Identifying polyamine related biomarkers in diagnosis and treatment of ulcerative colitis by integrating bulk and single-cell sequencing data.

Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon, and its pathogenesis remains unclear. Polyamine metabolic enzymes play a crucial role in UC. In this study, we aimed to identify pivotal polyamine-related genes (PRGs) and explore the underlying mechanism between PRGs and the disease status and therapeutic response of UC. We analyzed mRNA-sequencing data and clinical information of UC patients from the GEO database and identified NNMT, PTGS2, TRIM22, TGM2, and PPARG as key PRGs associated with active UC using differential expression analysis and weighted gene co-expression network analysis (WCGNA). Receiver operator characteristic curve (ROC) analysis confirmed the accuracy of these key genes in UC and colitis-associated colon cancer (CAC) diagnosis, and we validated their relationship with therapeutic response in external verification sets. Additionally, single-cell analysis revealed that the key PRGs were specific to certain immune cell types, emphasizing the vital role of intestinal tissue stem cells in active UC. The results were validated in vitro and in vivo experiments, including the colitis mice model and CAC mice model. In conclusion, these key PRGs effectively predict the progression of UC patients and could serve as new pharmacological biomarkers for the therapeutic response of UC.

Effect of Ferulic Acid Loaded in Nanoparticle on Tissue Transglutaminase Expression Levels in Human Glioblastoma Cell Line.

Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.

Non-biopsy Strategy for the Diagnosis of Celiac Disease in Adults: A Narrative Review.

Celiac disease (CeD) diagnosis is a complicated process, requiring a multi-step procedure and a high level of clinical knowledge. Some scientific societies, mainly from Europe and North America, have proposed appropriate guidelines for the diagnosis and management of CeD. Since duodenal biopsy is particularly challenging for children, guidelines of the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition, presented in 2012 and updated in 2020, have made it possible to avoid the biopsy in symptomatic pediatric patients with high levels of IgA anti-transglutaminase. Several parallel, similar studies in adults support the non-biopsy strategy. However, several pros and cons exist in applying such a strategy. The present narrative review reports the current evidence and the implication of omitting biopsy in the diagnosis of CeD in adults.

Diagnostic Accuracy of IgA Anti-Transglutaminase Assessed by Chemiluminescence: A Systematic Review and Meta-Analysis.

A new chemiluminescence immunoassay method (CLIA) for detecting IgA anti-transglutaminase (atTG IgA) in celiac disease (CD) has prompted inquiries into its diagnostic performance. We conducted a systematic review and meta-analysis comparing CLIA with traditional enzyme-linked immunosorbent assay (ELISA) and fluorescence enzyme immunoassay (FEIA). We searched PubMed, Medline, and Embase databases up to March 2024. The diagnostic references were intestinal biopsy and ESPGHAN guidelines. We calculated the sensitivity and specificity of atTG IgA assessed by CLIA and the odds ratio (OR) between the assays. Eleven articles were eligible for the systematic review and seven for the meta-analysis. Sensitivity and specificity of atTG IgA CLIA-assay were 0.98 (95% CI, 0.95-0.99) and 0.97 (95% CI, 0.94-0.99), respectively. The sensitivity of atTG IgA antibody detection did not significantly vary across the three assay modalities examined (CLIA vs. ELISA OR: 1.08 (95% CI, 0.56-2.11; p = 0.8); CLIA vs. FEIA OR: 6.97 (95% CI, 0.60-81.03; p = 0.1). The specificity of atTG IgA assessed by FEIA was higher than for CLIA (OR 0.17 (95% CI, 0.05-0.62); p < 0.007). According to the systematic review, normalization of atTG IgA levels in CD patients following a gluten-free diet was delayed when using CLIA compared to ELISA and FEIA methods. Conflicting findings were reported on the antibody threshold to use in order to avoid biopsy confirmation.

Distinct conformational states enable transglutaminase 2 to promote cancer cell survival versus cell death.

Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.

Characteristics of soy films as affected by transglutaminase cross-linking and inclusions of pectin and protein enhancers.

Previously, we showed that water extract (soymilk, except pH was increased to 8 from 6.5) of whole soybean could be used directly as a raw material for producing edible soy films by deposition of the film-forming solution (soy extract with enhancers). However, the strength of such soy films needed improvement because they were weak. The purpose of this study was to investigate how transglutaminase (TG) cross-linking reactions and film enhancers, including pectin (low- and high-methoxyl pectin), whey protein isolate (WPI), and soy protein isolate (SPI), improve the physical properties of soy films. Soy films prepared with TG had tensile strength (TS) of 3.01 MPa and puncture strength (PS) of 0.78 MPa, which were higher by as much as 51% and 30% than that of soy films without TG treatment, respectively. Pectin showed significant effects on the mechanical properties of TG-added soy films in terms of TS, PS, and % elongation. On the other hand, only TS and PS were increased by the addition of WPI or SPI. Heat curing had a significant effect on soy film's physical properties. TG treatment significantly reduced film solubility when soaked in water and various levels of acid (vinegar) and base (baking soda) solutions. Under the experimental conditions of 35 unit TG and 28 min of reaction, the degrees of cross-linking were evidenced by the disappearance of individual protein subunits, except the basic subunit of glycinin, and the reduction of 21% of lysine residues of the proteins. HIGHLIGHTS: Edible soy films were made with transglutaminase and about 21% lysine cross-linked. The mechanical strength of soy films was increased by incorporating film enhancers. Transglutaminase enhanced the mechanical properties of soy films.

Impact of Transglutaminase-Mediated Crosslinking on the Conformational Changes in a Dual-Protein System and IgE Reactivity of Soy Protein.

Transglutaminase (TGase)-catalyzed crosslinking has gained substantial traction as a novel strategy for reducing allergenic risk in food proteins, particularly within the realm of hypoallergenic food production. This study explored the impact of TGase crosslinking on conformational changes in a binary protein system composed of soy protein isolate (SPI) and sodium caseinate (SC) at varying mass ratios (10:0, 7:3, 5:5, 3:7 (w/w)). Specifically, the immunoglobulin E (IgE) binding capacity of soy proteins within this system was examined. Prolonged TGase crosslinking (ranging from 0 h to 15 h) resulted in a gradual reduction in IgE reactivity across all SPI-SC ratios, with the order of IgE-binding capability as follows: SPI > SPI5-SC5 > SPI7-SC3 > SPI3-SC7. These alterations in protein conformation following TGase crosslinking, as demonstrated by variable intrinsic fluorescence, altered surface hydrophobicity, increased ultraviolet absorption and reduced free sulfhydryl content, were identified as the underlying causes. Additionally, ionic bonds were found to play a significant role in maintaining the structure of the dual-protein system after crosslinking, with hydrophobic forces and hydrogen bonds serving as supplementary forces. Generally, the dual-protein system may exhibit enhanced efficacy in reducing the allergenicity of soy protein.

In silico studies of the open form of human tissue transglutaminase.

Human tissue transglutaminase (tTG) is an intriguing multifunctional enzyme involved in various diseases, including celiac disease and neurological disorders. Although a number of tTG inhibitors have been developed, the molecular determinants governing ligand binding remain incomplete due to the lack of high-resolution structural data in the vicinity of its active site. In this study, we obtained the complete high-resolution model of tTG by in silico methods based on available PDB structures. We discovered significant differences in the active site architecture between our and known tTG models, revealing an additional loop which affects the ligand binding affinity. We assembled a library of new potential tTG inhibitors based on the obtained complete model of the enzyme. Our library substantially expands the spectrum of possible drug candidates targeting tTG and encompasses twelve molecular scaffolds, eleven of which are novel and exhibit higher binding affinity then already known ones, according to our in silico studies. The results of this study open new directions for structure-based drug design of tTG inhibitors, offering the complete protein model and suggesting a wide range of new compounds for further experimental validation.

TG2 participates in pulmonary vascular remodelling by regulating the senescence of pulmonary artery smooth muscle cells.

Pulmonary hypertension (PH) is a severe cardiovascular disease characterised by pulmonary vascular remodelling. The pivotal role of cellular senescence in vascular remodelling has been acknowledged. Transglutaminase type 2 (TG2), a calcium-dependent enzyme, is intricately linked to both cellular senescence and PH. However, the precise mechanisms underlying the involvement of TG2 in PH remain unclear. In this study, we explored the expression of TG2 and the cellular senescence marker p16INK4a in the pulmonary vasculature of mice with PH induced by hypoxia combined with SU5416. Our findings revealed upregulation of both TG2 and p16INK4a expression in the pulmonary vasculature of PH mice. Additionally, a notable increase in TG2 expression was observed in senescent pulmonary artery smooth muscle cells (PASMC). To delve deeper, we employed proteomic sequencing to reveal seven genes associated with cellular senescence, with a subsequent focus on MAPK14. Our investigation revealed that TG2 regulates senescence in PASMC by modulating the phosphorylation levels of MAPK14. Additionally, in the context of hypoxia combined with SU5416, our observations revealed a noteworthy reduction in both pulmonary vascular remodelling and senescent manifestations in smooth muscle-specific TG2 knockout mice compared with their wild-type counterparts. In summary, our findings indicate that TG2 deficiency lowers the senescence levels of PASMC by inhibiting the activity of MAPK14. This inhibition of senescence in the pulmonary vasculature of PH mice helps to decelerate the progression of pulmonary vascular remodelling and consequently hinders the onset and development of PH.

Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state.

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

Transglutaminase 2 knockout mice are protected from bleomycin-induced lung fibrosis with preserved lung function and reduced metabolic derangements.

Pulmonary fibrosis is an interstitial scarring disease of the lung characterized by poor prognosis and limited treatment options. Tissue transglutaminase 2 (TG2) is believed to promote lung fibrosis by crosslinking extracellular matrix components and activating latent TGFß. This study assessed physiologic pulmonary function and metabolic alterations in the mouse bleomycin model with TG2 genetic deletion. TG2-deficient mice demonstrated attenuated the fibrosis and preservation of lung function, with significant reduction in elastance and increases in compliance and inspiratory capacity compared to control mice treated with bleomycin. Bleomycin induced metabolic changes in the mouse lung that were consistent with increased aerobic glycolysis, including increased expression of lactate dehydrogenase A and increased production of lactate, as well as increased glutamine, glutamate, and aspartate. TG2-deficient mice treated with bleomycin exhibited similar metabolic changes but with reduced magnitude. Our results demonstrate that TG2 is required for a typical fibrosis response to injury. In the absence of TG2, the fibrotic response is biochemically similar to wild-type, but lesions are smaller and lung function is preserved. We also show for the first time that profibrotic pathways of tissue stiffening and metabolic reprogramming are interconnected, and that metabolic disruptions in fibrosis go beyond glycolysis.

Transglutaminase-mediated proximity labeling of a specific Lys residue in a native IgG antibody.

The fusion protein of an engineered zymogen of microbial transglutaminase (EzMTG) with a protein G variant, EzMTG-pG, enabled the proximity-based, tag-free labeling of Lys65 in the heavy chain of a native IgG antibody (trastuzumab) with a Gln-donor peptidyl substrate functionalized with a fluorescent molecule.

Transcriptomic analysis of intestine following administration of a transglutaminase 2 inhibitor to prevent gluten-induced intestinal damage in celiac disease.

Transglutaminase 2 (TG2) plays a pivotal role in the pathogenesis of celiac disease (CeD) by deamidating dietary gluten peptides, which facilitates antigenic presentation and a strong anti-gluten T cell response. Here, we elucidate the molecular mechanisms underlying the efficacy of the TG2 inhibitor ZED1227 by performing transcriptional analysis of duodenal biopsies from individuals with CeD on a long-term gluten-free diet before and after a 6-week gluten challenge combined with 100 mg per day ZED1227 or placebo. At the transcriptome level, orally administered ZED1227 effectively prevented gluten-induced intestinal damage and inflammation, providing molecular-level evidence that TG2 inhibition is an effective strategy for treating CeD. ZED1227 treatment preserved transcriptome signatures associated with mucosal morphology, inflammation, cell differentiation and nutrient absorption to the level of the gluten-free diet group. Nearly half of the gluten-induced gene expression changes in CeD were associated with the epithelial interferon-γ response. Moreover, data suggest that deamidated gluten-induced adaptive immunity is a sufficient step to set the stage for CeD pathogenesis. Our results, with the limited sample size, also suggest that individuals with CeD might benefit from an HLA-DQ2/HLA-DQ8 stratification based on gene doses to maximally eliminate the interferon-γ-induced mucosal damage triggered by gluten.

Integrated strategies for efficient production of Streptomyces mobaraensis transglutaminase in Komagataella phaffii.

Transglutaminase (TGase) from Streptomyces mobaraensis commonly used to improve protein-based foods due to its unique enzymatic reactions, which imply considerable attention in its production. Recently, TGase exhibit broad market potential in non-food industries. However, achieving efficient synthesis of TGase remains a significant challenge. Herein, we achieved a substantial amount of a fully functional and kinetically stable TGase produced by Komagataella phaffii (Pichia pastoris) using multiple strategies including Geneticin (G418) screening, combinatorial mutations, promoter optimization, and co-expression. The active TGase expression reached a maximum of 10.1 U mL-1 in shake flask upon 96 h of induction, which was 3.8-fold of the wild type. Also, the engineered strain exhibited a 6.4-fold increase in half-life and a 2-fold increase in specific activity, reaching 172.67 min at 60 °C (t1/2(60 °C)) and 65.3 U mg-1, respectively. Moreover, the high-cell density cultivation in 5-L fermenter was also applied to test the productivity at large scale. Following optimization at a fermenter, the secretory yield of TGase reached 47.96 U mL-1 in the culture supernatant. Given the complexity inherent in protein expression and secretion, our research is of great significance and offers a comprehensive guide for improving the production of a wide range of heterologous proteins.

Prevalence and factors associated with celiac disease in high-risk patients with functional gastrointestinal disorders.

Celiac disease (CD) is an autoimmune disease of the small intestine triggered by the consumption of gluten-containing foods in individuals with a genetic predisposition. CD was a rare disease until 20 years ago, when the prevalence increased. Currently, there is no data on the prevalence of CD in high-risk adult populations in Indonesia, even though there is a trend of increasing gluten consumption. Therefore, basic research is needed to determine the magnitude of CD in high-risk adult patients in Indonesia while identifying clinical signs/symptoms, illness history, and lifestyle to determine factors associated with CD in Indonesia. This study is an observational study with a cross-sectional method.Two hundred eighty-three patients who fulfilled the selection and signed the informed consent were recruited from the gastroenterology clinic of Dr. Cipto Mangunkusumo General Hospital. Patients were asked to fill out a celiac disease-related questionnaire and then given anthropometry measurement and blood test for serologic examination with ELISA, consisting of IgA anti-tissue transglutaminase (anti-TTG) and IgG anti-deaminated gliadin peptide (anti-DGP). Statistical analysis was performed using Chi-square and Multivariate logistic regression tests with SPSS software ver. 26. Statistical test differences were considered significant if the p-values were < 0.05. Eight of 283 patients are serologically confirmed with CD (2,83%). On bivariate analysis, the significant variables are age (p < 0,05), constipation (p < 0,05) and history of autoimmune disease (p < 0,05). On multivariate analysis, the only significant variable is the history of autoimmune disease (p < 0,05). This study concluded that the prevalence of CD in high-risk patients with functional gastrointestinal disorder at Dr. Cipto Mangunkusumo Hospital is relatively high (2.83%). CD-associated factors are age, constipation, and history of autoimmune disease in patients. On simultaneous interaction between these factors, autoimmune is the only significant variable associated with CD.

Impacts of transglutaminase on the processing and digestion characteristics of glutinous rice flour: Insight of the interactions between enzymic crossing-linked protein and starch.

Glutinous rice is extensively consumed due to its nutritious content and wonderful flavor. However, glutinous rice flour has a high glycemic index, and the storage deterioration of sweet dumplingsissevere. Transglutaminase (TG) was used to cross-link glutinous rice protein and improve the characteristics of glutinous rice products. The findings demonstrated that TG significantly catalysed protein cross-linking to form a dense protein network, reduced the viscosity of glutinous rice paste and improved the thermal stability. The protein network may physically block the access of starch granules to digestive enzymes to lower the digestion rate of starch, and attenuate the damage of ice crystal molecules to the starch structure to improve the freezing stability of starch gels. The cracking rate and water loss of sweet dumplings prepared using glutinous rice flour with TG treated for 60 min reduced significantly. In conclusion, this study broadened the application of TG in starch products.

From waste to strength: Tailor-made enzyme activation design transformation of denatured soy meal into high-performance all-biomass adhesive.

Given the severe protein denaturation and self-aggregation during the high-temperature desolubilization, denatured soy meal (DSM) is limited by its low reactivity, high viscosity, and poor water solubility. Preparing low-cost and high-performance adhesives with DSM as the key feedstock is still challenging. Herein, this study reveals a double-enzyme co-activation method targeting DSM with the glycosidic bonds in protein-carbohydrate complexes and partial amide bonds in protein, increasing the protein dispersion index from 10.2 % to 75.1 % improves the reactivity of DSM. The green crosslinker transglutaminase (TGase) constructs a robust adhesive isopeptide bond network with high water-resistant bonding strength comparable to chemical crosslinkers. The adhesive has demonstrated high dry/wet shear strength (2.56 and 0.93 MPa) for plywood. After molecular recombination by enzyme strategy, the adhesive had the proper viscosity, high reactivity, and strong water resistance. This research showcases a novel perspective on developing a DSM-based adhesive and blazes new avenues for changes in protein structural function and adhesive performance.

Rapid aggregation of amyloid-like protein enhanced by mTGase to prepare functional wool fabrics for efficient and sustainable remove heavy metals from wastewater.

To counteract the increasing severity of water pollution and purify water sources, wastewater treatment materials are essential. In particular, it is necessary to improve the bonding strength between the adsorption material and the substrate in a long-term humid environment, and resist the invasion of microorganisms to prolong the service life. In this study, an amyloid-like aggregation method of lysozyme catalyzed by microbial transglutaminase (mTGase). Lysozyme self-assembles into an amyloid-like phase-transited lysozyme (PTL) in the presence of a reducing agent. Simultaneously, mTGase catalyzes acyl transfer reactions within lysozyme molecules or between lysozyme and keratin molecules, and driving PTL assembly on the wool fiber (TG-PTL@wool). This process enhances the grafting amount and fastness of PTL on the wool. Moreover, the tensile strength of wool fabric increased to 523 N. TG-PTL@wool achieves a 97.32 % removal rate of heavy metals, maintaining a removal rate of over 95 % after 5 cycles. TG-PTL@wool has excellent antibacterial property (99 %), and it remains above 90 % after 50 times of circulating washing. This study proved that mTGase can enhance the amyloid aggregation of lysozyme and enhance the bonding strength between PTL coating and substrate. Moreover, TG-PTL@wool provides a sustainable, efficient and cleaner solution for removing heavy metals from water.

3D printing of prawn mimics with faba proteins: The effects of transglutaminase and curdlan gum on texture.

With its capability for automated production of high-resolution structures, 3D printing can develop plant-based seafood mimics with comparable protein content. However, the challenge lies in solidifying 3D printed products to achieve the firmness of seafood. Targeting prawn, texturisation of its 3D printed mimic by curdlan gum was compared against incubation with a protein cross-linking enzyme, microbial transglutaminase. Faba bean protein extract (FBP) was selected for its lightest colour. To confer structural stability to the FBP-based ink without hindering extrudability, adding 1 % xanthan gum was optimal. Printed curdlan-containing mimics were steamed for 9 min, while printed transglutaminase-containing mimics were incubated at 55 °C before steaming. Either adding 0.0625 % or 0.125 % w/w curdlan or, incubating the transglutaminase-containing mimics for an hour achieved chewiness of 818.8-940.6 g, comparable to that of steamed prawn (953.13 g). Curdlan hydrogel penetrated and reinforced the FBP network as observed under confocal imaging. Whereas incubation of transglutaminase-containing mimics enhanced microstructural connectivity, attributable to transglutaminase-catalysed isopeptide cross-linkages, and the consequent increase in disulfide bonding and ß-sheet. Ultimately, transglutaminase treatment appeared more suitable than curdlan, as it yielded mimics with cutting strength comparable to steamed prawn. Both demonstrated promising potential to broaden the variety of 3D printed seafood mimics.