Accuracy of the No-Biopsy Approach for the Diagnosis of Celiac Disease in Adults: A Systematic Review and Meta-Analysis.

BACKGROUND & AIMS: Current international guidelines recommend duodenal biopsies to confirm the diagnosis of celiac disease in adult patients. However, growing evidence suggests that immunoglobulin A (IgA) anti-tissue transglutaminase (tTg) antibody levels ≥10 times the upper limit of normal (ULN) can accurately predict celiac disease, eliminating the need for biopsy. We performed a systematic review and meta-analysis to evaluate the accuracy of the no-biopsy approach to confirm the diagnosis of celiac disease in adults. METHODS: We systematically searched MEDLINE, EMBASE, Cochrane Library, and Web of Science from January 1998 to October 2023 for studies reporting the sensitivity and specificity of IgA-tTG ≥10×ULN against duodenal biopsies (Marsh grade ≥2) in adults with suspected celiac disease. We used a bivariate random effects model to calculate the summary estimates of sensitivity, specificity, and positive and negative likelihood ratios. The positive and negative likelihood ratios were used to calculate the positive predictive value of the no-biopsy approach across different pretest probabilities of celiac disease. The methodological quality of the included studies was evaluated using the QUADAS-2 tool. This study was registered with PROSPERO, number CRD42023398812. RESULTS: A total of 18 studies comprising 12,103 participants from 15 countries were included. The pooled prevalence of biopsy-proven celiac disease in the included studies was 62% (95% confidence interval [CI], 40%-83%). The proportion of patients with IgA-tTG ≥10×ULN was 32% (95% CI, 24%-40%). The summary sensitivity of IgA-tTG ≥10×ULN was 51% (95% CI, 42%-60%), and the summary specificity was 100% (95% CI, 98%-100%). The area under the summary receiver operating characteristic curve was 0.83 (95% CI, 0.77 - 0.89). The positive predictive value of the no-biopsy approach to identify patients with celiac disease was 65%, 88%, 95%, and 99% if celiac disease prevalence was 1%, 4%, 10%, and 40%, respectively. Between-study heterogeneity was moderate (I2 =30.3%), and additional sensitivity analyses did not significantly alter our findings. Only 1 study had a low risk of bias across all domains. CONCLUSION: The results of this meta-analysis suggest that selected adult patients with IgA-tTG ≥10×ULN and a moderate to high pretest probability of celiac disease could be diagnosed without undergoing invasive endoscopy and duodenal biopsy.

A new preparation method of covalent annular nanodiscs based on MTGase.

The preservation of the native conformation and functionality of membrane proteins has posed considerable challenges. While detergents and liposome reconstitution have been traditional approaches, nanodiscs (NDs) offer a promising solution by embedding membrane proteins in phospholipids encircled by an amphipathic helical protein MSP belt. Nevertheless, a drawback of commonly used NDs is their limited homogeneity and stability. In this study, we present a novel approach to construct covalent annular nanodiscs (cNDs) by leveraging microbial transglutaminase (MTGase) to catalyze isopeptide bond formation between the side chains of terminal amino acids, specifically Lysine (K) and Glutamine (Q). This methodology significantly enhances the homogeneity and stability of NDs. Characterization of cNDs and the assembly of membrane proteins within them validate the successful reconstitution of membrane proteins with improved homogeneity and stability. Our findings suggest that cNDs represent a more suitable tool for investigating interactions between membrane proteins and lipids, as well as for analyzing membrane protein structures.

Transglutaminases in fibrosis-overview and recent advances.

Transglutaminases (TGs) are a family of protein cross-linking enzymes that are capable of stiffening and insolubilizing proteins and creating protein networks, and thereby altering biological functions of proteins. Their role in fibrosis progression has been widely investigated with a focus on kidney, lung, liver, and heart where activity is triggered by various stimuli including hypoxia, inflammation, and hyperglycemia. TG2 has been considered one of the key enzymes in the pathogenesis of fibrosis mainly through transforming growth factor beta (TGF-beta) signaling and matrix cross-linking mechanisms. Although TG2 has been most widely studied in this context, the involvement of other TGs, TG1 and Factor XIII-A (FXIII-A), is beginning to emerge. This mini-review highlights the major steps taken in the TG and fibrosis research and summarizes the most recent advances and contributions of TG2, TG1, and FXIII-A to the progression of fibrosis in various animal models. Also, their mechanisms of action as well as therapeutic prospects are discussed.

Type 2 Transglutaminase in Coeliac Disease: A Key Player in Pathogenesis, Diagnosis and Therapy.

Type 2 transglutaminase (TG2) is the main autoantigen in coeliac disease (CD), a widespread inflammatory enteropathy caused by the ingestion of gluten-containing cereals in genetically predisposed individuals. As a consequence, serum antibodies to TG2 represent a very useful marker in CD diagnosis. However, TG2 is also an important player in CD pathogenesis, for its ability to deamidate some Gln residues of gluten peptides, which become more immunogenic in CD intestinal mucosa. Given the importance of TG2 enzymatic activities in CD, several studies have sought to discover specific and potent inhibitors that could be employed in new therapeutical approaches for CD, as alternatives to a lifelong gluten-free diet. In this review, we summarise all the aspects regarding TG2 involvement in CD, including its enzymatic reactions in pathogenesis, the role of anti-TG2 antibodies in disease management, and the exploration of recent strategies to reduce deamidation or to use transamidation to detoxify gluten.

Etiopathogenesis of dermatitis herpetiformis.

Dermatitis herpetiformis is a rare chronic, autoimmune bullous disease linked to gluten sensitivity with intense pruritus and characteristic skin eruptions. Etiopathogenesis is complex and not fully understood. It is currently considered to be a specific cutaneous manifestation of celiac disease. Genetic, environmental and immunological factors influence both conditions. Exposure to gluten is the starting point of an inflammatory cascade leading to the formation of circulating IgA antibodies against tissue transglutaminase and skin immune IgA deposition followed by skin lesions. Binding of the immune complex deposits of IgA transglutaminases and epidermal antibodies with enzymes in the papillary dermis stimulates complement activation, neutrophil influx, proinflammatory cytokine release and overproduction of matrix metalloproteinases. We have collected current knowledge of the pathogenesis of dermatitis herpetiformis.

TGFß-1 Induced Cross-Linking of the Extracellular Matrix of Primary Human Dermal Fibroblasts.

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFß signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFß contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFß-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFß-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFß-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFß-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFß signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.

Solid-Phase Synthesis of Selectively Mono-Fluorobenz(o)ylated Polyamines as a Basis for the Development of 18F-Labeled Radiotracers.

Polyamines are highly attractive vectors for tumor targeting, particularly with regards to the development of radiolabeled probes for imaging by positron emission (PET) and single-photon emission computed tomography (SPECT). However, the synthesis of selectively functionalized derivatives remains challenging due to the presence of multiple amino groups of similar reactivity. In this work, we established a synthetic methodology for the selective mono-fluorobenz(o)ylation of various biogenic diamines and polyamines as lead compounds for the perspective development of substrate-based radiotracers for targeting polyamine-specific membrane transporters and enzymes such as transglutaminases. For this purpose, the polyamine scaffold was constructed by solid-phase synthesis of the corresponding oxopolyamines and subsequent reduction with BH3/THF. Primary and secondary amino groups were selectively protected using Dde and Boc as protecting groups, respectively, in orientation to previously reported procedures, which enabled the selective introduction of the reporter groups. For example, N1-FBz-spermidine, N4-FBz-spermidine, N8-FBz-spermidine, and N1-FBz-spermine and N4-FBz-spermine (FBz = 4-fluorobenzoyl) were obtained in good yields by this approach. The advantages and disadvantages of this synthetic approach are discussed in detail and its suitability for radiolabeling was demonstrated for the solid-phase synthesis of N1-[18F]FBz-cadaverine.

Effect of enzyme-induced collagen crosslinking on porcine sclera.

This study aims to evaluate the effect of a new type of collagen crosslinking (CXL) mediated by microbial transglutaminases (Tgases) on sclera. Porcine eyes were divided into two groups according to the different crosslinking procedures used: the double-sided CXL group (D-CXL group) and the single-sided CXL group (S-CXL group). In the D-CXL group, 4.0 × 14.0 mm scleral strips harvested from 40 porcine eyeballs were incubated with 1 U/ml Tgases for 30 min at 37 °C. Parallel scleral strips from the same eyeball were incubated with PBS under the same conditions as the controls. In the S-CXL group, 80 whole globes were directly incubated with 1 U/ml Tgases and PBS as the controls for 30 min at 37 °C. After incubation, 4.0 × 14.0 mm scleral strips were cut from each eyeball. Biomechanical testing and light microscopy were used. In the D-CXL group, the general elastic modulus of the Tgases-treated scleral strips was 14.89 ± 6.05 MPa, and the controls was 6.72 ± 2.58 MPa, indicating an increase of 121% with Tgases treatment. In the S-CXL group, the general elastic modulus of the Tgases-treated scleral strips was 12.88 ± 4.29 MPa, and the controls was 7.00 ± 2.45 MPa, indicating an increase of 84% with Tgases treatment. In both the D-CXL and S-CXL groups, significant increases in scleral rigidity were observed compared to that of the respective controls (P < 0.05). The histology indicated increased collagen bundle density, decreased interfibrillar spaces and increased interlamellar spaces after CXL. In conclusion, scleral collagen crosslinking mediated by Tgases produced a significant increase in biomechanical strength.

From transglutaminases (TGs) to arylamine N-acetyltransferases (NATs): Insight into the role of a spatially conserved aromatic amino acid position in the active site of these two families of enzymes.

Transglutaminases (TG) and arylamine N-acetyltransferases (NAT) are important family of enzymes. Although they catalyze different reactions and have distinct structures, these two families of enzymes share a spatially conserved catalytic triad (Cys, His, Asp residues). In active TGs, a conserved Trp residue located close to the triad cysteine is crucial for catalysis through stabilization of transition states. Here, we show that in addition to sharing a similar catalytic triad with TGs, functional NAT enzymes also possess in their active site an aromatic residue (Phe, Tyr or Trp) occupying a structural position similar to the Trp residue of active TGs. More importantly, as observed in active TGs, our data indicates that in functional NAT enzymes this conserved aromatic residue is also involved in stabilization of transition states. These results thus indicate that in addition to the three triad residues, these two families of enzymes also share a spatially conserved aromatic amino acid position important for catalysis. Identification of residues involved in the stabilization of transition states is important to develop potent inhibitors. Interestingly, NAT enzymes have been shown as potential targets of clinical interest.

Assessment of expression and specific activities of transglutaminases TG1, TG2, and FXIII-A during osteoclastogenesis.

Osteoclasts are large multinucleated bone-resorbing cells derived from monocyte/macrophage lineage. Macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) drive the multi-stage osteoclastogenesis. Transglutaminases (TGs) are Ca2+- and thiol-dependent acyl transferases and protein crosslinking enzymes. TG enzyme family contains eight catalytically active enzymes TG1-7 and Factor XIII-A (FXIII-A). Recent studies have shown that TG1, TG2, and FXIII-A are present in osteoclasts and that TG2 and FXIII-A regulate osteoclastogenesis. In this study, we examined gene and protein expression and specific activities of TG1, TG2, and FXIII-A during osteoclastogenesis using "Hitomi peptides" in a day-by-day manner. We report that TG activities are highest in the differentiation and early fusion phases and then decrease dramatically. TG activities were upregulated by M-CSF and downregulated by addition of RANKL. FXIII-A was dramatically downregulated by RANKL, suggesting its involvement in M-CSF-mediated precursor commitment phase. TG1 and TG2 proteins were present throughout osteoclastogenesis, suggesting that they may have functions in both differentiation and fusion. In summary, the three TGs likely exert distinct functions at different stages of osteoclastogenesis. Our work also demonstrates that the "Hitomi peptides" are highly specific tools for detection of distinct TGs in a system where multiple TGs are present.

Transglutaminases: part I-origins, sources, and biotechnological characteristics.

The transglutaminases form a large family of intracellular and extracellular enzymes that catalyze cross-links between protein molecules. Transglutaminases crosslinking properties are widely applied to various industrial processes, to improve the firmness, viscosity, elasticity, and water-holding capacity of products in the food and pharmaceutical industries. However, the extremely high costs of obtaining transglutaminases from animal sources have prompted scientists to search for new sources of these enzymes. Therefore, research has been focused on producing transglutaminases by microorganisms, which may present wider scope of use, based on enzyme-specific characteristics. In this review, we present an overview of the literature addressing the origins, types, reactions, and general characterizations of this important enzyme family. A second review will deal with transglutaminases applications in the area of food industry, medicine, pharmaceuticals and biomaterials, as well as applications in the textile and leather industries.

Review transglutaminases: part II-industrial applications in food, biotechnology, textiles and leather products.

Because of their protein cross-linking properties, transglutaminases are widely used in several industrial processes, including the food and pharmaceutical industries. Transglutaminases obtained from animal tissues and organs, the first sources of this enzyme, are being replaced by microbial sources, which are cheaper and easier to produce and purify. Since the discovery of microbial transglutaminase (mTGase), the enzyme has been produced for industrial applications by traditional fermentation process using the bacterium Streptomyces mobaraensis. Several studies have been carried out in this field to increase the enzyme industrial productivity. Researches on gene expression encoding transglutaminase biosynthesis were performed in Streptomyces lividans, Escherichia coli, Corynebacterium glutamicum, Yarrowia lipolytica, and Pichia pastoris. In the first part of this review, we presented an overview of the literature on the origins, types, mediated reactions, and general characterizations of these important enzymes, as well as the studies on recombinant microbial transglutaminases. In this second part, we focus on the application versatility of mTGase in three broad areas: food, pharmacological, and biotechnological industries. The use of mTGase is presented for several food groups, showing possibilities of applications and challenges to further improve the quality of the end-products. Some applications in the textile and leather industries are also reviewed, as well as special applications in the PEGylation reaction, in the production of antibody drug conjugates, and in regenerative medicine.

Transglutaminase activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics.

Osteoclasts, bone resorbing cells, derive from monocyte/macrophage cell lineage. Increased osteoclast activity is responsible for bone destruction in diseases such as osteoporosis, periodontitis and rheumatoid arthritis. Transglutaminases (TGs), protein crosslinking enzymes, were recently found involved in osteoclastogenesis in vivo, however their mechanisms of action have remained unknown. In this study, we have investigated the role of TG activity in osteoclastogenesis in vitro using four TG inhibitors, NC9, Z006, T101, and monodansyl cadaverine. Our results showed that all TG inhibitors were capable of blocking the entire osteoclastogenesis process. The most potent of the inhibitors, NC9 when added to cultures at different phases of osteoclastogenesis, inhibited differentiation, migration, and fusion of pre-osteoclasts as well as resorption activity of mature osteoclasts. Further investigation into the mechanisms revealed that NC9 increased RhoA levels and blocked podosome belt formation suggesting that TG activity regulates actin dynamics in pre-osteoclasts. The inhibitory effect of NC9 on osteoclastogenesis as well as podosome belt formation was completely reversed with a Rho-family inhibitor Exoenzyme C3. Microtubule architecture, acetylation, and detyrosination of α-tubulin were not affected. Finally, we demonstrated that macrophages and osteoclasts expressed mRNA of three TGs:TG1, TG2, and Factor XIII-A which were all differentially regulated in these cells during differentiation. Immunofluoresence microscopic analysis showed that all three enzymes co-localized to podosomes in osteoclasts. Taken together, our data suggests that TG activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics and that this may involve contribution from all three TG enzymes.

Transglutaminases in autoimmune and inherited skin diseases: The phenomena of epitope spreading and functional compensation.

Transglutaminases (TGs) are structurally and functionally related enzymes that modify the post-translational structure and activity of proteins or peptides, and thus are able to turn on or switch off their function. Depending on location and activities, TGs are able to modify the signalling, the function and the fate of cells and extracellular connective tissues. Besides mouse models, human diseases enable us to appreciate the function of various TGs. In this study, skin diseases induced by genetic damages or autoimmune targeting of these enzymes will be discussed. TG1, TG3 and TG5 contribute to the cutaneous barrier and thus to the integrity and function of epidermis. TGM1 mutations related to autosomal recessive ichthyosis subtypes, TGM5 mutations to a mild epidermolysis bullosa phenotype and as novelty TGM3 mutation to uncombable hair syndrome will be discussed. Autoimmunity to TG2, TG3 and TG6 may develop in a few of those genetically determined individuals who lost tolerance to gluten, and manifest as coeliac disease, dermatitis herpetiformis or gluten-dependent neurological symptoms, respectively. These gluten responder diseases commonly occur in combination. In autoimmune diseases, the epitope spreading is remarkable, while in some inherited pathologies, a unique compensation of the lost enzyme function is noted.

Polyamine catabolism adds fuel to leaf senescence.

Leaf senescence is a terminal step in plant growth and development. Considerable information on processes and signals involved in this process has been obtained, although comparatively little is known about leaf senescence in monocotyledonous plants. In particular, little is known about players involved in leaf senescence imposed by a prolonged dark treatment. New information has now been unveiled on dark-induced leaf senescence in a monocot, barley. A close association has been found between ubiquitous polyamines, reactive oxygen species (ROS), and senescence of barley leaves during prolonged darkness. Although polyamines (putrescine, spermidine, and spermine) are absolutely essential for critical cellular functions, including regulation of nucleic acids and protein synthesis, macromolecular structural integrity, and signalling, a strong link between polyamines and dark-induced leaf senescence has been found using barley plant as a model of monocots. Interestingly, Arabidopsis polyamine back-conversion oxidase mutants deficient in the conversion of spermine to spermidine and/or spermidine to putrescine do not occur and have delayed entry into dark-induced leaf senescence. This review summarizes the recent molecular, physiological, and biochemical evidence implicating concurrently polyamines and ethylene in dark-induced leaf senescence and broadening our knowledge on the mechanistic events involved in this important plant death process.

A fluorescence anisotropy-based assay for determining the activity of tissue transglutaminase.

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2's transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein- and rhodamine B-conjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solid-phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published L-lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z' factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

Transglutaminase as polyamine mediator in plant growth and differentiation.

Transglutaminases (TGases) are ubiquitous enzymes catalyzing many biological reactions. The best-known TGase activity, namely the transamidation of specific proteins by polyamines (PAs), has been studied in plants to verify if TGase is a mediator of PAs mechanism of action to re-interpret some of PAs effects. Usually, the TGase activity is present at basal level in plant cells, but it can be induced by internal or external events or stresses, like rehydration, wounding, light, developmental differentiation and programmed cell death (PCD). Here, two models of induced growth are presented, namely pollen apical growth and dedifferentiation followed by reacquisition of the pluripotency of already differentiated cells. Moreover, PAs and TGase involvement during the differentiation and the activity of organelles and finally during the terminal organ differentiation or self-incompatibility-induced PCD are reported. In all of these models, TGase plays a role. The enzyme was detected in several cell compartments, like cytosol, chloroplasts and possibly mitochondria, microsomal fraction, cell wall and also extracellularly. The products of TGase catalysis, modified with PAs, mainly consist of high molecular mass complexes. Among the protein substrates until now identified we mention the cytoskeletal proteins, actin and tubulin, whose PA modification also affects their interaction with motor proteins and the dynamic of cytoskeleton. The most widely studied substrates are component of chloroplast photosystems, in particular light-harvesting complexes, whose modification is light dependent and whose differentiation and size are affected by TGase, thereby conditioning photosynthetic efficiency and photoprotection. Finally, modification of cell wall substrates affects wall growth and reinforcement.

Immunoglobulin A deficiency in celiac disease in the United States.

BACKGROUND AND AIMS: Multiple European studies report increased prevalence of selective immunoglobulin A deficiency (SIgAD) and partial immunoglobulin A deficiency (PIgAD) in patients with celiac disease (CD). However; prospective data representing North American adults are lacking. While SIgAD precludes the use of IgA-tissue-transglutaminase antibody (IgA-tTG), the effect of PIgAD on IgA-tTG sensitivity is not well documented. We aim to determine the prevalence and impact of IgA deficiency on CD presentation and diagnosis in North American adult patients. METHODS: We reviewed 1000 consecutive patients undergoing IgA-tTG testing and 243 healthy controls. Eligible sera were tested for IgA-tTG, serum immunoglobulins, and IgA/IgG-deamidated gliadin peptide (IgA/IgG-DGP). RESULTS: Prevalence of SIgAD was marginally higher in patients with CD (1.9%) compared with healthy controls (0.4%, P = 0.24) and patients without CD (0.7%, P = 0.173). Prevalence of PIGAD was similar in patients with CD (4.8%) compared with healthy controls (5.9%, P = 0.57) and patients without CD (7.2%, P = 0.22). One (16.7%) of 6 patients with CD with SIgAD and all 15 (100%) with PIGAD tested IgA-tTG positive prior to gluten-free diet initiation. Patients with CD with SIGAD showed lower frequency of gastrointestinal symptoms (33% vs 82%, P = 0.01) and more co-morbid autoimmune disease (67% vs 23%, P = 0.03) when compared with patients with CD with normal IgA. CONCLUSIONS: The prevalence of SIgAD in North American patients with CD is comparable with European data but not significantly different than control populations. Patients with CD with SIgAD exhibit decreased IgA-tTG sensitivity and lack of gastrointestinal symptoms. PIgAD is common in patients with gastrointestinal disorders but does not alter CD presentation or IgA-tTG sensitivity.

Coagulation factor XIIIa substrates in human plasma: identification and incorporation into the clot.

Coagulation factor XIII (FXIII) is a transglutaminase with a well defined role in the final stages of blood coagulation. Active FXIII (FXIIIa) catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide bonds between specific Gln and Lys residues. The primary physiological outcome of this catalytic activity is stabilization of the fibrin clot during coagulation. The stabilization is achieved through the introduction of cross-links between fibrin monomers and through cross-linking of proteins with anti-fibrinolytic activity to fibrin. FXIIIa additionally cross-links several proteins with other functionalities to the clot. Cross-linking of proteins to the clot is generally believed to modify clot characteristics such as proteolytic susceptibility and hereby affect the outcome of tissue damage. In the present study, we use a proteomic approach in combination with transglutaminase-specific labeling to identify FXIIIa plasma protein substrates and their reactive residues. The results revealed a total of 147 FXIIIa substrates, of which 132 have not previously been described. We confirm that 48 of the FXIIIa substrates were indeed incorporated into the insoluble fibrin clot during the coagulation of plasma. The identified substrates are involved in, among other activities, complement activation, coagulation, inflammatory and immune responses, and extracellular matrix organization.

Transglutaminase 2-dependent deamidation of glyceraldehyde-3-phosphate dehydrogenase promotes trophoblastic cell fusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein as well as a classic glycolytic enzyme, and its pleiotropic functions are achieved by various post-translational modifications and the resulting translocations to intracellular compartments. In the present study, GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift in two-dimensional electrophoresis after cell-cell fusion induction by forskolin. This post-translational modification was deamidation of multiple glutaminyl residues, as determined by molecular mass measurement and tandem mass spectrometry of acidic GAPDH isoforms. Transglutaminase (TG) inhibitors prevented this acidic shift and reduced cell fusion. Knockdown of the TG2 gene by short hairpin RNA reproduced these effects of TG inhibitors. Various GAPDH mutants with replacement of different numbers (one to seven) of Gln by Glu were expressed in BeWo cells. These deamidated mutants reversed the suppressive effect of wild-type GAPDH overexpression on cell fusion. Interestingly, the mutants accumulated in the plasma membrane, and this accumulation was increased according to the number of Gln/Glu substitutions. Considering that GAPDH binds F-actin via an electrostatic interaction and that the cytoskeleton is rearranged in trophoblastic cell fusion, TG2-dependent GAPDH deamidation was suggested to participate in actin cytoskeletal remodeling.