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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.

This chapter summarizes multiple points of convergence between chronic kidney disease and kidney senescence to highlight the proposition that both entities are deeply intertwined. Next, I describe geronic proteins using as an example the interactions between endostatin and tissue transglutaminase. I propose that the stress-induced premature senescence of kidney-resident cells constitutes a platform for progression of chronic kidney disease. The description of fate of senescent cells from enforced rejuvenation to a point of no-return or apoptosis follows. Local and systemic effects of senescent cells, including the release of senescence-associated secretory phenotype products, are delineated. The chapter concludes with the recounting of therapeutic strategies to limit the senescent cell burden and, by doing so, arresting the progression of chronic kidney disease.

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.

The objective of this research was to investigate the efficacy of procyanidin B2 rich cocoa extracts on interferon-gamma (IFN-γ) or gliadin peptide p31-43-induced transglutaminase-2 and interleukin-15 (IL-15) in Caco-2 cell model of in vitro celiac disease. Cysteamine was used as a positive control inhibitor of TG2. Procyanidin B2-rich extract reduced IFN-γ- and gliadin p31-43-induced TG2 activity by 77% and 45%, respectively. Cocoa extract containing 4.9–289 µg/mL of procyanidin B2 dose-dependently reduced IFN-γ-induced IL-15 secretion to the level of IL-15 in control cells. Similarly, cocoa extract containing 4.9–289 µg/mL of procyanidin B2 dose-dependently reduced gliadin p31-43-induced IL-15 to the level in control cells. Procyanidin B2-rich cocoa extracts reduced the activities of other inflammatory biomarkers including COX-2, IL-1β, IL-6, and IL-8 in both IFN-γ and p31-43-treated Caco-2 cells. Caffeine or theobromine, at the concentration found in the cocoa extracts, did not contribute to the activity of procyanidin B2 against TG2 or IL-15.

Dermatofibromas are common benign skin lesions, the etiology of which is poorly understood. We identified two unrelated pedigrees in which there was autosomal dominant transmission of multiple dermatofibromas. Whole exome sequencing revealed a rare shared heterozygous missense variant in the F13A1 gene encoding factor XIII subunit A (FXIII-A), a transglutaminase involved in hemostasis, wound healing, tumor growth, and apoptosis. The variant (p.Lys679Met) has an allele frequency of 0.0002 and is predicted to be a damaging mutation. Recombinant human Lys679Met FXIII-A demonstrated reduced fibrin crosslinking activity in vitro. Of note, the treatment of fibroblasts with media containing Lys679Met FXIII-A led to enhanced adhesion, proliferation, and type I collagen synthesis. Immunostaining revealed co-localization between FXIII-A and α4β1 integrins, more prominently for Lys679Met FXIII-A than the wild type. In addition, both the α4β1 inhibitors and the mutation of the FXIII-A Isoleucine-Leucine-Aspartate-Threonine (ILDT) motif prevented Lys679Met FXIII-A-dependent proliferation and collagen synthesis of fibroblasts. Our data suggest that the Lys679Met mutation may lead to a conformational change in the FXIII-A protein that enhances α4-integrin binding and provides insight into an unexpected role for FXIII-A in the pathobiology of familial dermatofibroma.

The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on the heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, suggesting that TG2-externalisation depends on an intact TG2 heparin binding site. Inhibition of N-ethylmaleimide sensitive factor (NSF) vesicle-fusing ATPase, which was identified in the recently elucidated TG2 kidney membrane-interactome, led to significantly lower TG2-externalisation, thus validating the involvement of membrane fusion in TG2 secretion. As cyclin-G-associated kinase (GAK) had emerged as a further TG2-partner in the fibrotic kidney, we investigated whether glucose-induced TG2-externalisation was accompanied by TG2 phosphorylation in consensus sequences of cyclin-dependent kinase (CDK). Glucose stress led to intense TG2 phosphorylation in serine/threonine CDK-target. TG2 phosphorylation by tyrosine kinases was also increased by glucose. Although the precise role of glucose-induced TG2 phosphorylation is unknown, these novel data suggest that phosphorylation may be involved in TG2 membrane-trafficking.

Background and aims Transglutaminase (TG) 2 and Factor (F) XIII-A have both been implicated in cardiovascular protection and repair. This study was designed to differentiate between two competing hypotheses: that TG2 and FXIII-A mediate these functions in mice by fulfilling separate roles, or that they act redundantly in this respect. Methods Atherosclerosis was assessed in brachiocephalic artery plaques of fat-fed mixed strain apolipoprotein (Apo)e deficient mice that lacked either or both transglutaminases. Cardiac fibrosis was assessed both in the mixed strain mice and also in C57BL/6J Apoe expressing mice lacking either or both transglutaminases. Results No difference was found in the density of buried fibrous caps within brachiocephalic plaques from mice expressing or lacking these transglutaminases. Cardiac fibrosis developed in both Apoe/F13a1 double knockout and F13a1 single knockout mice, but not in Tgm2 knockout mice. However, concomitant Tgm2 knockout markedly increased fibrosis, as apparent in both Apoe/Tgm2/F13a1 knockout and Tgm2/F13a1 knockout mice. Amongst F13a1 knockout and Tgm2/F13a1 knockout mice, the extent of fibrosis correlated with hemosiderin deposition, suggesting that TG2 limits the extravasation of blood in the myocardium, which in turn reduces the pro-fibrotic stimulus. The resulting fibrosis was interstitial in nature and caused only minor changes in cardiac function. Conclusions These studies confirm that FXIII-A and TG2 fulfil different roles in the mouse myocardium. FXIII-A protects against vascular leakage while TG2 contributes to the stability or repair of the vasculature. The protective function of TG2 must be considered when designing clinical anti-fibrotic therapies based upon FXIII-A or TG2 inhibition., Graphical abstract Image 1, Highlights • Double transglutaminase 2 and Factor XIII-A knockout exacerbates cardiac fibrosis. • Double knockout does not promote the growth of, or destabilise, brachiocephalic plaques. • FXIII-A in resident cardiac macrophages does not protect against cardiac fibrosis. • FXIII-A in inflammatory macrophages may contribute to protection against fibrosis. • Transglutaminase 2 and Factor XIII-A protect against extravasation of blood.

Chemically modified proteins are increasingly being tested and approved as therapeutic products. Batch-to-batch homogeneity is crucial to ensure safety and quality of therapeutic products. Highly selective protein modification may be achieved using enzymatic routes. Microbial transglutaminase (mTG) is a robust, easy to use and well-established enzyme that is used at a very large scale in the food industry such that its efficacy and its safety for human consumption are well established. In the context of therapeutic protein modification, mTG should crosslink one or more glutamines on the target protein with an aminated moiety such as a solubilizer, a tracer or a cytotoxic moiety. mTG has the advantage of being unreactive toward the majority of surface-exposed glutamines on most proteins, reducing sample heterogeneity. The caveat is that there may be no reactive glutamine on the target protein, or else a reactive glutamine may be found in a location where its modification compromises function of the target protein. Here we describe the glutamine-walk (Gln-walk), a straightforward method to create a glutamine-substrate site that is reactive to mTG in a target protein. Iterative substitution of single amino acids to a glutamine is followed by facile identification of reactivity with mTG, where covalent labeling of the target with an aminated fluorophore allows visualization of the most reactive modified targets. The approach is empirical; knowledge of the target protein structure and functional regions facilitates application of the method.

Objective Due to the high awareness of coeliac disease and improvement of serological tests, the number of requested laboratory tests has increased substantially over the years. In the current study we have evaluated the requesting behaviour of distinct clinical disciplines in relation to the prevalence of positive results and in the context of existing guidelines. Methods Data were retrospectively extracted from the laboratory information system over a time-span of 5 years in a tertiary hospital and compared with the situation in a secondary hospital. Results Data reveal that for initial testing (n=18,183) the percentage positive results for IgA anti-TTG is, Highlights • The prevalence of IgA anti-TTG for diagnosis of coeliac disease is very low. • The prevalence of IgA anti-TTG for diagnosis of paediatric patients is higher. • The request for IgA anti-TTG occurs more often in females than in males (2:1). • The request for IgA anti-TTG occurs as often in girls as in boys (1:1). • Positive IgA anti-TTG are not consistently confirmed in an independent sample.

WOS: 000535481200006 In this study, improvement of some properties of anchovy by-product protein (ABP-P) films by addition of transglutaminase (TGase), and the correlation between secondary structural results and mechanical properties were investigated. The solubility and water vapor permeability of films improved by the TGase treatment at low concentrations. The highest tensile strength value was obtained in films containing 5 % TGase, and the elongation at break values decreased with the TGase concentration increasing. Glass transition temperature (T-g) of films decreased depending on TGase concentration and the lowest Tg was observed from 5 % TGase treated film. Fourier transform infrared spectra of films displayed similar backbone structures with different band intensities. The secondary structure of films from Amide-I band was significantly affected by TGase addition and the results were well correlated with mechanical properties. TGase addition modified the film surface structure and 3 % concentration caused the lowest surface roughness. These results revealed that mechanical, barrier and surface properties of ABP-P films could be modified with 3 % TGase treatment.

Oat and faba bean protein isolates were treated with transglutaminase from Streptomyces mobaraensis and tyrosinase from Trichoderma reesei to modify the colloidal properties of protein particles in order to improve their colloidal stability and foaming properties. Transglutaminase crosslinked faba bean protein extensively already with 10 nkat/g enzyme dosage. Oat protein was crosslinked to some extent with transglutaminase with higher dosages (100 and 1000 nkat/g). Transglutaminase increased the absolute zeta-potential values and reduced the particle size of oat protein particles. As a result, the colloidal stability and foaming properties were improved. Tyrosinase had limited crosslinking ability on both plant protein materials. Tyrosinase greatly reduced the solubility of oat protein despite limited crosslinking. Tyrosinase did not have effect on zeta-potential or colloidal stability of either protein, but it impaired foaming properties of both. Thus, the crosslinking enzymes studied caused significantly different end product functionality, presumably due to the different mechanism of action.

The meat industry is experiencing a change due to an increase in consumer demand for quality products. The foremost approach is on animal management prior to slaughter and the postslaughter carcass handling. These seem to have effect on physiological and biochemical changes (involving a number of enzymes) evidenced in the conversion of muscle to meat, which impacts the quality. Another issue of concern is the tenderness of meat, the most desired sensory parameter in acceptance of the product. There are several endogenous enzymes, like calpains and lysosomal cathepsins, responsible for the reduction in toughness of meat during aging. However, the meat industry utilizes exogenous enzymes like papain, bromelain, and ficin from plant sources to achieve the desired tenderness in meat quality. Several other sources like bacteria and fungi have also been explored as tenderizing enzymes, especially proteases. Proteases can be used for the production of bioactive peptides to improve the quality of meat as these peptides have been associated with several pro-health effects. Similarly, an enzyme known as transglutaminase (meat glue), has found its importance in the meat industry, as it binds low-valued meat selections and creates large meat chunks of desired size and higher quality. Transglutaminases have been acclaimed to be widely used in the formulation of different meat products as it is responsible for the stable cross-links between protein molecules and acts as a binding agent.

Hemolymph coagulation is among the major arms of the humoral immune response in crustaceans. According to the current model, hemolymph clotting in decapod crustacean relies mostly on the polymerization of the plasmatic clotting protein (CP) which is directly promoted by calcium-depended transglutaminase (TGase) released from hemocytes upon microbial stimulus or injury. However, the type of hemocytes containing TGase, and hence how the TGase is released, might vary among species. Thus, we discourse here about possible mechanisms for clotting initiation. On the other hand, the initiation of coagulation reaction in the absence of microbial elicitors is poorly understood and seems to involve hemocytes lability, yet the mechanism remains unknown. A cellular clottable protein called coagulogen, different to the plasma CP, occurs in several species and could be related with the immune response, but the biological relevance of this protein is unknown. It is also demonstrated that the clotting response is actively involved in defense against pathogens. In addition, both TGase and the CP show pleiotropic functions, and although both proteins are relatively conserved, some of their physic-chemical properties vary significantly. The occurrence of differences in the clotting system in crustaceans is conceivable given the high number of species and their diverse ecology. Results from still non-studied decapods may provide explanation for some of the issues presented here from an evolutionary perspective.

Transglutaminases are cross-linking enzymes available for catalyzing covalent isopeptide bond formation among protein molecules of different origins. These enzymes, mainly the microbial molecular form, are considered a possible tool to improve physicochemical properties such as solubility, water binding or emulsifying capacity, foaming, viscosity, elasticity, and gelation of proteins intended for human consumption. In recent years, different biotechnological applications of transglutaminases have also come to light in areas ranging from material sciences to medicine. In this chapter, we review the most important applications of such biocatalysts in the preparation of different innovative biodegradable materials that are potentially useful as edible films and food coatings.

High intensity ultrasound (HIU) treated soy protein isolate (SPI) and non-HIU-treated SPI were cross-linked by transglutaminase to form hydrogels. SDS-PAGE showed that HIU increased the amount of high molecular weight aggregates, likely due to the formation of e-(γ-glutamyl) lysine bonds. Moreover, HIU pretreatment increased the hydrophobic nature of transglutaminase gels as demonstrated by FT-Raman. HIU changed the 3D-network structure of transglutaminase induced SPI gel with riboflavin (TSGR). Furthermore, 40 min HIU increased gel yield, riboflavin encapsulation efficiency and gel strength of TSGR. HIU decreased swelling and protein erosion of TSGR in simulated gastrointestinal fluids. It also resulted in reduced riboflavin release rate and altered the release mechanism in simulated gastrointestinal fluids both in the absence and presence of digestive enzymes. In conclusion, HIU may facilitate covalent cross-linking, increase hydrophobicity and change the 3D network of TSGR, leading to differences in hydrogel stability, as well as riboflavin encapsulation and release profiles.

Celiac disease (CD) is an immune-mediated disease affecting the small intestine secondary to gluten exposure. The currently available treatment is lifelong adherence to a gluten-free diet (GFD). Several disorders are known to be associated with celiac disease, including Down syndrome (DS). In several studies, the prevalence of CD in DS ranged between 4 and 17%. CD is prevalent in Arabs; however, few studies have been performed to determine the prevalence of CD in DS patients. Our study aimed to determine the prevalence of CD in Saudi Down syndrome patients using serological markers and small bowel biopsy. This is a retrospective study in which files relating to Down syndrome patients who were followed up in a general pediatric clinic at King Faisal Specialist Hospital and Research Center were reviewed regarding demographic data, serological markers and biopsy results. Of the total number of patients reviewed (91), 7 were excluded because data were missing; the remaining 84 patients included 35 females and 49 males. The age range of the patients at the time of screening was from 1 to 18 years. Patient demographic data are shown in Table 1. Among the studied patients, antigliadin antibody (AGA) IgA was high in 27 patients (32.14%), and AGA IgG was high in 44 patients (52.38%). Twelve patients (14.28%) tested positive and 58 (69.04%) tested negative for anti-endomysial antibodies. Anti-tissue glutaminase antibody IgA was found to be high in 13 patients (15.5%) and normal in 54 patients (64.28%). Serum IgA levels were normal in 36 patients (43%) and low in 1 patient (1.2%). Biopsy was performed in 22 patients who tested positive for anti-endomysial or anti-tissue transglutaminase antibodies. The biopsies provided positive results in 9 patients (10.7%). Our study showed a confirmed prevalence of 10.7% for celiac disease in Saudi patients with Down syndrome based on serology and biopsy; together with previous cases reported in the literature, this result indicates a need to screen these patients for celiac disease.

In this chapter, structure, gels and gelling processes, and emulsions and emulsifications involving various soy protein fractions are reviewed and structures in solid state are determined mainly by X-ray diffraction, solubility and aggregation in solution studied by light scattering and related techniques. Gels and gelling processes of defatted soy proteins (globulins) with and without various coagulants, calcium chloride, calcium sulfate, glucono-delta-lactone, transglutaminase, and the role of coexisting ingredients such as oil, phytate have been studied. Various factors controlling the emulsification, pH, ionic strength, temperature, mechanical stress, chilling, and freezing have been described. Methods to improve the gelling and emulsifying have been described. The process for the development of soy-based products such as yogurt, cheese, natto, tempeh, shoyu, miso, douchi, fibrous meat-like soy protein, frozen tofu, okara, and ice creams are also described.

peer-reviewed Angiotensin converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities of crosslinked and non-cross-linked sodium caseinate (NaCN) hydrolysates were studied. Three different samples were generated: NaCN hydrolysed with Prolyve 1000 (TM) (Prolyve), NaCN cross-linked with transglutaminase (TGase) pre-Prolyve hydrolysis and NaCN cross-linked post-Prolyve hydrolysis. Gel filtration and reverse phase HPLC analysis of the resulting samples indicated that the hydrolysates had similar peptide profiles. Hydrolysates showed higher (p 0.05) differences in activity were found between cross-linked and non-cross-linked hydrolysate samples. Hydrolysate IC50 values for ACE and DPP-IV inhibition ranged from 0.10 to 0.17 mg mL(-1) and 0.85-1.18 mg mL(-1), respectively. Simulated gastrointestinal digestion had no significant (p > 0.05) effect on the bioactivities of the hydrolysates. The results demonstrated that incubation with TGase before or after NaCN hydrolysis with Prolyve had no effect on ACE or DPP-IV inhibitory activities. (c) 2017 Elsevier Ltd. All rights reserved. peer-reviewed

Proteins hold a central role in medicine and biology, also confirmed by the several therapeutic applications based on biologic drugs. Such therapies are of great relevance thanks to high potency and safety of proteins. Nevertheless, many proteins as therapeutics might present issues like fast kidney clearance, rapid enzymatic degradation, or immunogenicity. Such defects implicate frequent administrations or administrations at high doses of the therapeutics, thus yielding or exacerbating potential side effects. A successful technology for improving the clinical profiles of proteins is the conjugation of polymers to the protein surface. The design of a protein-polymer conjugate presents critical aspects that determine the efficacy and safety of the final product. The control over stoichiometry and conjugation site is a strict criterion on which researchers have been intensively focused during the years, in order to obtain homogeneous and batch-to-batch reproducible products. An innovative site-specific conjugation strategy relies on the use of enzymes as tools to mediate polymer conjugation. Enzymatic approaches are attractive because they allow site-selective polymer conjugation at specific protein amino acids. In these reactions, the polymer is a substrate analog that replaces the native substrate. Furthermore, enzymes can count other advantages such as high yields of conversion and physiological conditions of reaction. This chapter provides a meaningful description of protein-polymer conjugation through transglutaminase-mediated and sialyltransferase-mediated enzymatic strategies, reporting the mechanism of action and some relevant examples.

The human transglutaminases (TGases) are a widely distributed and peculiar group of enzymes that catalyze the posttranslational modification of proteins by the formation of isopeptide bonds. Tissue or type 2 transglutaminase (TG2) represents the most ubiquitous isoform belonging to TGases family. The vast array of biochemical functions catalyzed by TG2 distinguishes it from the other members of the TGase family. In the presence of high calcium levels TG2 catalyzes a vast array of protein posttranslational modifications, including protein-protein cross-linking, incorporation of primary amines into proteins, as well as glutamine deamination. In the last few years, it has become evident that TG2 is involved in the final maturation of autolysosomes. The TG2 regulation of autophagy occurs by its transamidating activity and its inhibition results in the intracellular increase of ubiquitinated protein aggregates. In this chapter, we describe the methods used in our laboratories to assess the catalytic activity of TG2 in the autophagic process.

Whey proteins were hydrolyzed with pepsin to antioxidant peptides, cross-linked by transglutaminase and then particulated through ethanol addition. Reducing power assay indicated that cross-linking enhanced the antioxidant activity of hydrolysate. Particles prepared from non-hydrolyzed protein, peptides and cross-linked peptides were all of bimodal size distribution comprised from sub-micron (>100 nm) and nanoscalar (

Mitochondria produce the bulk of cellular energy and work as decisional "hubs" for cellular responses by integrating different input signals. The determinant in the physiopathology of mammals, they attract major attention, nowadays, for their contribution to brain degeneration. How they can withstand or succumb to insults leading to neuronal death is an object of great attention increasing the need for a better understanding of the interplay between inner and outer mitochondrial pathways residing in the cytosol. Of the latter, those dictating protein metabolism and therefore influencing the quality function and control of the organelle are of our most immediate interest and here we describe the Transglutaminase type 2 (TG2) contribution to mitochondrial function, dysfunction and neurodegeneration. Besides reviewing the latest evidences we share also the novel ones on the IF1 pathway depicting a molecular conduit governing mitochondrial turnover and homeostasis relevant to envisaging preventive and therapeutic strategies to respectively predict and counteract deficiencies associated with deregulated mitochondrial function in neuropathology. (C) 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

SummaryTissue transglutaminase (tTG) is a GTP-binding protein/acyltransferase whose expression is upregulated in glioblastoma and associated with decreased patient survival. Here, we delineate a unique mechanism by which tTG contributes to the development of gliomas by using two glioblastoma cell lines, U87 and LN229, whose growth and survival are dependent on tTG. We show that tTG significantly enhances the signaling activity and lifespan of EGF receptors (EGFRs) in these brain cancer cells. Moreover, overexpressing tTG in T98G glioblastoma cells that normally express low levels of tTG caused a marked upregulation of EGFR expression and transforming activity. Furthermore, we show that tTG accentuates EGFR signaling by blocking c-Cbl-catalyzed EGFR ubiquitylation through the ability of tTG to bind GTP and adopt a specific conformation that enables it to interact with c-Cbl. These findings demonstrate that tTG contributes to gliomagenesis by interfering with EGFR downregulation and, thereby, promoting transformation.

Microbial transglutaminase (mTGase; EC 2.3.2.13) belongs to the protein-glutamine γ -glutamyltransferase family and catalyzes the formation of covalent cross-links in protein-containing systems, which affect several technofunctional properties of the resulting foods. Milk proteins, especially the caseins, are excellent substrates. Also, enzyme-modified sodium caseinate may serve as an interesting adjunct in the food industry. In the dairy sector the fermented milks segment is of special interest for the application of mTGase. Set-style yogurt exhibits higher gel firmness and reduced syneresis after enzyme treatment, and stirred yogurt exhibits higher viscosity, allowing a reduction in dry matter fortification in the process of yogurt manufacturing. Possible product defects such as a gritty and coarse structure after cold storage in the case of a simultaneous cross-linking and fermentation step highlight the importance of carefully controlling the cross-linking process with mTGase.

Celiac disease is a multifactorial disease influenced by environmental and genetic factors. The most important environment factor is dietary gluten proteins, whereas HLA genes constitute the most important genetic factor. In individuals who are HLA-DQ2.5, HLA-DQ8, or HLA-DQ2.2, dietary gluten can induce chronic inflammation in the small intestinal mucosa which is reversible upon removal of gluten. A key event in the inflammatory processes instigated by gluten is the activation of CD4 + T cells. These T cells are specific to deamidated gluten epitopes presented by the disease-associated HLA-DQ molecules. Gluten-reactive CD4 + T cells appear to control the activation of intraepithelial cytotoxic T cells, as well as inducing B cell activation and plasma cell differentiation. The presence of serum IgA and IgG antibodies reactive to the autoantigen transglutaminase 2 is a specific feature of celiac disease. Celiac disease may serve as a model disease for understanding autoimmune diseases.

The mechanical properties and microstructure of 2.7% and 4.5% sodium caseinate gels chemically acidified by glucono-δ-lactone (GDL) and cross-linked by microbial transglutaminase (TG) were studied. The acidification was performed at different temperatures. According to SDS–PAGE TG clearly caused polymerisation of caseinate irrespective of the treatment temperature (4–50 °C), The cross-linking of the proteins was more extensive at temperatures 22–50 °C. Low amplitude viscoelastic measurements showed that 4.5% caseinate gels acidified at 50 °C were formed much faster than gels acidified at 22 °C. TG only slightly increased the time of gelling. Control gels prepared without TG at temperatures of 4, 22, 37 and 50 °C were mechanically weak. Examination of the control gels with a confocal laser scanning microscope showed that gels formed at 37 and 50 °C were coarse and porous with large cavities between particle aggregates, whereas those formed at 22 °C were much more homogeneous. The TG-treated and acidified sodium caseinate dispersions formed firm gels, indicating cross-linking of casein proteins. Interestingly, the strongest gels were formed at 22 and 37 °C. TG treatment improved the homogeneity of the gel structure at temperatures of 37 and 50 °C. The hardness of TG-treated gels acidified at 4 °C increased during 1 week of storage.

The texture of many dairy products is principally dependent on the properties and interactions of milk proteins, i.e., caseins and whey proteins, and their ability to form structures such as networks and gels when manipulated appropriately, e.g., by targeted enzymatic hydrolysis, acidification or heat-induced denaturation. The structures formed in this way lead directly to the structures associated with products such as cheese and yoghurt, as well as undesirable coagula which might occur for example heating or storage of sterilized or ultra-high-temperature (UHT)-treated milk. In addition, hydrolysis of milk proteins greatly influences dairy product structure, which may be problematic in the case of pre-processing hydrolysis of proteins by indigenous milk protease, or a positive and indeed critical role in the development of cheese texture during ripening. In recent years, significant attention has focused on new strategies for manipulating the texture of dairy products by exploiting the ability of certain enzymes to cross-link milk proteins, opening up new possibilities of manipulating the characteristics and stability of dairy products. Of these, the most well studied is probably transglutaminase, applications of which have been commercially implemented; other enzymes which have shown cross-linking activity against milk proteins include lactase, tyrosinases and peroxidases. The principal products for which these have been proposed to have benefit are yoghurt and cheese. In this chapter, the main strategies for enzymatic cross-linking of milk proteins, and consequent effects on dairy product texture, are reviewed. In addition, the significance of other enzymatic treatments, such as dephosphorylation, deglycosylation and deamidation, when applied to milk proteins is discussed, and the future perspective for enzymatic modification of dairy product texture considered.

The effects of β-lactoglobulin (βLg) hydrolysis with alcalase or bromelain and its association with polymerization by transglutaminase (TG) on both the protein antigenicity and resistance of epitopes to in vitro digestion were investigated. Despite the polymerization with TG altered the gastric and gastrointestinal (GI) digestion patterns of the hydrolysates with bromelain, no changes were observed with alcalase. Before and after in vitro digestion, native βLg showed the highest IgE-binding capacity, evaluated using sera from milk-allergic patients, while the enzymatic treatments—associated or not—reduced the βLg antigenicity response. Seven and four epitopes were found after gastric and GI digestion of native βLg, respectively. After gastric digestion of the hydrolysates with bromelain, two epitopes were identified, being Tyr42–Leu54 resistant to GI digestion. No epitope was found in GI digested products of hydrolysates with alcalase. Hydrolysis associated or not with polymerization reduced the number of epitopes and the IgE-binding capacity of the samples., This study received financial support from FAPESP (Brazil), CNPq (Brazil), AGL2011-24740. E. Molina is participant of the COST Action FA 1402. S. Benedé acknowledges the financial support of CSIC through JAE Pre. M. Villas-Boas and F. M. Netto also acknowledge their CNPq grants.

Transglutaminase (TG) modifies proteins by amine incorporation, crosslinking and deamination. It is used as an important tool in food processing. The aim of this work was to crosslink ovalbumin (OVA) and egg white (EW) with TG, either under high pressure (HP) or under atmospheric pressure on previously HP-treated proteins, to assess the allergenic potential of polymerized allergens. The susceptibility of OVA and EW to TG-mediated crosslinking was enhanced by HP (400 MPa, 40 °C, 30 min). Simultaneous or sequential HP and TG treatments led to the formation of high-molecular weight polymers, but left a substantial amount of monomeric proteins. Results of digestibility experiments, binding to immunoglobulin E (IgE) from egg allergic patients and stimulation of spleen and mesenteric lymph node cell cultures of EW-sensitized BALB/c mice showed that resistance to proteolysis, IgE-binding and immunostimulatory properties of the HP and TG-treated proteins were not substantially different from those of the native ones. [Industrial Relevance]: The use of transglutaminase as a crosslinking agent has been suggested for many industrial applications, as in the case of meat, fish, milk or soy proteins, which are good substrates for this enzyme. This work widens the possible use of transglutaminase, for modifying less suitable substrates for crosslinking, as egg proteins, through the use of high pressure before or during crosslinking. In our study, HP-assisted TG-induced OVA and EW crosslinking enhanced gastroduodenal digestion but did not reduce the allergenicity of egg proteins., Financial support was received from AGL2011-24740 project from MINECO (Spain). E. M, R. L-F and D. L-O are participants in the COST-Action ImpARAS FA1402. D. L-O is a recipient of an FPU fellowship from MECD (Spain). The author X.J. Ma thanks the financial support from the China Scholarship Council (CSC, 201306820013).

Among the adverse reactions caused by wheat, celiac disease (CD) is the longest studied and best-known pathology. The more recently defined non-celiac gluten sensitivity (NCGS) presents with symptoms which are often indistinguishable from CD. Diagnosis of CD is based on serologic, molecular, and bioptic testing. The IgA anti-transglutaminase (tTG) test is considered highly important, as it shows high sensitivity and specificity and its levels correlate to the degree of intestinal damage. Small bowel biopsy can be avoided in symptomatic patients with IgA anti-tTG levels above 10× the manufacturer's cut-off. Recently, tests of anti-deamidated peptides of gliadin (DGP) have replaced classic anti-native gliadin (AGA) tests. DGP assays have a considerably higher diagnostic accuracy than AGA assays, especially in the IgG class, and can replace anti-tTG tests in patients with selective IgA deficiency. The combination of IgG anti-DGP plus IgA anti-tTG assays show greater sensitivity than a single test, with very high specificity. EMA tests have great diagnostic accuracy but are not recommended by all the latest guidelines because they are observer dependent. Biopsy must still be considered the gold standard for CD diagnosis. HLA-DQ genotyping can be used to screen asymptomatic children and in cases of histology/serology disagreement. About half of NCGS patients are DQ2 positive and have IgG AGA. To diagnose NCGS, first CD and wheat allergy must be excluded; then the wheat dependence of symptoms must be verified by a gluten-free diet and subsequent gluten challenge.

Tyramine, histamine, putrescine and cadaverine, the most common biogenic amines indicating the food quality, were studied in the transglutaminase-catalyzed reaction. Transglutaminase (protein-glutamine gamma-glutamyltransferase EC 2.3.2.13) catalyzes an acyl transfer reaction between a donor substrate and an acceptor substrate (e.g. biogenic amine) and forms a cross-linkage between substrates with a release of ammonia. The reaction can be monitored by measuring the ammonia produced in the reaction. The concentration of produced ammonia was found to be proportional to the concentration of biogenic amine and could hence be used to determination of biogenic amines in food matrixes.

Lupine protein hydrolysates (LPHs) were obtained from a lupine protein isolate (LPI) by enzymatic hydrolysis using two proteases, Izyme AL and Alcalase 2.4 L, and their potential anti-inflammatory capacities were studied by determining their in vitro inhibition of the following enzymes that are involved in the inflammatory process: phospholipase A2 (PLA2), cyclooxygenase 2 (COX-2), thrombin, and transglutaminase (TG). The strongest inhibitory activities toward PLA2 and TG were found in the hydrolysates obtained by hydrolysis with Izyme and subsequently with Alcalase, with more than 70% inhibition obtained in some cases. All of the hydrolysates tested inhibited more than 60% of the COX-2 activity. In no case did the percentage of thrombin activity inhibition exceed 40%. The best inhibitory activities were found in the LPH obtained after 15 min of hydrolysis with Alcalase and in the LPH obtained after 60 min of hydrolysis with Izyme followed by 15 min of hydrolysis with Alcalase. Enzyme kinetic analyses were conducted to determine the Km and Vmax parameters of these two hydrolysates using the Lineweaver-Burk equation. Both hydrolysates competitively inhibited the thrombin and PLA2 activities. In the case of COX-2 and TG, the inhibition appeared to be the mixed type. © 2013 Elsevier Ltd. All rights reserved., This work was supported by the Grant AGL2012-40247-C02-01 from the Spanish Ministry of Economy and Competitiveness.

The transglutaminase enzyme (tTG) is the target antigen of autoantibodies found in the serum of patients with celiac disease (CD). The most physiologically important function of tTG is to deamidate the glutamine residues of the gliadin peptides contained in corn gluten and convert them into glutamic acid; this modification makes the gliadin negatively charged, allowing it to bind with HLA-DQ2/DQ8 antigens, with consequent exposure of the neopeptides to recognition by the T cells. In people who are genetically predisposed to CD, the development of a T and B immune response leads to synthesis of anti-tTG immunoglobulin (Ig)A antibodies and of proinflammatory cytokines, resulting in chronic inflammation and progressive destruction of the intestinal mucosa. The anti-tTG antibodies of class IgA are a very sensitive and specific marker for CD, while the anti-tTG antibodies of class IgG can be used to diagnose CD in subjects with an IgA deficiency. Anti-tTG antibodies recognize the same antigen as antiendomysial antibodies (EMA), from which they only differ in terms of detection method. EMA IgA antibodies are tested for by the indirect immunofluorescence method on cryostatic sections of monkey esophagus. The EMA IgA test is highly specific, but less sensitive than IgA anti-tTG, and should, therefore, preferably be used as a confirmation test. In symptomatic children, a high titer of anti-tTG IgA confirmed by positive EMA and HLA-DQ2 and/or DQ8 tests allows the diagnosis of CD without the execution of duodenal biopsy. Anti-tTG testing can also be used to monitor CD patients following a diet in which the autoantibodies gradually decline until they disappear.

After their discovery in the 1970s, immunoglobulin (Ig)A- and IgG-type antigliadin antibodies (AGA) were the only serologic test able to identify patients with celiac disease (CD) for many years. However, AGA no longer play a significant diagnostic role since the identification of antiendomysial and antitransglutaminase antibodies. Gliadin peptides, which are present in foods containing wheat, barley, and rye, are modified by the tissue transglutaminase enzyme (tTG) located in the extracellular space of the intestinal mucosa; conversion of glutamine residues into glutamic acid facilitates the binding of gliadin peptides to HLA antigens of class II DQ2 or DQ8 expressed on antigen-presenting cells. In individuals with this genetic makeup, an immune response leading to the synthesis of cytokines and specific anti-tTG and AGA antibodies can be triggered. The AGA IgA and IgG tests that use purified gluten extracts are unsatisfactory, and the different methods are not comparable. The recent identification of deamidated gliadin peptides (DGP) able to trigger a specific T-cell response in CD patients enabled far more specific and sensitive tests to be devised. In patients under 3 years old, it is useful to perform this test in association with anti-tTG IgA. Positivity for DGP IgG can also be important in patients with an IgA deficiency, in whom it may be the only positive serologic marker. Finally, testing for IgG- and IgA-type AGA using purified gluten extract, though no longer recommended for CD diagnosis, is still useful in diagnosing nonceliac gluten sensitivity in endomysial autoantibodies (EMA)-, anti-tTG-, and anti-DGP-negative patients with intestinal or extraintestinal symptoms suggestive of this gluten-related disorder.

Celiac disease (CD) is defined as a chronic immune-mediated enteropathy precipitated by exposure to dietary gluten (storage proteins of wheat, rye, barley, and oats) in genetically predisposed individuals. Recent epidemiologic data suggest a prevalence of approximately 1% in most Western countries. The genetic predisposition includes human leukocyte antigen (HLA) DQ2 and DQ8 genes as major factors and a number of non-HLA genes. In addition to gluten, other environmental triggers such as pathogenic microorganisms, timing of gluten introduction, and breastfeeding may be important for disease development. CD is classically characterized by small intestinal villous atrophy, crypt hyperplasia, and increased lymphocyte infiltration. The clinical features of CD include both intestinal symptoms, such as diarrhea or steatorrhea, as well as extraintestinal symptoms, such as bone pain or osteoporosis. CD is frequently associated with autoimmune diseases, such as type 1 diabetes, and neurological and psychiatric complaints, such as schizophrenia. Further gluten-related disorders are dermatitis herpetiformis, gluten ataxia, wheat-dependent exercise-induced anaphylaxis, non-celiac gluten sensitivity, and irritable bowel syndrome. The diagnostic scheme of CD is usually based on symptoms typical of CD, testing of serum antibodies, and histological judgment of small intestinal biopsies. The complex pathomechanism of CD consists of incomplete digestion of gluten, para- and transcellular passage of gluten peptides through the epithelium, and immune responses in the lamina propria. The adaptive immunity includes the deamidation and transamidation of gluten peptides by tissue transglutaminase, their binding to HLA-DQ molecules, their presentation to T cells, and the subsequent delivery of damaging cytokines and chemokines. Additionally, antibodies against gluten peptides and autoantibodies against transglutaminase are produced by B cells. The adaptive immunity is accompanied by the innate immunity characterized by the production of interleukin 15 and the expansion of intraepithelial lymphocytes.