Transglutaminase Research Articles

Neurological Complications of Coeliac Disease

Coeliac disease (CD) is a chronic autoimmune enteropathy of the small intestine that occurs in genetically predisposed individuals. It is characterized by atrophy of intestinal villi accompanied by an increase in the number of intraepithelial lymphocytes and crypt hypertrophy as well as the presence of specific endomysial antibodies (EMA), IgA tissue transglutaminase antibodies (IgA-tTG), and IgA and IgG deaminated gliadin peptide antibodies (DGP IgA and IgG). CD is associated with a wide spectrum of clinical signs and symptoms secondary to malabsorption (vitamin deficiency, anaemia, osteoporosis) or unrelated to the gastrointestinal tract. Neurological disorders are a common problem in patients with CD and are not always accompanied by gastrointestinal symptoms. The most common neurological manifestations of CD are cerebellar ataxia, epilepsy, and peripheral neuropathy.

Cell surface transglutaminase required for nodavirus entry into freshwater prawn hemocytes

To identify molecules involved in Macrobrachium rosenbergii nodavirus (MrNV) entry into hemocytes of the giant freshwater prawn M. rosenbergii, biotinylated prawn hemocyte membrane proteins were prepared, purified and separated by SDS-PAGE. The proteins were blotted on the nitrocellulose membrane before incubation with the MrNV capsid protein (MrNV-CP) by a VOPBA technique. Subsequent mass spectrometry and analysis of immune-reactive bands represent putative binding partners including transglutaminase (TG), actin, α2-macroglobulin, α1-tubulin, F1-ATP synthase β-subunit and a currently uncharacterized protein. The sequence of TG has been characterized and found 5 amino acids differences to a previously reported MrTG (ADX99580), mainly at its N-terminal part and thus, we named it MrTGII (KM008611). Recombinant MrTGII was prepared to produce a polyclonal antibody against it, which was successfully revealed the presence of MrTGII (100 kDa) in prawn hemocyte lysates. Using the pentylamine-biotin incorporation assay, an acyl transfer reaction was observed when hemocyte lysates were added to solutions containing MrNV-CP, suggesting that hemocyte MrTG could use MrNV-CP as the substrate. The expression levels of MrTGII were changed during the course of MrNV infection. By using immunostaining technique, location of MrTGII on the hemocyte surface was confirmed. Specific interaction between MrTGII with MrNV-CP in a dose-dependent manner was confirmed by in vitro ELISA assay. The highest binding activity of MrNV-CP was found with the N-terminal portion of the protein. In vitro neutralization using anti-MrTGII antibody resulted in inhibition of MrNV attachment to the hemocyte surface, accompanied by a dramatic reduction in viral replication. This is the first time that crustacean TG has been shown to be involved in viral entry, in addition to its roles in blood clotting and haematopoiesis.

Effect of enzymatic crosslinking on the handling properties of dough as a function of NaCl levels for CWRS varieties, Pembina and Harvest.

The effects of transglutaminase (TG) and glucose oxidase (GO) on the handling properties of model bread dough were examined at both normal (2% wt. by flour) and reduced (1% wt.) sodium chloride (NaCl) levels using two Canada Western Red Spring (CWRS) cultivars; Pembina and Harvest. The reduction of NaCl level had negative effects on dough rheology and stickiness, however, the inclusion of GO (0.001 and 0.01% by flour weight) or TG (only at the 0.5% by flour weight inclusion) was able to improve dough strength and reduce stickiness. GO appeared to be more effective than TG (at 0.01%) at equivalent concentrations for improving dough-handling properties. Flour cultivar had significant effects; Harvest flour (weaker) was more impacted by salt reduction and enzyme inclusion compared to Pembina flour (stronger). Crosslinking assays showed significant differences in glutenin macropolymer (GMP) content in dough prepared with GO, and dough prepared with different flours. Additionally, significantly fewer free thiol groups were found in dough produced with GO compared to dough without any enzymes and those with TG. GO appears to have potential for use as a bread improver to reduce stickiness and improve the strength of bread dough produced at lower salt concentrations, especially for dough prepared with weaker flour cultivars.

Bread staling: understanding the effects of transglutaminase and vital gluten supplementation on crumb moisture and texture using multivariate analysis

The objective of this study was to assess the contribution of transglutaminase (TG) and vital gluten (VG), alone or in conjunction, in preserving the quality of bread during storage (0, 3 and 9 days). The experimental design was set up to identify the best formulation to reduce changes in texture attributes and moisture content based on different levels of TG (0, 0.05, 0.1, 0.2 g/100 g flour), VG (0 and 1 g/100 g flour), and wheat flour strength (FS: weak—WF, medium—MF, strong—SF). Results revealed that, in most cases, TG and VG addition had a beneficial effect on preserving crumb moistness over storage. Likewise, TG and VG were able to reduce changes in textural attributes during storage. The best formulations to improve bread quality in storage were 0.1% TG and 1% VG for WF, 0.2% TG for MF and 0.2% TG and 1% VG for SF.

Shelf-life and sensory evaluation of pan breads produced with transglutaminase, bacterial xylanase and maltogenic alpha-amylase

The baking industry still has much to expand in the use of enzymes in its formulations but, to obtain the best results, it is necessary to increase the knowledge on how these processing aids affect the final product. Therefore, this study observed the effect of transglutaminase (TG), bacterial xylanase (XYL) and bacterial maltogenic alpha-amylase (AM) on pan bread. A 2³ Central Composite Rotatable Design was used and the bread quality parameters (dependent variables) were observed on the 1st, 5th and 9th days of shelf-life. The Response Surfaces obtained showed that, on day 1, all the enzymes affected the moisture and AM and XYL affected the water activity. On day 5 XYL affected the texture. Sensory analysis was carried out to evaluate the acceptance of the consumers. Differences between the samples were observed for "appearance", "color" and "texture" (α≤0,05). It is possible to conclude that enzymes can replace additives in "clean label" pan breads.

Long-term administration of diets containing Gracilaria tenuistipitata extract induce the expression of immune-related genes and increase the immune response and resistance against Vibrio harveyi in white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei juveniles (2.15 ± 0.16 g) fed diets containing Gracilaria tenuistipitata extract (GTE) at 0, 0.5, 1.0, and 2.0 g kg−1 for 16 weeks were examined for transcripts of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), integrin β (IB), prophenoloxidase (proPO) I, proPO II, α-2 macroglobulin (α-2 M), cytMnSOD, mtMnSOD, glutathione peroxidase (GPx), transglutaminase (TG) I, TG II, lysozyme, and heat shock protein 70 (HSP70) as well as immune parameters. Shrimp fed the 2.0 g kg−1 GTE diet significantly exhibited up-regulated expressions of these genes, increased hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total hemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, and earlier hemolymph clotting time. In a separate experiment, shrimp fed different diets for 16 weeks were challenged with Vibrio harveyi at 2 × 104 cfu shrimp−1 and then placed in seawater. The survival rate of shrimp fed the 2.0 g kg−1 GTE diet was significantly higher over 24–168 h post challenge. In conclusion, shrimp following long-term oral administration of a GTE-containing diet at 2.0 g kg−1 induced the expression of immune-related genes and exhibited increased their immune response and resistance against V. harveyi.

Effect of transglutaminase and acidification temperature on the gelation of reconstituted skim milk

Effects of transglutaminase (TG), acidification temperature and total milk solids level on the acid gelation of skim milk were investigated. Despite similar acidification kinetics, TG-treated milk acidified at ≥35 °C showed differences in elastic modulus (Gʹ) with acidification time, particularly by inhibiting the formation of the peculiar shoulder (Gʹshoulder) observed at an early stage of gelation in the control milk. Regardless of the milk solids content, the G'shoulder was absent in both types of milk at 30 °C. However, control milk above 2.5% (w/w) milk solids showed the G'shoulder at 45 °C. G'shoulder is proposed as the transition from the first increase in Gʹ, due to aggregation of soluble protein complexes at the early stage of acidification, to the second, due to further aggregation of casein micelles (and aggregated soluble complexes) as acidification progresses. The G'shoulder was absent in acidified TG-treated milk due to the lack of soluble protein complexes containing caseins.

Isotretinoin-associated Celiac Disease

27-yr old female marathon runner presented with profound fatigue and reduced exercise tolerance due to an iron deficiency anemia subsequent to treatment with isoretinoin for acne. Subsequent histologic and serologic studies revealed small bowel biopsies with histopathological features of celiac disease and an increased IgA tissue transglutaminase level. Isoretinoin use was terminated and after treatment with a gluten-free diet and oral iron, her symptoms resolved with normalization of her serological studies. Now, she remains completely well on a gluten-free diet alone and recently completed a full marathon.

False-negative IgA Anti-tissue Transglutaminase

The expanding knowledge about gluten-related autoimmunity led to a serology-based step-wised approach to diagnose celiac disease that might be misleading due to laboratory techniques. We present a rare case associating initial false-negative anti-transglutaminase IgA antibodies while CD was confirmed by IgG anti-transglutaminase and subsequent duodenal biopsy.

Skim yoghurt with microbial transglutaminase: evaluation of consumer acceptance

ABSTRACTThe decrease in the fat content of yoghurt causes sensory modifications. Microbial transglutaminase (TG) has been proposed as an alternative for reducing the problems caused by the fat reduction. This work deals with the evaluation of the sensory profile, acceptance and preferences of skim yoghurt produced with TG at pilot scale. A group of 124 consumers of yoghurts were randomly selected. A lower firmness has been observed in yoghurt without TG (NoTG). TG Yoghurt has shown a lower whey odor and less acid taste than NoTG yoghurt. Free whey was not observed in the TG yoghurt. Despite of those differences, consumers only observed minor textural differences. In terms of overall acceptance and preference, no statistically significant differences were observed. The use of TG avoids the addition of milk protein or other texture additives into the yoghurt, decreasing production cost with the same overall acceptance and preference by consumers.

Aldosterone nongenomically induces angiotensin II receptor dimerization in rat kidney: role of mineralocorticoid receptor and NADPH oxidase.

IntroductionPrevious in vitro studies demonstrated that aldosterone nongenomically induces transglutaminase (TG) and reactive oxygen species (ROS), which enhanced angiotensin II receptor (ATR) dimerization. There are no in vivo data in the kidney.Material and methodsMale Wistar rats were intraperitoneally injected with normal saline solution, or aldosterone (Aldo: 150 μg/kg BW); or received pretreatment with eplerenone (mineralocorticoid receptor (MR) blocker, Ep. + Aldo), or with apocynin (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, Apo. + Aldo) 30 min before aldosterone. Thirty minutes after aldosterone injection, protein abundances of dimeric and monomeric forms of AT1R and AT2R, and protein abundances and localizations of TG2 and p47phox, a cytosolic subunit of NADPH oxidase, were determined by Western blot analysis and immunohistochemistry, respectively.ResultsProtein abundances of dimeric forms of AT1R and AT2R were enhanced by 170% and 70%, respectively. Apocynin could block dimeric forms of both receptors while eplerenone inhibited only AT2R. Monomeric protein levels of both receptors were maintained. Aldosterone significantly enhanced TG2 and p47phox protein abundances, which were blunted by eplerenone or apocynin. Aldosterone stimulated p47phox protein expression in both the cortex and the medulla while TG2 was induced mostly in the medulla. Eplerenone or apocynin normalized the immunoreactivity of both TG2 and p47phox.ConclusionsThis is the first in vivo study demonstrating that aldosterone nongenomically increases renal TG2 and p47phox protein expression and then activates AT1R and AT2R dimerizations. Aldosterone-stimulated AT1R and AT2R dimerizations are mediated through activation of NADPH oxidase. Aldosterone-induced AT1R dimer formation is an MR-independent pathway, whereas the formation of AT2R dimer is modulated in an MR-dependent manner.

Diagnostic Accuracy of a Point-of-Care Test for Celiac Disease Antibody Screening among Infertile Patients

Background: Screening for celiac disease among infertile patients has been suggested. Several rapid point-of-care (POC) tests aimed at detecting celiac disease antibodies have been developed. It has been suggested that these POC tests can be implemented as a replacement for standard laboratory tests. Objective: To evaluatethe diagnostic accuracy of a POC test (Simtomax®) that detects celiac disease antibodies compared with standard laboratory tests when screening for celiac disease among patients referred for fertility treatment in 2 Danish fertility clinics. Methods: Serum samples were analyzed for IgA anti-tissue transglutaminase (TGA) as the reference standard test with a cutoff of ≥7 kU/L and by the index POC test based on IgA and IgG antibodies against deamidated gliadin peptides (DGP). In IgA deficiency, the reference standard test was IgG DGP with a cutoff of ≥7 kU/L. Participants answered a questionnaire on gluten intake, symptoms, and risk factors. Diagnostic confirmation was made by duodenal biopsies. IgA TGA/IgG DGP were used as the reference standard to calculate positive and negative predictive values. Results: A total of 622 men and women (51.6%) were enrolled during 2015. The reference standard IgA TGA/IgG DGP was positive in 7 participants (1.1% [95% CI 0.5–2.3]) and the POC test was positive in 84 participants (13.5% [95% CI 10.9–16.4]), 3 of whom also had positive reference standard tests. This yields a sensitivity of the index POC test of 42.9% (95% CI 9.9–81.6) and a specificity of 86.8% (95% CI 83.9–89.4). Positive and negative predictive values were 3.57% (95% CI 0.7–10.1) and 99.3% (95% CI 98.1–99.8). Conclusion: The sensitivity of the POC test was low; however, the specificity was moderately good. The POC test had a high negative predictive value in this low prevalent population but missed 1 patient with biopsy-confirmed celiac disease. However, because of many false-positive tests, it cannot be recommended as replacement for standard laboratory tests but rather as a triage test to decide if the standard serology tests should be performed.

Effect of incorporation of sodium caseinate, whey protein concentrate and transglutaminase on the properties of depigmented pearl millet based gluten free pasta

The current investigation reports the preparation of gluten free pasta from depigmented pearl millet flour blended individually with sodium caseinate (SC) (10 and 20%) and 1:1 combination of sodium caseinate and whey protein concentrate (SCW) (10 and 20%), both treated with and without transglutaminase (TG) enzyme (0.5 and 1.5%). SC addition (at 10 and 20%) maintained the cooking loss (10.38 ± 0.25 and 11.08 ± 1.71) and yellowness (b*) (16.11 ± 0.29 and 15.79 ± 0.32) as like control sample, decreased the water absorption (1.73 ± 0.19 and 1.47 ± 0.04), brightness (L*) (42.10 ± 0.49) (at 20%), pasting properties, and increased the redness (a*) (2.81 ± 0.10 and 3.58 ± 0.16) and sensory attributes (except colour and adhesiveness). The combination of SCW (at 10 and 20%) decreased the cooking loss (6.90 ± 0.00 and 5.99 ± 0.11), water absorption (1.92 ± 0.05 and 1.72 ± 0.08), pasting properties and increased L*, a*, b* (46.83 ± 0.45, 2.53 ± 0.09, 17.08 ± 0.20) (at 10%) and sensory attributes (except colour and adhesiveness). TG addition increased the cooking loss, water absoprtion and pasting properties (in all the samples). Protein solubility (%) decreased with increase in the enzyme concentration confirmed with electrophoretic patterns. Moreover, the TG treatment imparts tenderness and chewiness to pasta. Hence, the present combination could be useful for preparation of good quality of gluten free pasta.

Transglutaminase diseases: from biochemistry to the bedside.

In humans, 9 members of the transglutaminase (TG) family have been identified, of which 8 [factor XIII (FXIII)A and TG1-TG7] catalyze post-translational protein-modifying reactions, and 1 does not (protein 4.2). The TG enzymatic activities considered in our discussion of human disease include deamidation of glutamine (Gln) residues, amine incorporation into Gln residues, and protein crosslinking. Except for TG7, which remains poorly studied, all individual TG members have been correlated with disparate human diseases that arise from either TG function or lack of function. Loss of TG function is associated with numerous orphan diseases that affect a relatively small number of individuals: loss of FXIIIa (transamidase-activated form) crosslinking leads to defects in blood coagulation in FXIII deficiency; loss of TG1 and TG5 cross linking leads to defects in epidermal cornification in lamellar ichthyosis and acral peeling skin syndrome, respectively; loss of TG3 crosslinking in hair-cuticle formation leads to uncombable hair syndrome; the predicted loss of TG6 crosslinking leads to spinocerebellar ataxia-35; and loss of the structural erythrocyte membrane protein, protein 4.2, leads to hereditary spherocytosis type 5. The enzymatic activity of TG2 is involved in the exacerbation of celiac disease and in at least 1 case of hemoglobinopathy, characterized by shortened erythrocyte lifespan. TGs are also autoantigens in a number of immune diseases, resulting in the production of autoantibodies against FXIIIa in acquired FXIII deficiency, TG2 in celiac disease, TG3 in dermatitis herpetiformis, TG4 in autoimmume polyglandular syndrome type 1, and TG6 in gluten axonal neuropathy and gluten ataxia. Much still remains to be learned and confirmed with respect to disease mechanisms, particularly with respect to TG-related immune diseases, in which development of isozyme-specific inhibitors may be useful for treatment.-Lorand, L., Iismaa, S. E. Transglutaminase diseases: from biochemistry to the bedside.

Correction: Characterization of Transglutaminase 2 activity inhibitors in monocytes in vitro and their effect in a mouse model for multiple sclerosis.

[This corrects the article DOI: 10.1371/journal.pone.0196433.].

Electro-encapsulation of Lactobacillus casei in high-resistant capsules of whey protein containing transglutaminase enzyme

In this study, we aimed to evaluate the possibility of obtaining natural high-resistant protein capsules from aqueous solution of whey protein containing transglutaminase (TG) enzyme through electrohydrodynamic atomization (EHDA) as an appropriate system, and freeze-drying (FD) technique as the control method for encapsulation of Lactobacillus casei. A stable cone jet was achieved in a certain electrical conductivity, surface tension, and viscosity values equal to 1681 ± 8.49 μS, 48.97 ± 0.02 mN/m, and 51.96 ± 0.78 cP, respectively. Spherical water-resistant capsules with an average diameter of 3.09 ± 1.04 μm were obtained through the EHDA process. A comparison between the EHDA and FD revealed higher encapsulation efficiency of the electrospraying technique as well as greater viability of L. casei under the simulated gastrointestinal (GI) tract. DSC results showed that the enthalpy of cross-linked samples was decreased by incorporating TG enzyme. In contrast to freeze-dried structures, electrosprayed capsules revealed better protection during the storage, especially in the second and third months.

Effect of Partial Hydrolysis with Papain on the Characteristics of Transglutaminase-Crosslinked Tofu Gel.

The effects of partial enzymatic hydrolysis of soymilk on the characteristics of transglutaminase (TG)-crosslinked tofu gel were studied. SDS-PAGE showed that the molecular weight of the partially hydrolyzed soybean protein was reduced to that of a digested peptide (less than 43.0kDa) when papain was added at more than 50μL/100mL soymilk. The content of free sulfhydryls, β-sheets, and random coils in papain-treated soymilk increased. When TG was added to soy milk after papain treatment and tofu gel was formed, its storage modulus increased from 957.44 to 1241.39Pa. The gel strength, water-holding capacity, and nonfreezing water content of the tofu gel were greater than those without enzyme treatment. Scanning electron microscopy revealed that limited papain hydrolysis stimulated TG-catalyzed cross-linking of soymilk to form a dense gel network structure, whereas an extended enzymatic hydrolysis of soymilk did not promote crosslinking by TG. PRACTICAL APPLICATION: This work investigated the effect of partial hydrolysis on TG cross-linked tofu gel. Partial hydrolysis of soybean protein with papain can promote TG cross-linking reaction, thus form a dense network structure, increase gel strength, and water-holding capacity. Therefore, it can be used to produce a good gel product with higher gel strength, springiness, water-holding capacity, and a more dense microstructure.

Transglutaminase-mediated cross-linking of WDR54 regulates EGF receptor-signaling

WDR54 is a member of the WD40 repeat (WDR) domain-containing protein family that was recently identified as a novel oncogene in colorectal cancer. However, the molecular mechanism of WDR54 and its functional association with other molecules related to tumor cell growth are unknown. Here, we show that WDR54 can be cross-linked by the action of transglutaminase (TG) 2, which enhances the activation of EGF receptor-mediated signaling pathway. The most carboxyl-terminal WD domain was required for cross-linking. In addition, lysine 280 in WDR54, also in this WD domain, was an important residue for both cross-linking and ubiquitination. Cross-linked WDR54 was found in vesicles aggregated at the plasma membrane. The activated EGF receptor was co-localized with this vesicle, and the internalization of the EGF receptor into the cytosol was sustained. As a result, Erk activity in response to EGF stimulation was enhanced. Furthermore, the growth of the cells lacking WDR54 expression generated by genome editing was delayed compared with that in wild-type cells. Because TG2 is also has been proposed to activate the EGF receptor-signaling and proliferation of tumor cells, WDR54 might have a functional relationship with the EGF receptor and TG2.Our study on the mechanism of biological function of WDR54 may provide rationale for the design and development of a cancer drug based on inhibiting the post-translational modification of this oncogene product.

Effect of transglutaminase on rennet-induced gelation of skim milk and soymilk mixtures.

Protein gels made from cow milk and soymilk can yield products of exceptional value. Transglutaminase (TG) affect rennet-induced gelation of proteins, and improves the functionality of the final products. In this paper, TG and rennet were added to skim milk and soymilk mixtures simultaneously, and the rennet-induced coagulation was studied. Diffusing wave spectroscopy and rheology measurements were used to access the structural changes of the mixtures during renneting. Syneresis analysis and microscopy can give more information for understanding the system. Soymilk and TG have synergetic effects and inhibit rennet-induced gelation to a certain degree. With increasing soymilk and TG, elastic index and storage modulus decreased, gelation time was delayed, and curd yield and moisture content increased. At excess soymilk and TG, no curds can be formed. There were significant effects of soymilk and TG on curd microstructure. Soymilk inhibited the aggregation of casein micelles and contributed to more coarse and heterogeneous networks. TG limited reorganization of the proteins, leading to more homogenous networks with small pores. The use of soymilk and TG simultaneously impair rennet-induced gelation and curd syneresis, and consequently lead to a higher yield of high-moisture curd. © 2018 Society of Chemical Industry.

Improving the Water Resistance and Mechanical Properties of Feather Keratin/Polyvinyl Alcohol/Tris(Hydroxymethyl)Aminomethane Blend Films by Cross-Linking with Transglutaminase, CaCl₂, and Genipin.

The high moisture sensitivity of feather keratin/polyvinyl alcohol/tris(hydroxymethyl)aminomethane (FK/PVA/Tris) blend films hinders their application in the packaging field. Thus, in order to improve the water resistance and mechanical properties of such blend films, we attempted cross-linking the blend film with cross-linking agents such as transglutaminase (TG), CaCl2, and genipin. Obvious differences in the morphology of the blended films were observed by scanning electron microscopy before and after cross-linking, indicating that cross-linking can inhibit the phase separation of the blend film. Conformational changes in the blend films after cross-linking were detected by Fourier transform infrared spectroscopy. Importantly, from examination of the total soluble mass, contact angle measurements, and water vapor permeability tests, it was apparent that cross-linking greatly improved the water resistance of the blend films, in addition to enhancing the mechanical properties (i.e., tensile strength and elongation at break). However, cross-linking was also found to reduce the oxygen barrier properties of the blend films. Therefore, cross-linking appears to be an effective method for promoting the application of FK/PVA/Tris blend films in the packaging field.