its ability to act as trypsin inhibitor, we investigated the possibility of reducing the ovomucoid antiprotease activity and antigenic properties by covalently modifying its structure. The present paper reports data showing the ability of the Gln115 residue of ovomucoid to act as an acyl donor substrate for the enzyme transglutaminase and, as a consequence, to give rise to a covalent monodansylcadaverine conjugate of the protein in the presence of both enzyme and the diamine dansylated derivative. Moreover, we demonstrated that the obtained structural modification of ovomucoid significantly reduced the capability of the protein to inhibit trypsin activity, also having impact on its anti-ovomucoid serum-binding properties.

Keywords: Antigenic properties; Monodansylcadaverine; Ovomucoid; Transglutaminase; Trypsin inhibitory activity

Aloin enhances cisplatin antineoplastic activity in B16-F10 melanoma cells by transglutaminase-induced differentiation by Claudio Tabolacci; Stefania Rossi; Alessandro Lentini; Bruno Provenzano; Lorenzo Turcano; Francesco Facchiano; Simone Beninati (pp. 293-300).

Aloin, a natural anthracycline from aloe plant, is a hydroxyanthraquinone derivative shown to have antitumor properties. This study demonstrated that aloin exerted inhibition of cell proliferation, adhesion and invasion abilities of B16-F10 melanoma cells under non-cytotoxic concentrations. Furthermore, aloin induced melanoma cell differentiation through the enhancement of melanogenesis and transglutaminase activity. To improve the growth-inhibiting effect of anticancer agents, we found that the combined treatment of cells with aloin and low doses of cisplatin increases the antiproliferative activity of aloin. The results suggest that aloin possesses antineoplastic and antimetastatic properties, exerted likely through the induction of melanoma cell differentiation.

Keywords: Anthraquinone; Differentiation; Melanogenesis; Invasion; Metastasis

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #1)