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Objective Transglutaminase (TGM)2 and peroxisome proliferator-activated receptor (PPAR)γ are thought to participate in the pathogenesis of nasal polyp formation in cystic fibrosis (CF). We herein investigated expressions of cystic fibrosis transmembrane conductance regulator (CFTR), TGM2, PPARγ and isopeptide bonds, a reaction product of TGM, in non-CF nasal polyps. Methods Nasal polyps and inferior turbinates were collected from chronic rhinosinusitis patients without CF during transnasal endoscopic sinonasal surgery. Expressions of CFTR, TGM2, isopeptide bonds and PPARγ were examined by fluorescence immunohistochemistry and quantitative RT-PCR. Expression of CFTR was also analyzed by Western blot. Results Immunohistochemical fluorescence of the nasal polyp was significantly lower for CFTR and PPARγ, and significantly higher for TGM2 and isopeptide bonds than that of the turbinate mucosa. Lower expression of CFTR in the nasal polyp than in the turbinate mucosa was also observed in Western blot. Expression of PPARG mRNA was significantly lower in the nasal polyp than in the turbinate mucosa, whereas expressions of CFTR mRNA or TGM2 mRNA did not differ between the two tissues. Immunohistochemical fluorescence for CFTR showed significant negative correlation with that for TGM2 and isopeptide bonds, and significant positive correlation with that for PPARγ. The fluorescence for TGM2 was positively correlated with that for isopeptide bonds and negatively correlated with that for PPARγ. The fluorescence for isopeptide bonds tended to be negatively correlated with that for PPARγ. Conclusions These results suggest a possible role of the CFTR-TGM2-PPARγ cascade in the pathogenesis of nasal polyp formation in non-CF patients as in CF patients.

The surface modification of wool fibers is an important part of the textile industry to add functionality and stability to wool products. In this study, we developed a process utilizing a base/cati...

Milkfish is one type of fish that can be used as raw material for surimi. However, previous research indicated that the quality of surimi powder produced from milkfish needed some improvements in order to produce surimi with good texture and strength gel. The addition of microbial transglutaminase (MTGase) enzyme is expected to produce surimi with better gel characteristics. The research objectives were to determine the best enzyme concentration and reaction time in the formation of surimi gel and to determine the effect of enzyme addition on the physical and chemical characteristics of surimi gel produced. The research was divided into two stages: 1) preparation for making surimi, and 2) determination of enzyme concentration and reaction time for surimi gel formation. The research treatment consisted of two factors, which were enzyme concentration (0, 0.1, 0.3, 0.5 units/g of surimi) and reaction time (0, 30, 60 mins). The observation parameters consisted of yield, gel strength, whiteness, pH, water content, protein content, and fat content. The results showed that the best concentration of MTGase enzyme was 0.5 units/ g with a reaction time of 60 mins. The addition of transglutaminase enzyme gave an effect significantly on gel strength, whiteness, protein content, and fat content in the milkfish surimi gel produced.

Tissue-engineered vascular grafts (TEVGs) require adequate extracellular matrix (ECM) to withstand arterial pressure. Tissue transglutaminase (TG2) and lysyl oxidase (LOX) are enzymes that cross-link ECM proteins and play a pivotal role in the development of vascular stiffness associated with aging. The purpose of this study is to investigate the expression of ECM cross-linking enzymes and mechanisms of scaffold degeneration leading to vascular stiffness in TEVG remodeling. Fast- and slow-degrading electrospun TEVGs were fabricated using polydioxanone (PDO) and poly(L-lactide-co-caprolactone) (PLCL) copolymer, with a PDO/PLCL ratio of 9:1 for fast-degrading and 1:1 for slow-degrading graft. These grafts were implanted in rats (

В последние годы в Российской Федерации (РФ) появились сообщения об использовании в пищевой промышленности технологического вспомогательного средства (ТВС) - ферментного препарата на основе микробиальной трансглютаминазы (мТГ), который не входит в перечень разрешенных для использования в пищевой промышленности Евразийского экономического союза (ЕАЭС). Анализ данных показал, что используемые в настоящее время в пищевой промышленности штаммы-продуценты мТГ - Streptomyces mobaraensis DSM40587 и Streptomyces mobaraensis S-8112 - наряду с этим ферментом могут синтезировать антибиотики блеомицин, детоксин, пиерицидин А, а также ферменты антибиотикоустойчивости, такие как пенициллин ацилазу, бета-лактамазу, что служит развитию антибиотикоустойчивости у микроорганизмов. При поступлении в желудочно-кишечный тракт (ЖКТ) в составе пищевой продукции мТГ может вызывать изменение структуры белков слизистой оболочки кишечника. Под влиянием мТГ увеличивается проницаемость стенки кишечника, происходит активация иммунного ответа, увеличивается антигенная нагрузка иммунной системы, происходит нарушение связи ДНК с гистонами. Показано, что мТГ может явиться причиной увеличения частоты проявления симптомов целиакии у гипосимптомных с неустановленным диагнозом генетически восприимчивых лиц, нарушения адгезии белков при формировании синдрома Альцгеймера, аллергии, а также нарушений иммунного статуса организма и микробиоценоза кишечника. Данный фермент является привлекательным для производителей в связи с высокой эффективностью применения мТГ в технологическом процессе производства пищевой продукции из некачественного и некондиционного сырья, что обуславливает значимые экономические выгоды и возможность отказа от использования пищевых добавок. Последнее позволяет формировать у потребителя представление о высоком качестве вырабатываемой пищевой продукции. В связи с установленными рисками здоровью населения при использовании мТГ, отсутствием разрешения на ее использование в пищевой промышленности ЕАЭС, возможностью применения этого фермента в целях введения в заблуждение потребителей считаем необходимым проведение дополнительных оценок рисков здоровью и введение обязательного мониторинга на наличие остаточной активности этого фермента в пищевой продукции. In recent years, in the Russian Federation (RF), there have been reports about use in the food industry of processing aid (PA) - an enzyme preparation based on microbial transglutaminase (mTG), which is not included in the list of permitted for use in the food industry of the Eurasian Economic Union (EAEU). Analysis of the data showed that the currently used in the food industry strains-producer of mTG - Streptomyces mobaraensis DSM40587 and Streptomyces mobaraensis S-8112, along with this enzyme, have been synthesizing antibiotics bleomycin, detoxin, piericidin A, as well as such enzymes - penicillin acylases, beta-lactamazes, which could be reasons of antibioticoresistance of microorganisms. mTG when introduced into the gastrointestinal tract as part of food, could change the structure of proteins in the intestinal mucosa. Under the influence of mTG have increased intestinal permeability, immune system antigenic load, have been promoted the immune response, have been intensifying connection between DNA and histones. It has been shown that mTG could cause an increase the frequency of detection of celiac disease in hyposymptomatic / with an undetermined diagnosis of genetically susceptible individuals, impaired protein adhesion during the formation of Alzheimer's syndrome, allergies, as well as impaired of immune status and gut's microbiosis. This enzyme is attractive to manufacturers due to the high efficiency of the use of mTG in the technological process of food production from low-quality and substandard raw materials, which leads to significant economic benefits and the possibility of abandoning the use of food additives. The latter fact allows the consumer to form an idea of the high quality of the produced food. In connection with the established risks mTG for public health, lack of permission for its use in the food industry of the EAEU and the possibility of using this enzyme in order to mislead consumers, we consider it necessary to carry out additional health risk assessments and introduce mandatory monitoring for the presence of residual activity of this enzyme in food.

Introduction: Enzymatic cross-linking of the collagens within the extracellular matrix (ECM) catalyzed by enzymes such as lysyl oxidase (LOX) and lysyl oxidase like-enzymes 1–4 (LOXL), transglutaminase 2 (TG2), and peroxidasin (PXDN) contribute to fibrosis progression through extensive collagen cross-linking. Studies in recent years have begun elucidating the important role of collagen cross-linking in perpetuating progression of organ fibrosis independently of inflammation through an increasingly stiff and noncompliant ECM. Therefore, collagen cross-linking and the cross-linking enzymes have become new targets in anti-fibrotic therapy as well as targets of novel biomarkers to properly assess resolution of the fibrotic ECM. Areas covered: The enzymatic actions of enzymes catalyzing collagen cross-linking and their relevance in organ fibrosis. Potential biomarkers specifically quantifying proteolytic fragments of collagen cross-linking is discussed based on Pubmed search done in November 2020 as well as the authors knowledge. Expert opinion: Current methods for the assessment of fibrosis involve the use of invasive and/or cumbersome and expensive methods such as tissue biopsies. Thus, an unmet need exists for the development and validation of minimally invasive biomarkers of proteolytic fragments of cross-linked collagens. These biomarkers may aid in the development and proper assessment of fibrosis resolution in coming years.

The structures assembled by peptides have attracted great attention due to their unique physicochemical properties. Moreover, the co-assembly of peptides with additional components can endow the structures with extended functions. In this work, we have explored the co-assembly of peptides and carbon nanodots (CNDs) by taking advantage of their non-covalent binding; thus, the obtained structure may show both the recognition capability of peptides and the catalytic activity of CNDs. Therefore, we have further used the assembled structure for the sensitive analysis of transglutaminase 2 with a low detection limit of 0.25 pg/mL. By simply replacing the peptide sequences or the nanomaterials, the strategy proposed in this work can be developed as a universal model to build the co-assemblies of peptides and nanomaterials, thus leading to their broader applications in biological and biomedical research.

Összefoglaló. A glutén, alimentáris környezeti antigénként, különböző szervrendszereket érintő autoimmun betegségeket tud kiváltani. A kórképek hátterében a gluténtolerancia veleszületett hiánya vagy az élet során bekövetkező elvesztése áll. A gluténindukált autoimmun betegségek között a leggyakoribb a coeliakia, melyet különböző súlyosságú enteropátia jellemez, és melynek a szöveti, 2-es típusú transzglutamináz az autoantigénje. A coeliakia extraintestinalis tünetei között azonban néha olyan bőr- és idegrendszeri kórképek jellegzetességei is megtalálhatók, melyek hátterében további transzglutamináz-autoimmunitás kialakulása áll. Idesorolható a hevesen viszkető, polimorf autoimmun bőrbetegség, a dermatitis herpetiformis (transzglutamináz-3-autoimmunitás) és a centrális és/vagy perifériás neurológiai károsodások egy jellegzetes csoportja (transzglutamináz-6-autoimmunitás). Az indukált autoimmunitás reverzibilis, a szigorúan tartott gluténmentes diéta mellett a coeliakia és a bőrtünetek elmúlnak, de az idegrendszeri tünetek egy része maradandó. Az elmúlt évtizedben beszámoltak gluténérzékeny, transzglutamináz-6-pozitív, nem coeliakiás (transzglutamináz-2-negatív) betegekről is. A gluténszenzitivitás sokféle megjelenését ma is erősen kutatják. Fontos a korai felismerés és a kórképek interdiszciplináris szemléletű kezelése. A coeliakia családi szűrővizsgálatokkal való korai felismerése és a tünetmentes egyének diétás kezelése is nagy jelentőségű a gluténérzékenység által kiváltott hiányállapotok és a társuló egyéb betegségek kialakulásának megelőzésében. Orv Hetil. 2021; 162(28): 1107–1118. Summary. Autoimmune diseases induced by digestion of gluten, an environmental antigen, can affect different organ systems. The diseases develop in individuals with congenital or acquired loss of gluten tolerance for life. Amongst the gluten-induced autoimmune diseases, celiac disease is the most common one, characterized by an enteropathy of varying severity. Here the target autoantigen is tissue (type 2) transglutaminase. While the extraintestinal manifestations of celiac disease are complex, they may include characteristics of certain skin and nervous system disorders that develop due to additional transglutaminase autoimmunities. Such diseases are the severely pruritic, polymorphic autoimmune skin disease, dermatitis herpetiformis due to epidermal (type 3) transglutaminase autoimmunity, and a distinctive group of gluten-sensitive neuropathies with central and/or peripheral neurological involvement caused by type 6 transglutaminase autoimmunity. While the celiac and skin autoimmune diseases gradually get into remission under a strict gluten-free diet, some neurological symptoms may persist. In the last decade, gluten-induced transglutaminase 6 positive but non-celiac (transglutaminase 2 negative) patients were reported. Today, various manifestations of gluten sensitivity are under extensive research. Early detection and interdisciplinary treatment of these disorders are important. Family screenings are of particular relevance in early recognition and dietary treatment of latent disease forms in order to prevent enteropathy-induced, malabsorption-related and other associated co-morbidities. Orv Hetil. 2021; 162(28): 1107–1118.

Transglutaminases are protein cross‐linking and protein‐modifying enzymes that have attracted considerable interest due to their causal involvement in various diseases and versatility in industrial applications. In particular, microbial transglutaminases (MTG) from Streptomyces bacteria have managed in recent years to evolve from simple food additives to specialized enzymes for the site‐directed modification of therapeutic proteins. The review summarizes relevant studies from the beginning dealing with the occurrence, production, structure, catalysis, and substrate molecules of MTG enzymes. It also addresses biotechnological procedures with MTG from S. mobaraensis (SmMTG) as the most prominent representative in focus. Reassessment of the available data revealed unexpected insights into catalysis of SmMTG and other transglutaminases, suggesting selection of glutamine donor proteins by subsites at the front vestibule and the existence of distinct lysine pockets. Flexibility of the SmMTG‐accessible glutamine donor substrate regions seems to be more important than the glutamine environment. Nevertheless, residues in close vicinity to glutamines also determine interaction with the SmMTG subsites. The apparent lack of subsites for lysine donor proteins suggests self‐assembly of the substrate proteins prior to enzymatic cross‐linking. The study of natural substrate proteins, especially their mutual interaction, is proposed to further illuminate catalysis of SmMTG. To this end, structure and function of the characterized substrate proteins from S. mobaraensis are discussed in conclusion.

Present research aims to make labneh from different kinds of milk (buffalo , cow and goat)(BM,CM, and GM, respectively), and to compare the effect of the addition of transglutaminase (TG) on the examined treatments. Results show that the addition of TG did not considerably affect the development of acidity during the fermentation process, while the increase of acidity in B and C was faster than in G. On the other hand, an increase in the yield of labneh made with added enzyme, compared with samples without TG from was 43.16 to 50.55% ,38.78 to 48.16 % and 22.50 to 37.20%, in the same order. In addition, an improvement in the gel strength and decrease in the synersis were observed. The chemical composition and sensory properties was determined during 21 days showed an increase in the total solid (TS),total protein(TP) and total volatile fatty acid (TVFA) in the samples treated with TG, while there was no noticeable effect on acidity and PH. The addition of TG in all of the examined samples led to improvement in the sensory value and accessed to creamy body, particularly in the labneh made from cow milk with added TG.

A 16-year-old boy with nonresponsive celiac disease (NRCD), dermatitis herpetiformis, short stature, and failure to thrive, presented to this Functional Medicine practitioner because he had exceedingly high tissue transglutaminase (tTG) antibodies and poor growth, despite 10 months on a meticulous gluten-free diet (GFD). Immunological testing showed elevated antibody production against multiple peptides of wheat, food antigens, intestinal barrier dysfunction, lipopolysaccharide (LPS) antibodies, and polyreactive autoimmune reactions. An elimination diet, nutraceutical protocols to modulate the microbiome, address intestinal permeability, lower inflammation, and remove underlying bacterial infection were initiated. Global anti-inflammatory lifestyle modifications were recommended. Within 3 months of treatment, the patient’s tTG antibodies decreased by 14% for the first time since strict gluten elimination. Within 15 months, tTG IgG antibodies were nearly normal at 1.61 (0.03-1.60, ELISA Index). Test results improved dramatically in tandem with clinical progress. On a GFD and after initiating and maintaining these dietary and lifestyle changes, he gained 12 inches and 40 pounds. To our knowledge, this is the first published case of complete reversal of NRCD and failure to thrive by addressing endotoxin and lifestyle outside of a GFD.

BACKGROUNDS The structural and interfacial properties of soybean protein isolate (SPI) after glycosylation by the transglutaminase method were studied. It is hoped that preliminary explorations will find a new food ingredient and broader application of SPI in the food industry. RESULTS The contents of free amino proves that transglutaminase can insert glucosamine into SPI through its transamination, and realize the enzymatic glycosylated SPI. The results of structure properties showed that a decrease in the content of the α-helical structure indicates that the rigid structure of the protein is opened and the flexibility is increased. The blue shift of the maximum fluorescence intensity of soy protein isolate-glucosamine with transglutaminase (SPI-G) indicates the formation of a new substance; scanning electron microscopy shows that the SPI-G powder can be seen at a magnification of 2000×, and the protein structure becomes soft. The results of interfacial properties found that enzymatic protein glycosylation exposes the internal hydrophobic groups of SPI, resulting in increased surface hydrophobicity, increased emulsification and emulsification stability, and reduced surface tension. CONCLUSION It shows that SPI-G effectively improves the interfacial properties of SPI, providing a theoretical basis for the application of enzymatic glycosylation of SPI in the food industry. © 2021 Society of Chemical Industry.

Nowadays, celiac disease is known as an autoimmune disorder, the only cure for whom is consuming gluten-free foods. In this study, potato flour (Konbak variety) was used as a gluten-free resource in bread baking and texture improvement. Nevertheless, industrial challenges and strategies to facilitate formulation development still exist in terms of gluten-free bread characteristics such as texture, flavor, and extension of the products shelf life. As additives, guar gum, sodium caseinate and microbial transglutaminase enzyme at four levels (0.0, 0.5, 1.0 and 1.5) were used. Principal component analysis (PCA) was applied to evaluate type and level of additives in three bread formulations. The multivariate analysis allowed us to recognize different effect of additives from each other. The results showed that these additives increased the porosity, adhesion and elongation of the treatments and reduced the amount of a*, L* and b* dye component in the bread crust. Moreover, they reduced the specific volume, firmness of the tissue, adhesion and wrinkle of the final product. Besides that, all optimal formulation showed an acceptability quality level, indicating the potential to be used on an industrial scale.

Enzymatic deamidation, the conversion of glutamine (Gln) into glutamic acid (Glu) residues, mediated by tissue transglutaminase enzymes, can provoke autoimmunity by generating altered self-epitopes, a process well-known in celiac disease and more recently also described in type 1 diabetes (T1D). To identify deamidated proteins, liquid chromatography-tandem mass spectrometry is the method of choice. However, as nonenzymatic deamidations on asparagine (Asn) and to a minor extent on Gln are frequently induced in vitro during proteomics sample preparation, the accurate detection of in vivo deamidation can be hampered. Here we report on the optimization of a method to reduce in vitro generated deamidation by 70% using improved trypsin digestion conditions (90 min/pH 8). We also point to the critical importance of manual inspection of MS2 spectra, considering that only 55% of the high quality peptides with Gln deamidation were assigned correctly using an automated search algorithm. As proof of principal, using these criteria, we showed a significant increase in levels of both Asn and Gln deamidation in cytokine-exposed murine MIN6 β-cells, paralleled by an increase in tissue transglutaminase activity. These findings add evidence to the hypothesis that deamidation is occurring in stressed β-cell proteins and can be involved in the autoimmune process in T1D.

Celiac disease is an immune-mediated systemic disorder caused by gluten in people with genetic predisposition. Celiac disease is characterized by wide range of clinical manifestations (both gastroenterological and extraintestinal), that can complicate the diagnosis. Thus, celiac disease often remains undiagnosed. ESPGHAN has published updated clinical guidelines with adjusted coeliac disease diagnosis algorithms in 2020. It is proposed to determine antibodies to tissue transglutaminase (TGA-IgA) and total IgA within normal content of gluten-containing products in the diet on the first stage of children screening. The diagnosis of celiac disease can be established without small intestine biopsy in case of increased levels of TGA-IgA ≥ 10 of upper limit of normal and presence of antibodies to endomysium (EMA-IgA) in secondary serum. In such cases, ESPGHAN does not recommend any additional genetic testing to confirm celiac disease as it does not increase the reliability of the diagnosis. Antigen tests on class G or A antibodies against native gliadin are not specific and are not recommended for use in the diagnosis of celiac disease.

Celiac disease is an autoimmune mediated enteropathy caused by ingestion of gluten and affects approximately 1% of the population. It has significant deleterious effects on health and mortality. Helicobacter pylori is the major causative agent of peptic ulcer and gastric cancer. The aim of this study was to assess the prevalence of both H. pylori (HP) and celiac disease (CD) as mono and co-infections and to find out the possible association between CD and presence of HP. A total of 172 blood samples from individuals suspected of CD and HP infection were collected during March to December, 2015 from Hyderabad. Serum analyzed for presence of anti-HP antibodies for evaluation of HP infection and tissue transglutaminase IgA antibodies (tTGA) for detection of CD. The serodiagnosis data revealed that out of 172 samples tested 19.19% were positive for CD which included 6.98% (n=12) male patients, whereas 12.21% (n=21) were females. The HP was detected in 44.77% (n=77) including 22.09% (n=38) males and 22.67% (n=39) females out of the 33 CD positive cases, HP was detected in 17 (9.88%). HP in CD positive cases was found to be 6.60% in males 9.43% in females respectively. The HP prevalence in non-CD patients appeared to be high such as 18.02% and 16.86%. In conclusion, the data of the present study indicates that HP is more prevalent than CD.

Improvements of melting resistance and quality by modification of pork‐skin jelly through enzymatic cross‐linking were studied, and the mechanism of quality improvement was discussed in this work. Gel strength, springiness and chewiness of modified gel increased significantly (P

Transglutaminase (TGase) is inextricably associated with plant growth and development. However, the mechanism by which TGase enhances salt tolerance of higher plants under salt stress is poorly understood. In this study, we investigated the effects of NaCl stress and exogenous o-phenanthroline (o-Phen, a metalloprotease inhibitor) on TGase activity, chlorophyll fluorescence parameters, carbohydrates contents, the reactive oxygen species (ROS) scavenging system, and endogenous polyamines (PAs) contents of salt-sensitive ‘Jinyou No. 4′ and salt-tolerant ‘Inbred Line 9930’ cucumber. Salt stress significantly inhibited plant growth of the two cultivars, as well as hindered carbohydrates transport, which was more evident in the salt-sensitive cultivar. TGase activity and expression, ROS scavenging capacity, and bound PAs content were up-regulated by salt stress to some extent, which was more distinct in the salt-tolerant cucumber cultivar. However, o-Phen treatment significantly inhibited TGase expression, and further decreased plant growth and the actual photochemical efficiency of photosystem II in the two cultivars. In addition, application of o-Phen significantly decreased endogenous PAs content in leaves of ‘Jinyou No. 4’ and ‘Inbred Line 9930’ seedlings by 9.60% and 42.32% under NaCl stress, respectively. These results suggested that high activity of TGase increases the salt stress tolerance of cucumber plants by increasing endogenous PAs content and ROS scavenging capacity, and promoting carbon assimilation and photosynthetic products.

International audience; This study used a new psychrophilic transglutaminase (TGase) to catalyze the formation of protein and peptide hydrogels by forming isopeptide bonds. The psychrophilic recombinant TGase (rTGase) was overexpressed in E. coli and purified using a Ni-Sepharose column followed by stepwise dialysis to recover the cold-active enzyme (specific activity of 1.63 U/mg). The rTGase catalyzed the formation of cold-set hydrogels at 4 °C, which were comprised of high molecular weight gelatin and antioxidant peptides. The antioxidant bioactivity of the cold-set gelatin–peptides composite hydrogels was significantly improved with rTGase at 4 °C for 10 days. To understand the mechanism of the psychrophilic rTGase on the enhanced bioactivity of cross-linked hydrogels, in silico molecular docking studies were performed to assess the binding of antioxidant peptides into the rTGase active pocket, as well as to analyze the interactions of Gln and Lys residues in the antioxidant peptides with amino acid residues in the rTGase catalytic site. The formation of covalent cross-linkages in the gelatin–gelatin, gelatin–peptide and peptide–peptide matrices, and possible structures of composite hydrogels formed by rTGase catalysis in cold conditions, were deduced. The new psychrophilic rTGase enabled stabilization of the antioxidant bioactivity of protein- and peptide-containing cold-set hydrogels formed at low temperatures.

The gluten-free diet (GFD) has proven to be an imperfect treatment for celiac disease (CeD) for many subjects. Additional therapeutic options may be useful in combination with the GFD for persistent/recurrent CeD symptoms, for preventing acute symptomatic reactions, and decreasing disease burden by perhaps one day allowing CeD patients to include a certain amount of gluten in their diet. Therapeutic strategies include 1) decreasing intestinal mucosal exposure from gluten's toxic and immunogenic peptides (Glutenases [Latiglutenase and Kuma]), AGY, single chain variable fragment (scFv), polymer P(HEMA-co-SS), and probiotics 2) decreasing intestinal permeability (the Zonulin antagonist Larazotide) and 3) inhibiting the immune proinflammatory response (inhibitors/antagonists of tissue transglutaminase, IL-15, CCR9, γc Receptor, elafin, and cathepsin). Immune tolerizing techniques are a new, promising approach, for which CeD with its very well described antigen may be a perfect model; some of the therapies tested include the vaccine NexVax2, and the nanoparticle-based TIMP-Glia. This chapter further analyzes the advantages and pitfalls of these therapies, and details the broad principles for drug testing, including outcome measures, gluten challenge, use of gluten immunogenic peptide testing, animal testing, and clinical trial designs.

Reporting matrix metalloproteinase (MMP) activity directly from the extracellular matrix (ECM) may provide critical insights to better characterize 2D and 3D cell culture model systems of inflammatory diseases and potentially leverage in vivo diagnosis. In this proof-of-concept study, we designed MMP-sensors, which were covalently linked onto the ECM by co-administration of the activated transglutaminase factor XIIIa (FXIIIa). Elements of the featured MMP-sensors are the D-domain of insulin-like growth factor I (IGF-I) through which co-administered FXIIIa covalently links the sensor to the ECM followed by an MMP sensitive peptide sequence and locally reporting on MMP activity, an isotopically labeled mass tag encoding for protease activity, and an affinity tag facilitating purification from fluids. All sensors come in identical pairs, other than the MMP sensitive peptide sequence, which is synthesized with l-amino acids or d-amino acids, the latter serving as internal standard. As a proof of concept for multiplexing, we successfully profiled two MMP-sensors with different MMP sensitive peptide sequences reporting MMP activity directly from an engineered 3D ECM. Future use may include covalently ECM bound diagnostic depots reporting MMP activity from inflamed tissues.

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50°C and 8.0, respectively. MTG activity increased 1.42- fold in the presence of β-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8°C. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.

Protein ubiquitination is one of the most prevalent post-translational modifications, controlling virtually every process in eukaryotic cells. Recently, the Legionella effector MavC was found to mediate a unique ubiquitination through transglutamination, linking ubiquitin (Ub) to UBE2N through UbGln40 in a process that can be inhibited by another Legionella effector, Lpg2149. Here, we report the structures of MavC/UBE2N/Ub ternary complex, MavC/UBE2N-Ub (product) binary complex, and MavC/Lpg2149 binary complex. During the ubiquitination, the loop containing the modification site K92 of UBE2N undergoes marked conformational change, and Lpg2149 inhibits this ubiquitination through competing with Ub to bind MavC. Moreover, we found that MavC itself also exhibits weak deubiquitinase activity towards this non-canonical ubiquitination. Together, our study not only provides insights into the mechanism and inhibition of this transglutaminase-induced ubiquitination by MavC, but also sheds light on the future studies into UBE2N inhibition by this modification and deubiquitinases of this unique ubiquitination., The Legionella effector MavC catalyses the ubiquitination of the E2 enzyme UBE2N in the host cell through a transglutamination reaction, which can be inhibited by the Legionella effector Lpg2149. Here, the authors provide mechanistic insights into these processes by determining the crystal structures of the MavC/UBE2N/Ub ternary complex, the MavC/UBE2N-Ub product complex and the MavC/Lpg2149 complex.

Recent describing of atypical, silent and latent form of celiac disease (CD) increased the preocupation for screening methods.To perform a comparative study of immunoglobulin A (IgA) anti tissue-transglutaminase (tTG) antibodies (Ab) assessment using chemiluminescence versus Enzyme Linked Immunosorbent Assay (ELISA).The study included two lots.The first lot consisted in 35 biopsy confirmed CD children aged between two and 18 years.The control lot included 40 children with normal duodenal morphology on intestinal biopsy that underwent upper digestive endoscopy for different gastrointestinal symptoms.Serum samples were provided from all subjects for IgA measurement, IgA anti tTG and EMA assessment. Immulite 2500 Anti tTG IgA (Siemens Co, LA, USA) and ImmuLisa anti-hu tTG antibody IgA ELISA (Immco Diagnostics Inc, NY, USA) kits were used for tTG-Ab assessment. All children underwent HLA typing for DQ2/DQ8. The sensitivity for IgA tTG assessment was greater for chemiluminescence (93,3%) versus ELISA (86,6%), p[0,05, while specificity, positive and negative predictive value didn�t register significant differences. Statistical comparison of the two tested methods revealed a better sensitivity for chemiluminescence. Diagnosis algorithm optimisation may be obtained by associating IgA anti tTG-Ab assessment using chemiluminescence followed by EMA confirmation by indirect immunofluorescence for CD screening.

The microbial TGase from Streptomyces mobaraensis has used in various food industries. However, the detailed substrate specificities of TGases from the Streptomyces species toward the natural peptides remains to be unclear. In this study, we conducted the comparison of two different TGases from Streptomyces mobaranensis (SMTG) and Streptomyces cinnamoneus (SCTG). To clarify the region associated with the characteristics of enzymes, we constructed a chimeric enzyme of CM, of which is consisted of N-terminal half of SCTG and C-terminal half of SMTG. To reveal the differences in the substrate specificity between SCTG and SMTG toward natural peptides, we investigated the time dependence of TGase activity on the productivity of cross-linking peptide with tryptic casein and lysine by using LC-MS. We identified two peptides of “VLPVPQK” and “AVPYPQR” as substrates for both of the TGases.

The functional and instrumental textural properties of reduced-salt meat emulsions (RSMEs) containing konjac gel, with combined transglutaminase (TG), isolated soy protein (ISP), and alginate were investigated to determine optimal quality characteristics. Control groups were produced using 1.2% salt (-) and 1.5% salt (+). RSMEs (1.2% salt) formulations contained T1 (konjac gel), T2 (konjac gel + TG), T3 (konjac gel + ISP), T4 (konjac gel + TG + ISP), T5 (konjac gel + alginate), and T6 (konjac gel + TG + alginate). The moisture content and myofibrillar protein solubility of all RSMEs (from T1 to T6) were significantly higher than that of control (-) (P

Introduction: Enzyme-induced corneal crosslinking (CXL) may be a promising and effective method to stiffen the cornea. However, there have been no reports on the effect of corneal CXL with enzyme in vivo. Objective: To evaluate the biomechanical efficacy after in vivo enzyme-induced corneal CXL in rabbits using noninvasive imaging. Methods: The eyes of 10 white New Zealand rabbits were unilaterally treated with CXL enzyme (transglutaminases [Tgases]). The contralateral eyes served as negative controls. In every rabbit, a 1 U/mL solution of Tgases was applied to the corneal surface. Corneal dynamic deformation by an air puff was measured using high-speed Scheimpflug imaging on day 14 after the procedure. Biomechanical parameters, central corneal thickness, and intraocular pressure were recorded and assessed. Then, corneal buttons were excised from euthanized animals for hematoxylin and eosin staining to evaluate changes in corneal collagen distribution and cell damage. Results: Some biomechanical parameters showed statistically significant changes after in vivo CXL. The deformation amplitude and maximum corneal velocity during the second applanations were significantly lower than those of the untreated control group. The second applanations were significantly increased compared to the control group. No changes were found in other parameters. The morphology of the corneal stroma was similar in CXL-treated and untreated corneas. No obvious cell apoptosis was observed. Conclusions: Corneal stiffness increased after in vivo CXL induced by Tgases. This CXL method may be useful for corneal ectasia.

The increasing sensitivity to gluten has aroused interest in gluten-free products like bread. However, one of the biggest challenges of producing gluten-free bread is to get a good quality structure. We hypothesize that using chitosan along with transglutaminase, a network of crosslinks would be generated, guaranteeing a better structure. Thus, in the present work, we produced gluten-free bread using red rice flour and cassava flour, transglutaminase, and chitosan at concentrations of 0%, 1%, and 2%. Loaves of bread were characterized, and the instrumental texture properties during five days were determined. Bread produced with chitosan and transglutaminase presented lighter brown coloration due to incomplete Maillard reaction and low specific volumes varying from 1.64 to 1.48 cm(3)/g, possibly due to chitosan interfering with yeast fermentation. Rheological tests revealed increases in viscosity before and after fermentation when chitosan was used. Bread with chitosan presented high initial firmness but a lower rate of staling, possibly due to water retention. According to results, a possible network involving chitosan and other proteins promoted by transglutaminase was formed and after optimization could yield better gluten-free bread. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-019-04223-5) contains supplementary material, which is available to authorized users.

Celiac disease is a chronic small bowel disease induced by ingestion of gluten in genetically susceptible individuals affecting 1% among Caucasians in the Western world. The prevalence of celiac disease is still unknown in most developing countries, especially in Africa which suffer from lack of resources to perform screening of the general population. The aim of this study was to determine the prevalence of celiac disease autoimmunity in women undergoing antenatal care in selected Ethiopian health institutes. A total of 1942 pregnant women were included at median 25 (range 15-45) years of age who were attending antenatal care at 14 health centers of Central and South-East Oromia regional state of Ethiopia. Serum samples were analyzed for both IgA and IgG autoantibodies against tissue transglutaminase (tTG) using radioligand binding assays. Celiac disease autoimmunity defined as testing positive for both of IgA-tTG and IgG-tTG. In all, 4 of 1942 (0.2%) were positive for IgG-tTG of whom one participant (0.05%) was positive for both IgA-tTG and IgG-tTG and defined as having celiac disease autoimmunity. Based on these results, it was concluded that celiac disease autoimmunity is expected to be less common among the female adult Ethiopian population compared with the expected prevalence in Caucasians.

peer-reviewed The salt-induced gelation behavior of soy protein isolate (SPI) emulsions was markedly influenced by microbial transglutaminase (TGase) pre-crosslinking. Rheological data showed that when SPI emulsions were incubated with TGase at low concentrations (1 and 3 U/g protein) at 50 °C for 30 min prior to gelation, no change in storage modulus (G′), but enhanced resistance to deformation of the gels was observed. Extensive crosslinking by TGase (5 U/g protein) resulted in severe decreases in gel firmness and fracture properties (yielding stress and strain), likely due to the impairment of hydrophobic bonds and the formation of coarse networks. The water-holding capacity of the gels was significantly enhanced by increased concentrations of TGase. Interactive force analysis indicated that non-covalent interactions and disulfide bonds are the primary forces involved in CaSO4-induced SPI emulsion gel, but TGase treatment may limit hydrophobic interactions within the gel network. These results are of great potential value for the application of TGase in the food industry.

Bovine split hide is the subcutis layer with the low percentage of collagen so that the quality of the gelatin is different from that of the gelatin from cattle’s skin. This study aims to improve the characteristics of bovine split hide gelatin combined with soy protein isolate (SPI) using transglutaminase (TGase) enzyme as a protein cross-linking agent. The study was conducted using a completely randomized design with 3 x 3 factorial pattern consisting of three levels of a mixture of bovine split hide gelatin : SPI at the ratios of 90 : 10, 80 : 20, 70 : 30, and three concentrations of transglutaminase enzyme, i.e. 10, 20, and 30 U. The results showed that treatments significantly affected viscosity, gel strength, the moisture, ash, fat, and protein content of gelatin. Electrophoresis of gelatin protein showed bands distribution between 60-190 kDa. The amino acid profile of the gelatin was similar to that of collagen with a high level of aspartate, glutamate, cysteine, and proline. Morphology of gelatin was observed by Scanning Electronic Microscope (SEM) and showed a compact distributed collagen crosslink. The combination of gelatin bovine split hide and SPI at the ratio of 90 : 10 provides the best physicochemical characteristic.

Background Terminally differentiated keratinocytes acquire corneocyte protein envelopes (CPE) complexed with corneocyte lipid envelopes (CLE). These two structural components of the corneocyte envelopes (CEs) undergo maturation by gaining in hydrophobicity, rigidity and surface area. Linoleoyl acylceramides are processed by 12R‐lipoxygenase (12R‐LOX) and other enzymes before transglutaminase (TG) attaches ω‐hydroxyceramides to involucrin in the CPE. Concurrently, structural proteins are cross‐linked by TG that has been activated by cathepsin D (CathD). Objectives The primary aim of this work was to demonstrate the impact of relative humidity (RH) during ex vivo CE maturation. Low, optimal and high RH were selected to investigate the effect of protease inhibitors (PIs) on CE maturation and TG activity; in addition, 12R‐LOX and CathD activity were measured at optimal RH. Finally, the effect of glycerol on ex vivo CE maturation was tested at low, optimal and high RH. Methods The first and ninth tape strip of photo‐exposed (PE) cheek and photo‐protected (PP) post‐auricular sites of healthy volunteers were selected. Ex vivo CE maturation was assessed via the relative CE maturity (RCEM) approach based on CE rigidity and hydrophobicity. The second and eighth tapes were exposed to RH in the presence of inhibitors. Results Irrespective of tape stripping depth, CEs from PE samples attained CE rigidity to the same extent as mature CEs from the PP site, but such improvement was lacking for CE hydrophobicity. 70% RH was optimal for ex vivo CE maturation. The inhibition of 12R‐LOX activity resulted in enhanced CE rigidity which was reduced by the TG inhibitor. CE hydrophobicity remained unchanged during ex vivo maturation in the presence of TG or 12R‐LOX inhibition. CE hydrophobicity was enhanced in the presence of glycerol at 44% RH and 100% RH but not at 70% RH. Furthermore, TG activity was significantly diminished at 100% RH compared to the commercial inhibitor LDN‐27219. However, a protease inhibitor mix reversed the negative effect of overhydration. Conclusion The study adds to the understanding of the roles of 12R‐LOX and TG activity in CE maturation and gives further insight into the effect of glycerol on the SC., A cocktail of protease inhibitor prior to ex vivo maturation shows a humidity‐dependent effect on TG activity with the biggest impact on samples ex vivo matured at 100% RH. Data are shown in mean ± SD (n = 6) with one‐way ANOVA with Holm–Sidak's post‐test; *P ≤ 0.05, **P ≤ 0.01 or ***P ≤ 0.001.

Even though the role of gluten had been identified in the late 40’s, it was not until the mid-80’s that the long-sought after pathogenesis of celiac disease (CD) was finally unveiled. After years of theories (enzymatic deficiencies, lectin-type effect of gluten…) it became finally clear that celiac disease is an autoimmune disorder. A unique type of autoimmune disease, as a matter of fact: indeed, the only one where both, the environmental trigger and the autoantigen have been well identified: gluten, a complex of protein found in wheat, barley, and rye being the environmental trigger, and tissue Transglutaminase (tTG) the auto antigen. Autoimmune diseases tend to share to various extent their genetic bases, so it is no surprise that CD goes hand in hand with other autoimmune disorders, the best known being Type 1 Diabetes (T1D) and autoimmune thyroid diseases (AITD - Hashimoto thyroiditis and Grave’s disease). But the association with autoimmune disorders goes beyond that with endocrine organs, affecting the heart, the joints, the liver, the connective tissue, the skin. We’ll review such associations and their meaning in terms not only of the possible role played by gluten intake before diagnosis to elicit them, but also in practical terms of diagnosis and follow-up.

Transglutaminases are protein modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is frequently associated with pathological symptoms and particularly with cancer invasiveness. Although rodent TG4 is well characterised, bio-chemical characteristics of human TG4 that could help the understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtiter plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.

Microbial transglutaminase (mTG) is a heavily used food additive and its industrial transamidated complexes usage is rising rapidly. It was classified as a processing aid and was granted the GRAS (generally recognized as safe) definition, thus escaping full and thorough toxic and safety evaluations. Despite the manufacturers claims, mTG or its cross-linked compounds are immunogenic, pathogenic, proinflammatory, allergenic and toxic, and pose a risk to public health. The enzyme is a member of the transglutaminase family and imitates the posttranslational modification of gluten, by the tissue transglutaminase, which is the autoantigen of celiac disease. The deamidated and transamidated gliadin peptides lose their tolerance and induce the gluten enteropathy. Microbial transglutaminase and its complexes increase intestinal permeability, suppresses enteric protective pathways, enhances microbial growth and gliadin peptide’s epithelial uptake and can transcytose intra-enterocytically to face the sub-epithelial immune cells. The present review updates on the potentially detrimental side effects of mTG, aiming to interest the scientific community, induce food regulatory authorities’ debates on its safety, and protect the public from the mTG unwanted effects.

Microbial transglutaminase (MTGase) is an enzyme widely used in the dairy sector to improve the functional properties of protein-based products via the formation of a network between protein molecules. The aim of this study involving cheese from the milk of donkeys was to evaluate the effects of treatment with MTGase at the concentrations of 0 (control), 5, 8 and 10 U/g milk protein on the cheese-making process parameters, as well as the physical and chemical characteristics of the resulting cheese. MTGase influenced the time of gel formation from rennet addition (P < 0.05), with a delay at the two highest concentrations, accompanied by a lower (P < 0.01) pH of cheese and the lowest (P < 0.01) loss in cheese weight at 24 h of storage. The highest gel viscosity (P < 0.01) was observed at the highest concentration of the enzyme, reaching the value of 70 mPa⋅sec after 60 min. The chemical composition and color of the cheeses were not significantly affected by the inclusion of MTGase, regardless of the enzyme concentration. These findings may be of relevance in adapting the cheese-making process and might help in the design of new dairy products from donkey's milk.

Background: In celiac disease, small intestinal transglutaminase 2 causes deamidation of glutamine residues in gluten peptides, which enhances stimulation of T cells and leads to mucosal injury. Inhibition of transglutaminase 2 is a potential treatment for celiac disease. Methods: In a proof-of-concept trial, we assessed the efficacy and safety of a 6-week treatment with ZED1227, a selective oral transglutaminase 2 inhibitor, at three dose levels as compared with placebo, in adults with well-controlled celiac disease who underwent a daily gluten challenge. The primary end point was the attenuation of gluten-induced mucosal damage, as measured by the ratio of villus height to crypt depth. Secondary end points included intraepithelial lymphocyte density, the Celiac Symptom Index score, and the Celiac Disease Questionnaire score (for assessment of health-related quality of life). Results: Of the 41 patients assigned to the 10-mg ZED1227 group, the 41 assigned to the 50-mg group, the 41 assigned to the 100-mg group, and the 40 assigned to the placebo group, 35, 39, 38, and 30 patients, respectively, had adequate duodenal-biopsy samples for the assessment of the primary end point. Treatment with ZED1227 at all three dose levels attenuated gluten-induced duodenal mucosal injury. The estimated difference from placebo in the change in the mean ratio of villus height to crypt depth from baseline to week 6 was 0.44 (95% confidence interval [CI], 0.15 to 0.73) in the 10-mg group (P = 0.001), 0.49 (95% CI, 0.20 to 0.77) in the 50-mg group (P

The method of tumor microenvironment (TME)-responsive aggregation has become a promising approach to enhance treatment effect by improving the accumulation of nanoparticles in tumors. The enzymatic cross-linking strategy has widely attracted attention owing to its good aggregation stability and biocompatibility. However, the enzymes in nontumor tissue can also catalyze the cross-linking reaction and reduce accumulation of nanoparticles in tumor. In this work, a "dual key"-responsive strategy is utilized to construct a transglutaminase (TGase)/pH-responsive radiosensitizer (Au@TAcoGal) with specific aggregation behavior in hepatic tumor cells. Au@TAcoGal can retain its stability in blood circulation (pH 7.4) even in the presence of TGase in plasma. On reaching tumor sites, it can be endocytosed by hepatoma cells by the active targeting of phenylboronic acid (PBA) and aggregated under acidity and overexpression of TGase in cells. Due to its specific accumulation in hepatoma cells, radiotherapy can be operated under a lower dose of X-ray. The results show that the cellular accumulation of Au@TAcoGal increases by 30-70%, and the cell survival rate is less than 25% under X-ray irradiation. The antineoplastic results show that Au@TAcoGal exhibits a higher therapeutic effect, and the tumor inhibition rate can reach 84.21%.

Transglutaminase 2 (TG2) is the most abundant crosslinking enzyme in murine and human cornea, while retinoids are well-known inducers of TG2 expression. This study aims to determine if the retinoic acid supplementation can increase corneal stiffness by crosslinking through upregulating the corneal TG2 expression. The right eyes of C57BL/6 mice were treated with 2×10−2M retinol palmitate (VApal) eyedrops or control eyedrops and hold for 30 min, once a day for 28 consecutive days. The WB and qPCR results showed increased expression of TG2 in murine cornea with the prolongation of VApal eyedrop application. After 28 days of VApal eyedrop treatment, the increased TG2 were found catalytically active and distributed in corneal epithelium and stroma as detected by 5-(biotinamido) pentylamine (5-BP) incorporation method and immunofluorescence staining. The transmission electron microscope image revealed that VApal treated cornea manifested with increased collagen density in anterior and middle layer of stroma. The higher elastic module was found among VApal treated cornea by nano-indentation test. In cultured corneal epithelial cells and keratocytes, all-trans retinoid acid (ATRA) treatment increased the content of TG2 in cell lysis and in culture medium. These results indicate that retinoic acid induce the reinforcement of the cornea by TG2 mediated crosslinking via increasing the TG2 expression in corneal epithelium and keratocyte. As TG2 was found to be less in the cornea of keratoconus patients in several RNA-sequencing studies, retinoic acid could serve as a non-invasive prevention method for keratoconus progression.

The changes in digestibility of TG-treated myofibrillar protein (MP), soybean protein isolate (SPI) and mixed proteins were evaluated by measuring liberation of primary amino groups, monitoring structural changes and investigating peptide fingerprints. TG treatment generally increased gastric digestion of treated proteins, possibly due to the structural changes occurred during TG treatment. In contrast, the initial intestinal digestion was suppressed by TG treatment. Compared with MP, the digestibility and peptide composition of SPI were affected by TG treatment to a larger degree, possibly due to the higher level of glutamine in SPI. Peptidomics analysis indicated that the changes in peptide composition of digests of TG-treated samples were related with the loss of Lys residues during TG treatment. Larger quantities of bioactive peptides KIEFEQFLPM, EVHEPEEKPRPK and TVKEDQVFPMNPPK were released after digestion of TG-treated MP. These results highlighted the complex and substantial influence of TG treatment on the digestibility of dietary proteins.

For a whole century, citrate has been used as an in vitro anticoagulant via chelation of calcium. Later, also EDTA was introduced as an anticoagulant. An often overlooked fact is that zinc is bound to citrate and EDTA with affinities much greater than that for calcium, imposing problems in biomedical research. In vivo, proteins of the S100 family are released from leukocytes and known to bind calcium. Some of them, e.g., calprotectin, also chelate zinc. Thus, at an inflamed site, the ratio between Ca2+ and Zn2+ is changed. This mechanism is of importance for the modulation of the activation of a fascinating family of post-translationally acting calcium-dependent thiol enzymes, the transglutaminases, which are inhibited by zinc. This presentation illustrates the complexity of in vitro studies with zinc. Moreover, it exemplifies the role of Zn2+ in pathophysiological situations such as celiac disease and neurodegeneration., Highlights • Citrate, EDTA and DTT bind zinc as well as calcium. • At inflammation, calprotectin binds Zn2+, which leads to low concentrations of the ion. • Zn2+ inhibits the activation of transglutaminases and peptidylarginine deiminases.

Poor solubility of proteins negatively affects their functional properties and greatly limits their application. Enzymatic cross-linking with polysaccharides can improve solubility and functional properties of proteins. The enzymes used include transglutaminase, laccase and peroxidase. Therefore, this work introduces the cross-linking mechanisms of these enzymes and the characterization techniques, the improved properties and the potential applications of the enzymatically-synthesized protein-polysaccharide conjugate. Transglutaminase catalyzes the formation of a new peptide bond and thus works on amino-containing polysaccharides to conjugate with proteins. However, laccase and peroxidase catalyze oxidation of various compounds with phenol and aniline structures. Therefore, these two enzymes can catalyze the conjugate reaction between proteins and feruloylated polysaccharides which are widely distributed in cereal bran. Compared with the unmodified protein, the enzymatically-synthesized protein-polysaccharide conjugate usually has higher solubility and better functional properties. Thus, it is inferred that enzymatic conjugation with polysaccharide molecules can extend the application of proteins.

Transglutaminases (TGs) are a family of crosslinking enzymes catalyzing the formation of intra- and inter-molecular glutamine-lysine isopeptide bonds in a calcium dependent manner. Activation of transglutaminases is pathogenically associated with severe human diseases including neurodegenerations, cardiovascular diseases, and autoimmune diseases. Although continuous efforts determining the enzymes’ substrate preference have been witnessed, a high-throughput assay platform with the “omic” efficiency is still missing for the global identification of substrate-specific TG modification sites. In this study we report a protein microarray-based in vitro TG assay platform for rapid and large-scale (up to 30000 reactions per chip) determination of the glutamine (Q)-bearing TG modification motifs. With this platform we identified the Q16 in superoxide dismutase 1 and Q109 in alpha-synuclein as the primary modification sites for tissue transglutaminase (TG2), the most ubiquitous member of the enzyme family. Of particular interest, we found a close match between the modification motifs and published vaccine epitope sequences in alpha-synuclein, implying an essential and intrinsic role transglutaminase might play in the determination of immunodominant epitopes. Our data collectively suggest the glutamine and its follow-up five residues on the C terminal of a protein compose a minimal determinant motif for TG2 modification and, more importantly, the TG2 modification motifs determined by our platform could finally correspond to the substrate’s immunodominant epitope sequences in antigen processing. To establish an efficient approach to optimize the enzyme modification motifs and screen for site-specific interfering peptides, we scanned the known TG2 modification motifs with onchip amino-acid swapping and glutamine repeat addition, and obtained multiple variants with significantly upregulated TG2 reactivity. This approach could also be employed to improve the target peptide’s immunogenicity. Taken together, our synthetic transglutaminase assay platform might be able to deliver a precise epitope blueprint for immunotherapeutic targeting and provide pilot and directional studies for TG-based peptide discovery and immunogen design.

Fibrotic lesions accompany several pathological conditions including tumors. We show that expression of a dominant-active form of the Ras oncogene in Drosophila salivary glands (SGs) leads to redistribution of components of the basement membrane (BM) and fibrotic lesions. Similar to several types of mammalian fibrosis, the disturbed BM attracts clot components including insect transglutaminase and phenoloxidase. SG epithelial cells show reduced apico-basal polarity accompanied by a loss of secretory activity. Both the fibrotic lesions and the reduced cell polarity are alleviated by ectopic expression of the antimicrobial peptide Drosomycin (Drs), which also restores secretory activity of the SGs. In addition to ECM components, both Drs and F-actin localize to fibrotic lesions.

Celiac disease is a chronic inflammatory condition of the small intestine caused by aberrant adaptive immune response to gluten protein from wheat and related cereal plants. Over 90% of celiac disease patients carry the HLA-DQ2.5 allotype and HLA-DQ2.5 presents gluten peptides to gluten-reactive CD4+ T cells in celiac disease patients. A large number of HLA-DQ2.5-restricted gluten T cell epitopes have been identified over the years. These epitopes are in general proline-rich and contain at least one glutamic acid residue that is generated from glutamine in the native gluten protein by deamidation. The deamidation is mediated by the enzyme transglutaminase 2 (TG2). It has been shown that the same T cell could recognize several different HLA-DQ2.5-restricted gluten T cell epitopes due to sequence similarities. In this paper, we demonstrate that three T cell clones derived from duodenal biopsies of different celiac disease patients are able to respond to at least five different gluten T cell epitopes within the DQ2.5-glia-γ4 epitope family, including two novel epitopes.

Transglutaminase is an effective enzyme that catalyzes the crosslinking of various meats, improves meat product quality, and is widely used in the meat industry. In this study, microbial transglutaminase (MTG) was expressed successfully in Pichia pastoris strain GS115, and the enzyme activity was approximately 0.70 U/ml. The recombinant MTG expressed in P. pastoris was used in the investigation of restructured pork and crosslinking of soy protein isolate (SPI) and chicken myofibrillar protein (MP). Results showed that the hardness, chewiness, and F1 of the restructured pork increased, and the adhesiveness decreased after MTG treatment, However, high temperature had greater effect on the texture of restructured pork after MTG treatment than that of the control. MTG can crosslink SPI component acidic subunits, subunits of β-conglycinin and MP component myosin heavy chain, and actin. MTG, as a food additive, can be successfully heterologously expressed, and the recombinant MTG has potential application in restructured meat products.

Clinical trials suggested that the vascular system can remember episodes of poor glycemic control through a phenomenon known as hyperglycemic memory (HGM). HGM is associated with long-term diabetic vascular complications in type 1 and type 2 diabetes, although the molecular mechanism of that association is not clearly understood. We hypothesized that transglutaminase 2 (TGase2) and intracellular reactive oxygen species (ROS) play a key role in HGM-induced vascular dysfunction. We found that hyperglycemia induced persistent oxidative stress, expression of inflammatory adhesion molecules, and apoptosis in the aortic endothelium of HGM mice whose blood glucose levels had been normalized by insulin supplementation. TGase2 activation and ROS generation were in a vicious cycle in the aortic endothelium of HGM mice and also in human aortic endothelial cells after glucose normalization, which played a key role in the sustained expression of inflammatory adhesion molecules and apoptosis. Our findings suggest that the TGase2-ROS vicious cycle plays an important role in HGM-induced endothelial dysfunction.—Lee, J.-Y., Lee, Y.-J., Jeon, H.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory–induced endothelial dysfunction.

The hallmark of fibrosis is an excessive accumulation of collagen, ultimately leading to organ failure. It has become evident that the deposited collagen also exhibits qualitative modifications. A marked modification is the increased cross-linking, leading to a stabilization of the collagen network and limiting fibrosis reversibility. Not only the level of cross-linking is increased, but also the composition of cross-linking is altered: an increase is seen in hydroxyallysine-derived cross-links at the expense of allysine cross-links. This results in irreversible fibrosis, as collagen cross-linked by hydroxyallysine is more difficult to degrade. Hydroxyallysine is derived from a hydroxylysine in the telopeptides of collagen. The expression of lysyl hydroxylase (LH) 2 (LH2), the enzyme responsible for the formation of telopeptidyl hydroxylysine, is universally up-regulated in fibrosis. It is expected that inhibition of this enzyme will lead to reversible fibrosis without interfering with the normal repair process. In this review, we discuss the molecular basis of collagen modifications and cross-linking, with an emphasis on LH2-mediated hydroxyallysine cross-links, and their implications for the pathogenesis and treatment of fibrosis.