Transglutaminase 2 serves as a pathogenic hub gene of KRAS mutant colon cancer based on integrated analysis

BACKGROUND Colon cancer is acknowledged as one of the most common malignancies worldwide,ranking third in United States regarding incidence and mortality.Notably,approximately 40%of colon cancer cases harbor oncogenic KRAS mutations,resulting in the continuous activation of epidermal growth factor receptor signaling.AIM To investigate the key pathogenic genes in KRAS mutant colon cancer holds considerable importance.METHODS Weighted gene co-expression network analysis,in combination with additional bioinformatics analysis,were conducted to screen the key factors driving the progression of KRAS mutant colon cancer.Meanwhile,various in vitro experiments were also conducted to explore the biological function of transglutaminase 2(TGM2).RESULTS Integrated analysis demonstrated that TGM2 acted as an independent prognostic factor for progression-free survival.Immunohistochemical analysis on tissue microarrays revealed that TGM2 was associated with an elevated probability of perineural invasion in patients with KRAS mutant colon cancer.Additionally,biological roles of the key gene TGM2 was also assessed,suggesting that the downregulation of TGM2 attenuated the proliferation,invasion,and migration of the KRAS mutant colon cancer cell line.CONCLUSION This study underscores the potential significance of TGM2 in the progression of KRAS mutant colon cancer.This insight not only offers a theoretical foundation for therapeutic approaches but also highlights the need for additional clinical trials and fundamental research to support our preliminary findings.

Transglutaminase inhibition:A therapy to protect cells from death in neurodegeneration?

Transglutaminases(TGs;E.C.2.3.2.13)are ubiquitous enzymes which catalyze post-translational modifications of proteins.TGs and TG-catalyzed post-translational modifications of proteins have been shown to be involved in the molecular mechanisms responsible for several human diseases.In particular,TG activity has been hypothesized to also be involved also in the molecular mechanisms responsible for human neurodegenerative diseases.In support of this hypothesis,Basso et al recently demonstrated that the TG inhibition protects against oxidative stress-induced neuronal death,suggesting that multiple TG isoforms participate in oxidative stress-induced cell death and that nonselective TG isoform inhibitors will be most effective in fighting oxidative death in neurological disorders.In this commentary,we discuss the possible molecular mechanisms by which TG activity could be involved in the pathogenesis of neurological diseases,with particular reference to neurodegenerative diseases,and the possible involvement of multiple TG isoforms expressed simultaneously in the nervous system in these diseases.Moreover,therapeutic strategies based on the use of selective or nonselective TG inhibitors for the amelioration of thesymptoms of patients with neurological diseases,characterized by aberrant TG activity,are also discussed.

Physio-pathological roles of transglutaminase-catalyzed reactions

Transglutaminases(TGs) are a large family of related and ubiquitous enzymes that catalyze post-translational modifications of proteins.The main activity of these enzymes is the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate.In addition to lysyl residues,other second nucleophilic co-substrates may include monoamines or polyamines(to form mono-or bi-substituted/crosslinked adducts) or-OH groups(to form ester linkages) .In the absence of co-substrates,the nucleophile may be water,resulting in the net deamidation of the glutaminyl residue.The TG enzymes are also capable of catalyzing other reactions important for cell viability.The distribution and the physiological roles of TG enzymes have been widely studied in numerous cell types and tissues and their roles in several diseases have begun to be identified."Tissue" TG(TG2) ,a member of the TG family of enzymes,has definitely been shown to be involved in the molecular mechanisms responsible for a very widespread human pathology:i.e.celiac disease(CD) .TG activity has alsobeen hypothesized to be directly involved in the pathogenetic mechanisms responsible for several other human diseases,including neurodegenerative diseases,which are often associated with CD.Neurodegenerative diseases,such as Alzheimer's disease,Parkinson's disease,supranuclear palsy,Huntington's disease and other recently identified polyglutamine diseases,are characterized,in part,by aberrant cerebral TG activity and by increased cross-linked proteins in affected brains.In this review,we discuss the physio-pathological role of TG-catalyzed reactions,with particular interest in the molecular mechanisms that could involve these enzymes in the physio-pathological processes responsible for human neurodegenerative diseases.

Development and Physical Properties of Film of Wheat Gluten Cross-linked by Transglutaminase

Films were developed from the modified wheat glutens by microbial transglutamina se(MTGase, [E/S]=10u/g,15u/g and 20u/g) in order to improve physical and barri er properties of the films.Glycerol was used as a plasticizer.The films prepared from the modified-glutens by MTGase show a lower elongation at break(E) and a water vapor permeability(WVP), and a higher tensile strength(TS) than the nati ve gluten films.When the modified gluten films by different concentrations of MT Gase are immersed in water at 25℃,their weight losses decreased significantly, and their water resistance increases obviously as expected, compared with the c ontrol gluten films. Moreover, an addition of glycerol as plasticizer greatly mo dified water vapor barrier and mechanical properties of the films.

Microbial transglutaminase should be considered as an environmental inducer of celiac disease

Due to the recent interest in food additives that can act as triggering factors in autoimmune diseases including celiac disease(CD),the present letter to the editor expands on the microbial transglutaminase(mTG).It is heavily consumed by a plethora of food processing industries as“glue of proteins”thus improving product’s stability,texture and shelf life.However,more and more information is accumulated lately,questioning its safety.Its cross-linked gliadin complexes are immunogenic in CD.The enzyme increases gliadin uptake,is transported in a trans-epithelial way and deposited below the enterocyte’s line,has antiphagocytic activity,enhances intestinal permeability and creates luminal resistant isopeptide bonds.No doubt that mTG is beneficial to food industries but a caveat to public health is highly recommended.

Erythrocytic transglutaminase inhibition hemolysis at presentation of celiac disease

Celiac disease (CD) is a common autoimmune condition.Previously it was considered to be a rare childhood disorder,but is actually considered a relatively common condition,present at any age,which may have multiple complications and manifestations.Hematological disorders of the disease are not uncommon.Among these disorders,the most frequently reported are anemias as a result of iron deficiency,often associated with folate and/or B12 deficiency.Anemias caused by hemolysis are very rarely reported in celiac patients.An 11-year-old girl with a previous uneventful medical history presented with severe hemolytic anemia.Hemolysis was Coombs negative,accompanied by inappropriate low reticulocyte count,despite exaggerated bone marrow hyperplasia of the erythroid precursors which showed normal maturation.Serology for recent infections,including EpsteinBarr virus,parvovirus B19,cytomegalovirus and mycoplasma,were all negative.Levels of serum IgA,IgG and IgM,were all within normal ranges for age.Screeningfor anti-DNA,antinuclear,antineutrophil cytoplasmic,antimicrosomal,antithyroglobulin,and antimitochondrial antibodies and lupus anticoagulants,was negative.She was also negative for human immunodeficiency virus.Conventional therapy with corticosteroids and intravenous immunoglobulin failed.CD was serendipitously discovered upon screening for anti-tissue transglutaminase autoantibodies.The disease was confirmed by biopsy of the small intestine mucosa.The patient recovered with gluten-free diet.A unique case of CD is presented.CD should be serologically screened in each patient with Coombs negative "immune"hemolytic anemia,particularly if accompanied by "reticulocytopenia".A new hemolytic mechanism and very speculative explanation for "reticulocytopenia"are discussed.

Inhibitory Effect of Tissue Transglutaminase (tTG) Antisense Oligodeoxynucleotides on tTG Expression in Cultured Bovine Trabecular Meshwork Cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides （tTG-ASDON） on tTG expression in cultured bovine trabecular meshwork cells （BTMCs） in vitro and explore a new treatment alternative for primary open angle glaucoma （POAG）, the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTCr-ASDON2 were significantly decreased as compared with that of the controls （P〈0.05）. On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

The Impact of Microbial Transglutaminase on the Quality and Antioxidant Activity of Camel-Milk Soft Cheese

This study aimed at investigating the impact of adding microbial transglutaminase (MTGase) after rennet addition on some properties of fresh soft cheese made from camel milk. MTGase was added to milk at concentration of 80, 100 and 120 U/L after 20 and 30 min of renneting. The chemical composition, yield, hardness, antioxidant activity and sensory properties of cheese were estimated. Enzymatic protein crosslinking was analyzed by SDS-PAGE. Results revealed that MTGase-treated cheeses were higher in moisture and lower in protein content compared to control. In addition, the concentration of MTGase and time of addition significantly (P 0.05) impacted these parameters. Among treated cheeses, samples with 80 U of MTGase and addition time of 20 min were the highest in total solids and protein content. Adding MTGase significantly (P 0.05) increased the cheese yield, however, increased MTGase concentration at any time of addition did not improve it. The electrophoretic patterns of MTGase-cheese proteins showed a reduction in the intensity of caseins bands and the appearance of new protein fractions with high molecular weights. However, the changes in the intensity of the whey proteins bands were not sufficiently clear as caseins. The cheese hardness was significantly (P 0.05) affected by adding MTGase. Cheese containing 80 U of MTGase had the highest hardness value compared to control and other treated samples. The antioxidant activity of cheese was negatively influenced by adding the enzyme. The use of MTGase enhanced the mouthfeel, texture and overall acceptability of cheese. However, the effect of MTGase concentration and addition time was not significant (P > 0.05) on the sensory attributes. In conclusion, adding MTGase to milk at concentration of 80 U after 20 min of renneting is recommended to improve the yield, textural and some sensory properties of fresh soft cheese made from camel milk.

Variability of anti-human transglutaminase testing in celiac disease across Mediterranean countries

AIM To verify the precision and accuracy of transglutaminase antibodies(TGA)assays across Mediterranean countries.METHODS This study involved 8 referral centres for celiac disease(CD)in 7 Mediterranean countries.A central laboratory prepared 8 kits of 7 blinded and randomized serum samples,with a titrated amount of Human TGA Ig A.Each sample was analysed three times on three different days,with each centre running a total of 21tests.The results were included in a blindly coded report form,which was sent to the coordinator centre.The coordinator estimated the mean coefficient of Variation(Co Var=σ/μ),the mean accuracy(Accur=Vobserved-Vreal)and the mean percent variation(Var%=[(Vobserved-Vreal)/Vreal]×100).RESULTS The analysis showed that 79.17%of the mean variation fell between-25%and+25%of the expected value,with the accuracy and precision progressively increasing with higher titres of TGA.From values 1.25 times greater than the normal cut-off,the measurements were highly reliable.CONCLUSION TGA estimation is a crucial step for the diagnosis of CD;given its accuracy and precision,clinicians could be confident in establishing a diagnosis.

Expression of Tissue Transglutaminase in Cultured Bovine Trabecluar Meshwork Cells

Summary: To study whether cultured bovine trabecluar meshwork cells (BTMC) are capable of expressing tTG in protein and at mRNA level, BTMC were cultured in vitro and passaged three times, then the cells were transferred onto or cultured on sterile cover or submitted to isolation of RNA with Trizol, and the expression of tTG was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR) respectively. Our results showed that tTG immunostaining was positive in the cytoplasm and rarely in the nucleus of cultured BTMC. No immunostaining was seen in the negative control. Moreover, a single RT-PCR amplified product whose sequence and size were in accordance with our known parameters was obtained. The expression of tTG in cultured BTMC was confirmed in protein and at mRNA level. BMTC is available more readily for the investigation of the relationship between tTG and primary open-angle glaucoma.

A peptide of 17 aminoacids from the N-terminal region of maize plastidial transglutaminase is essential for chloroplast targeting

Transglutaminases (TGases, EC 2.3.2.13) catalyse posttranslational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines. In plants, the functionality of these enzymes is scarcely known. The maize transglutaminase gene (tgz), the only cloned plant TGase, produces major alterations in thylakoid membrane architecture when the transglutaminase (chlTGZ) protein was over-expressed in tobacco chloroplasts, significantly increasing the number of grana stacked layers. Here we demonstrate that nuclear transformation of rice plants starting from a tgz gene truncated in 17 N-terminal aas (tgzt) non altered chloroplast thylakoid structures. F3 transformed plants were analysed for TGase activity, chlTGZ presence and tgzt transcription levels. Transformed plants exhibited double the in vitro TGase activity of the non-transformed plants. Immunoblot and quantitative RT-PCR analysis results of tgzt-rice plants grown under different illumination periods revealed that chlTGZ maintains its differential expression depending on the light regime. Nevertheless, the maize protein was localised by confocal microscopy in the cell wall of transformed rice cells. TEM analyses of the transformed cells showed normal, non-altered chloroplast thylakoid structures with the maize protein preferentially located in the cell walls. The results confirmed that the tgz eliminated sequence is essential for chloroplast targeting, being its absence sufficient to the lack of protein expression in its original plastidal compartment. Interestingly, the immunolocalization of a putative endogenous rice TGase protein is also showed. These data give further information on plant TGase functionality and its relationship to photosynthetic membranes.

Baicalein and Salvia officinalis Extract Upregulate Transglutaminase 1 mRNA Expression via the Activation of Transient Receptor Potential Channel V4

Background: It is important to maintain skin homeostasis for cosmetic and medical reasons. Many ceramide-related ingredients and cosmetics have been developed to improve the skin barrier function and skin hydration. Similar to extracellular lipids, the cornified envelope, which is a structure formed beneath the plasma membrane, contributes to the skin barrier function as a scaffold for extracellular lipids. Therefore, in this study, we focused on transglutaminase 1 (TGM1) which is the key enzyme for formation of the cornified envelope Objective: The objectives of this study were to identify compounds that could upregulate the expression of TGM1 and evaluate their underlying action mechanisms. Methods: Expression of the transient receptor potential channel vanilloid subfamily member 4 (TRPV4) at the mRNA and protein levels was estimated by PCR and western blotting. Effects of baicalein and Salvia officinalis (SO) extract on TGM1 mRNA expression were measured by PCR. The involvement of TRPV4 in TGM1 mRNA expression was evaluated by the inhibition and silencing of TRPV4. Results: TRPV4 was expressed in both basal cell-like HaCaT cells and suprabasal cell-like HaCaT cells. Baicalein and SO extract upregulated TGM1 mRNA expression in basal cell-like HaCaT cells. However, inhibition and silencing of TRPV4 abrogated the effects of baicalein and SO extract. Conclusion: Baicalein and SO extract upregulated the expression of TGM1 mRNA via the activation of TRPV4, suggesting that it may improve the skin barrier function by enhancing cornified envelope formation.

Autoantibodies related to ataxia and other central nervous system manifestations of gluten enteropathy

Gluten ataxia and other central nervous system disorders could be linked to gluten enteropathy and related autoantibodies.In this narrative review,we focus on the various neuro-logical manifestations in patients with gluten sensitivity/celiac disease,immunological and autoimmune mechanisms of ataxia in connection to gluten sensitivity and the autoantibodies that could be used as a biomarker for diagnosing and following.We focused on the anti-gliadin antibodies,antibodies to different isoforms of tissue transglutaminase(TG)(anti-TG2,3,and 6 antibodies),anti-glycine receptor antibodies,anti-glutamine acid decarboxylase antibodies,anti-deamidated gliadin peptides antibodies,etc.Most studies found a higher prevalence of these antibodies in patients with gluten sensitivity and neurological dysfunction,presented as different neurological disorders.We also discuss the role of a gluten-free diet on the clinical improvement of patients and also on imaging of these disorders.

Novel hemostatic agents based on gelatin-microbial transglutaminase mix

Hemostasis is a major challenge in surgical procedures and traumas. Conventional hemostatic methods have limited efficacy and may cause additional tissue damage. In this study, we designed a novel hemostatic agent based on the in situ gel formation of gelatin cross-linked by a novel microbial transglutaminase(mTGase), in which the amino acid sequences differed from commercial mTGases. The new hemostatic agent showed the same biochemical crosslinking chemistry as the final stages of the blood coagulation cascade while using gelatin as a "structural" protein(rather than fibrin) and a calcium-independent mTGase as the crosslinking catalyst(rather than factor XIIIa). In rat liver hemostasis models, the hemostatic agent not only showed a similar hemostatic effect as that of SURGIFLO~(positive control), but also stronger adhesion strength and elasticity than SURGIFLO~.Therefore, this biomimetic gelatin-mTGase mix hemostatic is a novel and effective surgical sealant.

Effects of nuclear translocation of tissue transglutaminase and the release of cytochrome C on hepatocyte apoptosis

Objective To assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes. Methods Hepatocytes isolated from tissue transglutaminase gene knock-out mice and rats were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by 14C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.Results TTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.Conclusions Increased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.

亲水多糖对谷氨酰胺转氨酶交联大豆分离蛋白凝胶特性的影响

目的 基于谷氨酰胺转氨(transglutaminase, TG)酶交联法研究亲水多糖对TG酶交联大豆分离蛋白凝胶特性的影响。方法 以大豆分离蛋白为主要原料,TG酶交联法为基础进行响应面优化,得到最优凝胶弹性的工艺参数,在此参数条件下将大豆分离蛋白与亲水多糖混合,制备亲水多糖-大豆分离蛋白复合凝胶,并对凝胶的质构特性、持水性、热力学性质以及结构进行表征。结果 在酶交联pH 7.3、酶交联时间2.3 h、酶交联温度48℃条件下,制备的大豆分离蛋白凝胶弹性最佳。添加了亲水多糖后,凝胶的质构特性和持水性显著提高,热稳定性增强,蛋白质二级和三级结构发生变化,凝胶的微观结构变得更致密,孔径变小。结论 亲水多糖的添加能够改善大豆分离蛋白的凝胶特性,该研究为大豆分离蛋白凝胶深加工提供了理论基础。

谷氨酰胺转氨酶热稳定剂优化

谷氨酰胺转氨酶(Transglutaminase,TGase)能够催化蛋白质分子发生交联,被广泛应用于食品、医药和纺织等领域。为提高其热稳定性,研究了糖、盐及醇类等稳定剂对TGase在55℃下的半衰期[t_(1/2)(55℃)]的影响。结果表明,山梨醇、小麦蛋白、Na Cl和葡萄糖能延长TGase的t_(1/2)(55℃)。在此基础上进行正交实验,获得最佳的稳定剂配方:山梨醇50 g/L、小麦蛋白50 g/L、Na Cl 50 g/L、葡萄糖50 g/L。在该复合稳定剂保护下,TGase的t_(1/2)(55℃)和最适反应温度较对照(无稳定剂)分别提高59.73倍和10℃;室温(25℃)储存80 d的残余酶活率为73.84%,较对照提高62.79%。此研究结果将促进TGase的生产改进和应用拓展。

南极磷虾肉糜制作研发

为开发南极磷虾食品,开展南极磷虾肉糜制作研究。研究采用谷氨酰胺转氨酶、组织化植物蛋白与南极磷虾肉糜进行反应,考察NaCl、CaCl2以及反应时间对凝胶的影响;结果表明:添加2%NaCl,4%组织化植物蛋白,0.5%转谷氨酰胺酶、0.1%CaCl2充分混匀,于20℃下放置2h,制备得到的虾肉糜结构紧密,凝胶强度高,同时在外观上色泽红润,并通过食品质量安全检测。综上所述,该研发技术,可以为南极磷虾肉糜开发与生产起到理论指导作用。

Novel method for extracting exosomes of hepatocellular carcinoma cells

AIM:To develop a novel method for the rapid and efficient extraction of exosomes secreted by tumor cells.METHODS:Unlike the traditional extraction method,the supernatants of cell cultures were concentrated,and the exosomes were isolated promptly and effectively using a novel nanomaterial called ExoQuick.Coomassie brilliant blue staining was used for protein quantification,and the morphology of the exosomes extracted by both methods was visualized by transmis-sion electron microscopy.Exosome marker proteins were detected by Western blot analysis.Two potential hepatoma-associated proteins,tissue transglutaminase2(TGM2)and annexin A2,were analyzed.RESULTS:The exosomes separated by the new extraction assay based on the nanomaterial were discshaped,intact vesicles with lipid bilayer membranes.They were approximately 30-100 nm in diameter,which is similar to the diameter of exosomes isolated by the traditional method.The protein concentration of exosomes extracted by the new method was approximately780μg/108 cells,and therefore,it was 19 times higher than that of exosomes extracted in the traditional manner.There were differences between the total proteins of Huh-7 cells and the exosomal proteins.Typical exosome proteins,such as the transmembrane protein CD63 and heat shock protein 70,were confirmed.Two potential hepatoma-associated proteins were also identified.TGM2 was first found to exist in the exosomes of human liver cancer cells,but annexin A2 was not secreted into exosomes.CONCLUSION:The new extraction method based on the nanomaterial is quick and efficient.The cancerassociated protein TGM2 can be secreted through an exosome-mediated non-classical secretion pathway,and it may be a valuable tumor marker.