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Background: The currently available, clinically approved factor XIII (fXIII) activity assays rely on indirect measurement of activated fXIII transglutaminase activity by detecting "ammonia-release" during an intermediate step of the reaction. Unfortunately, this assay is also sensitive to fXIII-independent ammonia-producing reactions that may take place physiologically in plasma, rendering it prone to overestimate fXIII activity, especially when levels are Methods: A standard curve was established by diluting known concentrations of recombinant factor XIII-A (rFXIII-A; Tretten) into (1) fXIII-A congenitally deficient and (2) fXIII immunodepleted plasma to determine if our assay is able to detect variable fXIII concentrations from 1 to 200% (0.01 - 2 IU/mL). Results: We demonstrated that α2-AP incorporation is dependent upon rfXIII-A concentration in the starting sample and was linear from 1-12% (0.01 - 0.12 IU/mL; Figure). Further expansion of the standard curve to include all concentrations within the normal physiologic range demonstrated that the assay remains strongly linear from 1-20% (0.01 - 0.2 IU/mL; R2 0.966). Moderate linearity (R2 0.908) extended to 50% (0.5 IU/mL). The curve plateaued between 50%-200% (0.5 IU/mL- 2 IU/mL). Thus, the dynamic range of the Enzyme Capture-ELISA is expected within 1-50% (0.01 - 0.5 IU/mL). Conclusion: We conclude that Enzyme Capture-ELISA is a promising novel method for accurate detection of FXIII activity and is expected to improve sensitivity at low levels ( Disclosures No relevant conflicts of interest to declare.

Candida albicans is the most common opportunistic fungal pathogen which can cause life-threatening bloodstream infections known as candidaemia. It is very important to discover new drugs and targets for the treatment of candidaemia. In this study, we first investigated the combination antifungal effects of the small molecule ENOblock and fluconazole (FLC) against FLC-resistant C. albicans. A checkerboard microdilution assay showed that ENOblock has a significant synergistic effect in combination with FLC against FLC-resistant C. albicans. The time-kill curve further confirmed the synergism of this compound with FLC against FLC-resistant C. albicans. Moreover, we demonstrated the significant inhibitory effects of ENOblock alone and in combination with FLC against C. albicans hypha and biofilm formation. Furthermore, the XTT assay showed that ENOblock has relatively low toxicity to human umbilical vein endothelial cells. The in vivo antifungal efficacy of ENOblock was further assessed in a murine model of systemic C. albicans infection. Although ENOblock alone was not sufficient to treat C. albicans infection, the combination of FLC and ENOblock showed significant in vivo activity against FLC-resistant C. albicans. Finally, using surface plasmon resonance analysis as well as an inhibition assay, we determined that ENOblock directly interacted with CaEno1 and significantly inhibited the transglutaminase activity of this enzyme, which is involved in the growth and morphogenesis of C. albicans. In summary, these results demonstrate the synergistic effects of FLC and ENOblock against FLC-resistant C. albicans, and indicate that inhibition of the transglutaminase activity of CaEno1 by ENOblock might confer an advantage for the synergism of FLC and ENOblock, suggesting the potential of ENOblock as a new antifungal candidate.

In order to demonstrate transglutaminase activity in biological samples immunological as well as glutamine- and amine-donor based assays are commonly used. However, the identification of the transglutaminase reaction product, i. e. the isopeptide cross-linked peptides/proteins or the deamidated protein/peptide are often neglected. This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples.

Transglutaminase (TGase:E.C. 2.3.2.13) catalyzes the acyl-transfer reaction between one or two primary amino groups of polyamines and protein-bound Gln residues giving rise to post-translational modifications. One increasing the positive charge on a proteins surface and the other results in the covalent crosslinking of proteins. Pioneering studies on TGase in plants started in the middle of the 1980’s but the methodology designed for use with animal extracts was not directly applicable to plant extracts. Here we describe radioactive and colorimetric methods adapted to study plant TGase, as well as protocols to analyze the involvement of TGase and polyamines in the functionality of cytoskeletal proteins.

Acquired FXIII deficiencies caused by autoantibodies against FXIII subunits represent rare but very severe bleeding diatheses. Alloantibodies in FXIII-deficient patients also cause life-threatening bleeding complications, but they develop extremely rarely. In this review we provide an overview of the diagnosis and classification of anti-FXIII antibodies and analyze 48 patients with autoimmune FXIII deficiency and four additional FXIII-deficient patients who developed anti-FXIII alloantibody. The patients were collected from peer-reviewed publications from which relevant data could be extracted. With the exception of two cases the antibodies were directed against FXIII-A. The difficulties in the diagnosis of FXIII deficiency in the presence of anti-FXIII antibodies are discussed and a scheme for the functional classification of the anti-FXIII antibodies is recommended. The three main categories are neutralizing and non-neutralizing antibodies and antibodies with combined effect. The methods being used for detecting and quantifying the inhibitory effect on FXIII activation and on the transglutaminase activity of activated FXIII are summarized and techniques for the classification of neutralizing anti-FXIII antibodies are outlined. The importance of clearance studies in these cases is emphasized. Binding assays, useful for the identification of non-neutralizing and combined type antibodies, were collected from the literature and their informative power is demonstrated by examples. The most frequently occurring bleeding symptoms in patients with anti-FXIII antibodies were soft tissue bleeding; intracranial bleedings also occurred, but less frequently than in inherited FXIII deficiency. Treatment of such patients is extremely challenging; the main aim should be eradication of the antibody.

Transglutaminase (TGase) has been implicated in maintaining the undifferentiated stage of hematopoietic stem cells (HSC) in the crayfish Pacifastacus leniusculus. TGase activity has been reported to be regulated by astakine1, an essential crayfish cytokine for inducing new hemocyte synthesis in hematopoietic tissue (HPT). Here, the role of astakine1 in TGase activity regulation and clotting protein (CP) cross-linking was characterized. A reduction in TGase activity was observed by the addition of purified astakine1 in vitro for both endogenous crayfish TGase and a commercial purified guinea pig liver TGase. As a result, we observed that astakine1 inhibits TGase enzyme activity and acts as a non-competitive inhibitor for the TGase enzyme. Additionally, the clotting reaction was impaired in the presence of astakine1. A decrease in TGase-mediated crosslinking of ε(γ-glutamyl)-lysine bonds was also observed in the presence of astakine1. In conclusion, this study shows that astakine1 acts as an inhibitor of TGase activity and that it also affects CP cross-linking during crayfish hematopoiesis.

Transglutaminases are ubiquitous enzymes which catalyze post-translational modifications of proteins. The main activity of these enzymes is the cross-linking of glutaminyl residues of a protein/peptide substrate to lysyl residues of a protein/peptide co-substrate. In addition to lysyl residues, other second nucleophilic co-substrates may include monoamines or polyamines (to form mono- or bi-substituted /crosslinked adducts) or -OH groups (to form ester linkages). In absence of co-substrates, the nucleophile may be water, resulting in the net deamidation of the glutaminyl residue. Transglutaminase activity has been suggested to be involved in molecular mechanisms responsible for both physiological or pathological processes. For example, 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. This review focuses on the possible molecular mechanisms responsible for such diseases and on the possible therapeutic effects of selective transglutaminase inhibitors for patients with diseases characterized by aberrant transglutaminase activity. The article presents some promising patents on the transglutaminase activity.

19 p.- 5 fig., The A subunit of plasma factor XIII (FXIII-A) is thought to function as an intracellular transglutaminase (TG) in the monocyte/macrophage lineage to regulate certain intracellular processes involving cytoskeleton remodeling, but its precise role and the functional consequences of its absence remain poorly understood. In the present study, we show that cellular FXIII (cFXIII) expression is largely upregulated during in vitro differentiation of monocytes into dendritic cells (DCs). Monodansyl-cadaverine, a competitive substrate of TG activity, inhibited basal and CCL19-stimulated migration of mature DCs. In agreement, FXIII-A-deficient DCs showed a reduced chemotactic response to CCL19. Consistent with these findings, CHO cells stably expressing human FXIII-A showed enhanced motility in transwell and scratch-wound assays. These cells displayed increased formation of membrane blebs, dynamic cell protrusions implicated in cell movement that were also observed in DCs. The results provide evidence suggesting that upregulation of cFXIII in DCs has a role in regulating cell motility, This work was supported by grants from the Dirección General de Investigación del Ministerio de Educación y Ciencia (BFU2006-00914) and Fundación Rodríguez Pascual. A. Jayo was recipient of a fellowship from the Ministerio de Educación y Ciencia. I. Conde holds a research contract from the Consejo Superior de Investigaciones Científicas

Molecular mechanisms underlying gingival and periodontal inflammation caused by dental alloys are still poorly understood. Recently, it has been demonstrated that tissue transglutaminase can be involved in inflammatory cell response. The aim of this study was to evaluate effects of exposure to orthodontic materials on transglutaminase in cultured human gingival fibroblasts. The incubation with Ni–Ti heat-activated (T3) or Ni–Ti super-elastic (T4), and with Ni–Cr–Co (T2) alloys produced respectively 2.5-fold and 8-fold increases in IL-6 release compared with control cultures. Transglutaminase activity was significantly increased in cells exposed to T3 and T4 alloys (about 170% of control; p < 0.05), where it was mainly localized close to inner part of cell membrane. The exposure to T3 and T4 specimens significantly up-regulated also tTG expression compared with control cultures. These data first show an association between IL-6 release and tissue transglutaminase increases, suggesting that TGase-mediated reactions may play a major role in periodontal inflammation.

Flaky snack, a popular Taiwanese snack was prepared and subjected to force deformation tests, to study the functional roles of the intact starch granule and different levels of flour gluten on texture development of the snack. The role played by the enzyme, transglutaminase, was also investigated. Instron results showed that at different levels of flour gluten, there was significant distribution of hardness and fracture intensities. Transglutaminase activity exhibited and confirmed the expected binding or crosslinking activity in the gluten-protein network matrix of the snack structure. Scanning micrographs showed intact starch granules dispersed within the network matrix of the snack samples. The role of the intact starch granule dispersed throughout the gluten matrix system, significantly increases the crispness of the flaky snack.

A novel electrochemical assay to quantify transglutaminase activity is reported. The assay is based on the enzyme-controlled diffusion of Fe(CN)6(3-/4-) through amino-functionalized nanochannels of a mesoporous silica thin film on a Au surface in the presence of N-benzyloxycarbonyl-L-glutaminylglycine.

A common feature of polyglutamine (polyQ) diseases is the presence of aggregates in neuronal cells caused by expanded polyglutamine tracts. PolyQ proteins are the substrates of transglutaminase 2, and the increased activity of transglutaminase in polyQ diseases suggests that transglutaminase may be directly involved in the formation of the aggregates. We previously identified the transglutaminase 6 gene to be causative of spinocerebellar ataxia type 35 (SCA35), and we found that SCA35-associated mutants exhibited reduced transglutaminase activity. Here we report that transglutaminase 6 interacts and co-localizes with both normal and expanded polyQ proteins in HEK293 cells. Moreover, the overexpression of transglutaminase 6 promotes the formation of polyQ aggregates and the conversion of soluble polyQ into insoluble polyQ aggregates. However, SCA35-associated mutants do not affect their interactions with polyQ proteins. These data suggest that transglutaminase 6 could be involved in polyQ diseases and there may exist a common pathological link between polyQ associated SCA and SCA35.

Keratinocyte growth factor (KGF) exhibits paracrine action on numerous epithelia, including skin. We have found that cultures of normal human keratinocytes must attain confluence before KGF promotes an increase in cell number relative to untreated controls. In postconfluent cultures, treatment with KGF promoted tight packing of keratinocytes with a small basal cell morphology. Based on these observations, we hypothesized that KGF increased cell number in postconfluent cultures by affecting the ability of normal keratinocytes to undergo terminal differentiation and/or programmed cell death. In support of this hypothesis, keratinocytes treated with KGF produced fewer cross-linked envelopes and exhibited reduced membrane-associated transglutaminase activity relative to cells treated with epidermal growth factor or untreated controls. We also found that nucleosomal fragmentation was reduced in postconfluent cultures of KGF-treated keratinocytes. Furthermore, KGF-treated keratinocytes were more resistant to suspension-induced nucleosomal fragmentation than control or epidermal growth factor-treated cultures. Therefore, it appears that KGF modulates aspects of keratinocyte terminal differentiation which share features with programmed cell death. We propose that stromally-derived KGF may act as a paracrine survival factor in skin and perhaps other renewal tissues.

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.

Transglutaminases catalyze the covalent cross-linking of protein and are involved in the mechanism of polyamine-dependent mucosal healing. The current study examined the effect of polyamines on transglutaminase activity in gastrointestinal mucosa. Rats were fasted 22 hr before experiments and enzyme activity was measured as the Ca(++)-dependent covalent incorporation of [3H]-putrescine into acid-precipitable protein. In some of the experiments, mucosal ornithine decarboxylase (ODC) activity and polyamine levels were also examined. Transglutaminase activity in both gastric and duodenal mucosa increased significantly after polyamine administration. Treatment with alpha-difluoromethylornithine (DFMO) decreased both basal ODC activity and putrescine levels in the duodenal mucosa. DFMO also significantly decreased mucosal transglutaminase activity. In stress or hypertonic NaCl-induced gastric mucosal injury models, increased polyamine biosynthesis was associated with increased transglutaminase activity, which was completely prevented by DFMO. Exogenous polyamines returned transglutaminase activity toward control levels in the presence of DFMO. In conclusion, these results indicate that: (i) luminal polyamines increase transglutaminase activity in gastric and duodenal mucosa; (ii) polyamine depletion caused by the inhibition of ODC is accompanied by a significant decrease in transglutaminase activity; and (iii) exogenous polyamines significantly reverse the decrease in transglutaminase activity caused by polyamine depletion.

Transglutaminases are ubiquitous enzymes which catalyze posttranslational modifications of proteins. Recently, transglutaminase-catalyzed post-translational modification of proteins has been shown to be involved in the molecular mechanisms responsible for human diseases. Transglutaminase activity has been hypothesized to be involved also in the pathogenetic mechanisms responsible for several human neurodegenerative diseases. Alzheimer's disease and other neurodegenerative diseases, such as 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. This paper focuses on the possible molecular mechanisms by which transglutaminase activity could be involved in the pathogenesis of Alzheimer's disease and other neurodegenerative diseases, and on the possible therapeutic effects of selective transglutaminase inhibitors for the cure of patients with diseases characterized by aberrant transglutaminase activity.

Salted surimi pastes were prepared either with each of NaCl, KCl, and NH4Cl, or with a mixture of the two salts at pH 7.0 and a constant ionic strength (I=0.6) in the presence or absence of monodansyl cadaverine (MDC). They were incubated at 25°C for several hours (suwari gel) and then heated at 90°C for 20 min (cooked gel). The gels produced were analyzed by measuring the breaking strength and amounts of cross-linked myosin heavy chain and incor-porated MDC as a probe of transglutaminase activity. The breaking strength of the directly heated gels containing NaCl and KCl at various ratios was almost constant, whereas those of the suwari and cooked gels increased in propor-tion to the increasing ratio of NaCl to KCl. A similar NaCl-dependent increase was found in the amounts of cross-linked myosin heavy chain and incorporated MDC. The breaking strength of suwari and cooked gels was strongly depressed by a small amount of NH4Cl (in the mM range) contained in NaCl at a total ionic strength of I=0.6 with a con-comitant decrease in the amounts of cross-linked myosin heavy chain and incorporated MDC.

Epidermal transglutaminase (TGase) is known to catalyse cross-linking of several precursor proteins in the formation of cornified cell envelope at the terminal differentiation of keratinocytes. Expression of TGase activity was studied using an in situ TGase activity assay in human fetal skin samples of 49-163 days estimated gestational age (EGA). In the early two-layered epidermis (49-56 days EGA), in situ TGase activity was not observed in the periderm cells or the basal cells. In the late two-layered epidermis 57-65 days EGA), in situ TGase activity became weakly positive in the periderm cells, but not in the basal cells. In the three-layered ( 66-95 days EGA) and in four- or more layered (96-135 days EGA) stratified epidermis, in situ TGase activity was still restricted only to the periderm cells. After keratinization occurred in the interfollicular epidermis (163 days EGA ), in situ TGase activity was expressed in the granular and cornified layers. This unique localization of TGase activity further support the hypothesis that periderm cells form cornified cell envelope during their regression process in human fetal skin development.

25 p.-6 fig., Background: The A subunit of factor XIII (FXIII-A) functions as an intracellular transglutaminase (TG) in the megakaryocyte/platelet lineage, where it probably participates in the cytoskeletal remodeling associated with cell activation. However, so far, the precise role of cellular FXIII (cFXIII) and the functional consequences of its absence in FXIII-A-deficient patients are unknown. Objectives and methods: In this study, we used platelets from four patients with congenital deficiency of FXIII-A to study the role of cFXIII in platelet functions. Results: We found that FXIII-A represents the only detectable source of TG activity in platelets and that the binding of fibrinogen in response to thrombin receptor agonist peptide (TRAP) stimulation was significantly reduced in platelets from the patients. In agreement with this, in control platelets, monodansyl-cadaverine (MDC), a competitive amino-donor for TGs, inhibited fibrinogen binding induced by TRAP in a dose-dependent manner. Moreover, upon adhesion to fibrinogen, normal platelets incubated with MDC as well as FXIII-A-deficient platelets showed a distinct extension pattern with reduced lamellipodia and increased filopodia formation, suggesting a delay in spreading. Conclusions: These findings provide evidence for the direct involvement of cFXIII-dependent TG activity in the regulation of platelet functions, This work was supported by grants from the Dirección General de Investigación del Ministerio de Educación y Ciencia (BFU2006-00914) and Fundación Rodríguez Pascual. A. Jayo was recipient of a fellowship from the Ministerio de Educación y Ciencia. I. Conde holds a research contract from the Consejo Superior de Investigaciones Científicas

Background Celiac disease (CD) occurs in as many as 1 in 80 pregnant women and is associated with poor pregnancy outcome, but it is not known if this is an effect on maternal nutrient absorption or, alternatively, if the placenta is an autoimmune target. The major autoantigen, tissue transglutaminase (tTG), has previously been shown to be present in the maternal-facing syncytiotrophoblast plasma membrane of the placenta. Methods ELISA was used to demonstrate the presence of antibodies to tissue transglutaminase in a panel of CD sera. Immunohistochemistry was used to evaluate the binding of IgA autoantibodies from CD serum to term placenta. In addition, novel direct binding and activity assays were developed to mimic the in vivo exposure of the villous placenta to maternal autoantibody. Results and Discussion CD IgA autoantibodies located to the syncytial surface of the placenta significantly more than IgA antibodies in control sera (P < 0.0001). The distribution of antigen was similar to that observed using a monoclonal antibody to tissue transglutaminase. Staining was reduced by pre-absorption of CD serum with recombinant human tissue transglutaminase. In direct binding assays, autoimmune immunoglobulin A (IgA) from the maternal compartment became associated with antigen at the syncytial surface of the placenta, as a result of which transglutaminase activity at this site was inhibited. Conclusion These data indicate that direct immune effects in untreated CD women may compromise placental function.