The industrial food additive, microbial transglutaminase, mimics tissue transglutaminase and is immunogenic in celiac disease patients

    The industrial food additive microbial transglutaminase, mimics the tissue transglutaminase and is immunogenic in celiac disease patients T. Matthias, P. Jeremias, S. Neidh¨ofer, A. Lerner PII: DOI: Reference:

S1568-9972(16)30206-3 doi:10.1016/j.autrev.2016.09.011 AUTREV 1922

To appear in:

Autoimmunity Reviews

Received date: Accepted date:

26 July 2016 3 August 2016

Please cite this article as: Matthias T, Jeremias P, Neidh¨ofer S, Lerner A, The industrial food additive microbial transglutaminase, mimics the tissue transglutaminase and is immunogenic in celiac disease patients, Autoimmunity Reviews (2016), doi:10.1016/j.autrev.2016.09.011

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Matthias T1, Jeremias P1, Neidhöfer S1, Lerner A1, 2

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The industrial food additive microbial transglutaminase, mimics the tissue transglutaminase and is immunogenic in celiac disease patients

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1. AESKU.KIPP Institute, Wendelsheim, Germany 2. B. Rappaport School of Medicine, Technion-Israel Institute of Technology, Haifa, Israel2.

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Corresponding Author: Lerner A.

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Actual address: AESKU.KIPP Institute, Mikroforum Ring 2, Wendelsheim 55234, Germany. Tel: 49-6734-9622-1010

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Fax: 49-6734-9622-2222

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Mail: [email protected]

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Key words: celiac disease, microbial transglutaminase, tissue transglutaminase, antibodies, immunogenic, immunoreactivity, food additive, food processing. Short title: microbial transglutaminase is a bio-marker for celiac disease

Not grant supported.

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Abstract

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Microbial transglutaminase (mTg) is capable of cross-linking numerous molecules. It is a family member of human tissue transglutaminase (tTg), involved in CD. Despite declarations of mTg industrial use safety, direct evidence for immunogenicity of the enzyme is lacking.

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The serological activity of mTg, tTg, gliadin complexed mTg (mTg neo-epitope) and gliadin complexed tTg (tTg neo-epitope) were studied in: 95 pediatric celiac patients (CD), 99 normal children (NC) 79 normal adults (NA) and 45 children with nonspecific abdominal pain (AP). Sera were tested by ELISAs, detecting IgA, IgG or both IgA and IgG (check): AESKULISA® tTg (tTg), AESKULISA® tTg New Generation (tTg neo-epitope (tTg-neo)), microbial transglutaminase (mTg) and mTg neo-epitope (mTg-neo). Marsh criteria were used for the degree of intestinal injury. Parallel, mTg and tTg neo-epitopes were purified by asymmetric field flow fractionation, confirmed by multi light scattering and SDS-page, and analyzed on the adult CD and controls group by competition ELISAs.

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No sequence homology but active site similarity were detected on alignment of the 2 Tgs. Comparing pediatric CD patients with the 2 normal groups: mTg-neo IgA, IgG and IgA+IgG antibody activities exceed the comparable mTg ones (p<0.0001). All mTg-neo and tTg-neo levels were higher (p<0.001). tTg IgA and IgG+IgA were higher than mTg IgA and IgA+IgG (p<0.0001). The levels of tTg-neo IgA/IgG were higher than tTg IgA/IgG (p<0.0001). The sequential antibody activities reflecting best the increased intestinal damage were: tTg-neo check>tTg-neo IgA ≥ mTg-neo IgG > tTg-neo IgG>mTg-neo check > mTg-neo IgA. Taken together, tTg-neo check, tTgneo IgA and mTg-neo IgG correlated best with intestinal pathology (r2=0.6454, r2=0.6165,r2=0.5633 p<0.0001, p<0.0001, p<0.0001, respectively). Purified mTg-neo IgG and IgA showed an increased immunoreactivity compared to single mTg and gliadin (p<0.001) but similar immunoreactivity to the tTg-neo IgG and IgA ELISA. Using a competition ELISA, the mTg neo-epitopes and tTg neo-epitopes have identical outcomes in CD sera both showing a decrease in optical density of 55±6%, (p<0.0002). mTg is immunogenic in children with CD and by complexing to gliadin its immunogenicity is enhanced. Anti-mTg-neo-epitope IgG antibodies correlate with intestinal damage to a comparable degree as anti-tTg-neo IgA. mTg and tTg display a comparable immunopotent epitope. mTg-neo IgG is a new marker for CD. Further studies are needed to explore the pathogenic potential of anti-mTg antibodies in CD.

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Introduction

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Celiac disease (CD) is an inflammatory enteropathy characterized by a harmful immune response to dietary gluten ingestion. The incidence of CD is increasing, parallel to a general worldwide surge in autoimmune diseases (1,2). Similarly to this trend, the food industry is expanding towards new technologies, introducing additives and ingredients to the processed food, thus changing the composition and antigenicity of modern food products (3,4). Microbial transglutaminase (mTg) is one of the enzymes that deamidates/transamidates proteins and is capable of cross-linking numerous molecules, thereby revolutionizing food product qualities. In fact, it is used as a major industrial glue, connecting proteins to improve products’ qualities such as gelation, solubility, foaming, viscosity, water-holding, emulsion stability, texture, time shelves, etc.(5,6) It belongs to a large family of Tgs with multifunctional related proteins, widely distributed in all living organisms. The predominant and classical function of these enzymes is protein crosslinkers, however, as more is discovered about their biology additional roles, complicate our understanding of their function in human biology and diseases. There is a rapidly expanding literature describing dysregulation of these enzymes in multiple diseases and how this contributes to the pathogenesis of human diseases. Tissue fibrosis, apoptosis, cancer and metastasis, celiac disease, neurodegenerative disorders such as Parkinson's disease, and skin diseases are just a few examples of where Tgs have been implicated (7-9). Human tissue transglutaminase (tTg) is the autoantigen and anti-tTg antibodies are the corresponding specific serological markers in CD (7-10). Both enzymes, the tTg and mTg de/transamidate gluten, known until now to be the main nutritional environmental factor inducing CD. Previously, it was hypothesized that mTg is a new environmental enhancer of CD, based on the multiple observations and scientific data, but direct proofs for immunogenicity of the enzyme or its complexes in celiac patients is lacking (3,11). Celiac disease (CD) is an autoimmune inflammatory disorder of the small intestine, triggered by the ingestion of prolamines contained in wheat, barley or rye, in genetically susceptible individuals. Specific amino acid sequences in gluten are the driving force in CD development through activation of T cells via the HLA presenting grove. These immunogenic/toxic peptides are taken up intact through

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intra- and inter-enterocyte routs, into the lamina propria where they interact with tTg which is the autoantigen of the disease (7). A major step in the pathogenic cascade, which will increase the immunogenicity of the gluten peptides is their deamidation/transamidation at the subepithelial level. The tTg plays a crucial role in the in vivo gluten preparation before being incorporated and processed by the antigen presenting cells and exposed to immune effector cells (7,8).

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The repertoire of environmental triggers of CD beyond that of gluten is expanding. The genetic determinants of CD cannot alone explain the changing phenotype expression of the disease in an individual nor the recent surge in CD incidence (1,2,12,13). Furthermore, the classical intestinal clinical picture of malnutrition, chronic diarrhea and nutritional deficiencies are disappearing and extraintestinal presentations are emerging. We are witnessing actually an epidemiological shift in the disease phenotype towards a more advanced age, and increased prevalence of latent, hypo-symptomatic or asymptomatic presentations (12-14). It is logical that such changes are triggered by environmental exposures, because genetic alterations are too slow to drive these phenomena. Except for the major role of prolamines in CD induction, multiple environmental factors have been reported as enhancers of the disease. Infections like Rotavirus in infants and Campylobacter jejuni in adults are associated with an increased risk of CD (15,16). The infectome-autoimmune diseases relationship is congruent with the hygiene hypothesis, which states that decreased exposure to microbes may be driving the rise of autoimmune diseases. Additional environmental factors that have been associated with increased risk for celiac disease include: a short period of breast feeding, the timing and increased amount of gluten ingestion, prescription of antibiotics and proton pump inhibitors, elective cesarean section, socioeconomic factors and most recently, maternal iron supplementation to pregnant woman(16). Given the uncertainty regarding causality, these associations between CD and environment mandate further investigations to test the mechanistic pathways by which modern exposures contribute to the induction of CD. Recently it was hypothesized that mTg is a new environmental enhancer of CD, based on the following observations and scientific data: It de/transamidates gluten like the endogenous human tTg. Being less substrate sensitive, it is capable of crosslinking many more proteins and other macromolecules, changing their

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structure/composition/antigenicity/physical and chemical characteristics resulting in an increased intestinal luminal load presented to the immune system. It increases stability of protein to proteinases, thus diminishing nutrient digestion and foreign protein elimination. Intestinal permeability is increased in CD and gluten and infections are major contributors to the intestinal leakage. Gluten is changed and cross-linked to many food constituents by industrial mTg. These mTg-mediated processes can potentially open the inter-enterocyte tight junction, allowing more immunogenic foreign molecules to induce CD (3,4,11).

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mTg is a frequent industrial food additive spanning a variety of industrial purposes: improvement of meat texture, appearance, hardness and shelf life, increase in fish product hardness, improved quality and texture of milk and dairy products, decrease calories, improved texture and elasticity of sweet foods, stability of protein films and improve texture and volume in the bakery industry ( 5,6, 17).

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In order to further investigate the role of the mTg in relation to CD induction the two enzymes were aligned for sequence similarity and the immune reactivity of the enzyme complexed to gliadin were investigated in CD patients’ sera compared to controls. The current hypothesis is that if the ingested mTg has a deleterious effect on celiac patients, it has to be absorbed and get in contact and stimulate the local and/or the systemic immune system, resulting in production of specific antibodies.

Material and methods: 1. Patient populations 1.1.Four groups of patients were investigated: 95 pediatric CD patients, mean age 8.3±4.4 y, F/M ratio 1: 09. The CD group was divided according to the degree of intestinal injury, using March criteria, to 6 groups M0, M1, M2, and M3a-c. M0 represents a normal intestinal biopsy and M3c-total villous atrophy (16). Those histological CD sub-groups contained M1 =7, M2=13, M3a=41, M3b=27, M3c=7 children, respectively. 1.2. 45 children with nonspecific abdominal pain (AP), mean age 7.3±5.1 y, F\M ratio 1:0.9, served as a normal gastrointestinal symptomatic control group, having M0 or M1 intestinal morphology.

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1.3. 99 normal children recruited from a normal school (NC), mean age 8.5 ±4.2 years, F\M ratio 1:0.7, served as a normal, asymptomatic pediatric control group. 1.4.79 normal adults (NA) mean age 28.1 ±5.1 years, F/M ratio 1:0.7, comprised the fourth group. The adults were normal young blood donors and serves as a normal asymptomatic control group.

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Only the CD and the AP groups underwent esophago-gastro-duodenoscopy using GIF-xp 20 endoscope (pentax, Tokyo, Japan). At least 5 biopsies were obtained: 4 from the second part of the duodenum for the diagnosis or exclusion of CD and 1 from the antrum. The biopsies were immediately fixed in buffered formalin and embedded on edge in paraffin. Sections were stained with hematoxylin-eosin and Giemsa, analyzed by the pathologist and graded according to Marsh criteria, as previously described (18) Celiac disease was diagnosed according to the revised criteria of the European Society for Pediatric Gastroenterology and nutrition, based on specific serology (anti-tTg antibodies, by ELISA) and duodenal biopsies (19). All the participants were on a gluten containing diet and had physical examination, laboratory workup, celiac serology. On the day of endoscopy, 5 ml of peripheral blood was withdrawn, centrifuge 5000 c/sec for 10 minutes and the serum was frozen in 80 °C until assayed for serology. The sera for groups 3 and 4 were an in-house bio-bank and were acquired commercially from in.vent DIAGNOSTICA GmbH, Germany. The local ethical committee of Carmel Medical Center, Haifa, Israel, approved the study and the patients or legal guardians signed the informed consent. 2. ELISA All the sera were tested by the following ELISAs, detecting IgA, IgG or both, IgA and IgG (check): AESKULISA® tTg (RUO), AESKULISA® tTg New Generation (tTG complexed to gliadin peptides: tTg neo-epitope (tTg-neo)), mTg (ZEDIRA GmbH, Germany) and mTg complexed to gliadin peptides (mTg neo-epitope). The kit uses a neo-epitope that is formed by a complex of mTg and gliadin peptides, the main antigens in CD. The basic idea is that mTg does not only deamidate gliadin peptides but also has a high catalyzation rate in crosslinking reactions. The results were compared to the degree of intestinal injury, using revised Marsh criteria.

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3. Sequence and structural alignment of mTg and tTg

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For the structural alignment the program YASARA was used which is a molecular graphics, modeling and simulation program. The structural alignment was performed with the basic structures of mTg (PDB-ID: 1IU4) and tTg (PDB-ID: 2Q3Z) by using the MUSTANG-algorithm (20). The structures were aligned in a way, that the amino acids of the catalytical triade fit together as good as possible. The sequence-conservation in the structural alignment is shown in shades between blue and yellow. The color refers to the apparent degree of sequence similarity as deduced from a Needleman-Wunsch alignment, performed with standard settings of the program “needle” from the EMBOSS software so that the higher the alignment is stained in the yellow color, the higher is the conservation.

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The docking experiment was also performed by YASARA, using an integrated autodocking program with a flexible approach for induced fit docking. The resulted complexes were stained among their electrostatic profile and the color refers to the apparent degree of sequence similarity as deducted from a Needleman-Wunsch alignment (21).

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4. Creation of tTg and mTg complexes The mTg and tTg stock solutions (ZEDIRA GmbH, Germany) were diluted to a final concentration of 0.1-0.5 U/ml in suitable reaction buffers. Gliadin peptides were added and the reaction mixtures were now allowed to react over night at room temperature. The complex formation was proven using SDS-PAGE and SEC-MALS (size exclusion chromatography combined with multi angle light scattering, supplied by Wyatt Technology Corp., Germany) analysis and stored at -20 °C. SECMALS analyses were performed using a SuperdexTM 75 (10/300 GL, supplied by GE Healthcare) column and 1x PBS or Tris buffer was used as mobile phase. To avoid bacterial contamination a UV lamp (Solaris supplied by ©Wyatt Technology Corp., Germany) was applied under stirring conditions in the mobile phase. A typical separation run including fractionation (fractionation steps were set up in 0.4 min steps at a detector flow rate of 0.5 ml/min) was completed in 50-60 min. The single fractions were analyzed on SDS-PAGE, concentrated and stored at 20 °C. 5. Competition Assay

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Competition ELISA was used to test whether the different single antigenic components (mTg, tTg, gliadin, DGP, mTg-neo and tTg-neo) influence the immunoreactivity of the patients’ sera in an ELISA experiment. The aim was to look for cross-reactivity between the single isolated enzymes and the formed neoepitope complexes.

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Sera were selected according to their antibody activities as determined by their corresponding ELISAs. For competition of the neo-epitopes, sera with high titer in mTg and tTg neo-epitope antibodies but low titer in the single antigens like DGP, Gliadin, mTg or tTg uncomplexed, were chosen. The selected sera were diluted according to the assay instructions and pre-incubated with different increasing concentrations of single antigenic components and the purified tTg and mTg neoepitopes. After pre-incubation, the sera were tested for immunoreactivity on a tTg neo-epitope ELISA plate, the outcomes were compared to the sera without added antigen (100% immunoreactivity).

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6. Statistics

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As an internal control, sera which showed positive results in the tTg-neo (IgA or IgG) assay from AESKU, but negative results to the self-performed tTg (RUO) were used.

Scatter diagrams and regression analysis comparing the antibodies’ OD activities to the degrees of the intestinal damage were used. Mean values of optical density (immunoreactivity against different antigens in patient sera) at different Marsh degrees were compared using the t-test and a p<0.05 was defined statistically significant. For analysis and graphical presentation, the statistical software MedCalc Version 12.7.1.0 was used. Sensitivity, specificity and the Area Under the Curve (AUC) values were calculated. The third and fourth groups’ antibody determination data were included in the statistical workup of the antibody performance comparisons.

Results: 1. Sequence alignment and three dimensional comparison

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Sequence alignment of mTg and tTg did not show analogy and just small identities, presenting more by chance (used sequences for alignment PDB ID: 3S3J vs. 3IU0). The yielded identity and similarity was smaller than 1%.

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The three dimensional structure of the two enzymes is shown in Fig 1a. Since the two enzymes differ structurally, tTg having 4 domains (molecular weight of 78 kDa), while mTg only one domain (molecular weight of 38 kDa), the expected homology was quite low. Indeed, the yellow segment, representing homology, is short. However, the structural alignment of the catalytical triade as shown in Fig 1b is more similar between the two enzymes, whereby, the 3 amino acids are equivalent (Cys – Asp– His), though, not in the same localization along the proteins.

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When the enzymes are docked by gliadin, the schematic 3 dimensional complexes of the two enzymes appear similar, when the electrical charges are taken in account. The stronger the red staining, the higher the negative charges, on both complexes (figure 1c)

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Taken together, there is no similarity in sequence or 3D structure alignment (figure 1 A) that could come along with immunopotency. The highest surface homology was found at the active center of the Tgs after binding of the gliadin peptides (PDB-ID: 1NNA), shown in figure 1 B, in the molecular docking experiment. Both enzymes show an adjacent electrostatically similar surface as shown by the relatively large hydrophobic areas of the gliadin peptides and the schematic, 3D similar binding properties of mTg and tTg (Figure 1c). Both neoepitope complexes show a superimposed epitope similarity at the catalytical site, even though the homology at the entrance to the active center is substantially low.

1. Antibodies performances Comparing pediatric CD patients with the 2 normal groups: (Figure No 2a, b) mTgneo IgA and IgG antibody activities exceed significantly the comparable mTg ones (p<0.0001). All mTg-neo and tTg-neo levels were significantly higher, than the single antigens’ titers (p<0.001). The levels of tTg-neo IgA/IgG were higher than tTg IgA/IgG (p<0.0001). It is clear that there is immunoreactivity against the single peptides alone but a much higher one when the Tgs are docked to gliadin. It

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seems that the IgG isotype against mTg-neo are much higher compared to the IgA isotypes. The opposite is true concerning the tTg-neo, where the IgA isotype has higher titers (Fig No 2,b)

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When IgA and IgA sera immunoreactivity against the enzymes alone or to their gliadin docked neo-epitope complexes, were compared between the 4 groups, the CD patients had a much higher reactivity (Figure 3). Once again the mTg-neo IgG and the tTg-neo IgA had the highest immune reactivities.

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Comparing antibody levels of the individual CD patients, sera with high antibody titers [U/ml] against the tTg neo-epitope ones- show similar high antibody activities to the mTg neo-epitope and vice versa, indicating the presence of similar epitopes within the Tg-gliadin complexes (Figure No 4). 2. Antibodies’ reflection of intestinal damage

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The sequential antibody activities, reflecting best the increased intestinal damage, going from M0 to M3c were: tTg-neo check ≥ tTg-neo IgA > mTg-neo IgG > tTgneo IgG >mTg check> mTg-neo IgA. (Table 1) Taken together, mTg-neo IgG, tTgneo IgA and tTg neo IgA+ IgG correlated best with intestinal pathology. (Figure No 5 A, B, C and Table 1). As depicted in Table 1, the higher the correlation factor, the better the correlation of the antibody to the intestinal atrophy of CD patients.

3. Antibodies’ competitive assaysThe dynamic behavior of the different antibodies, when competing on the corresponding antigens, is shown in Figure No 6. Identical outcomes of the competitive ELISA of the mTg neo-epitope and tTg neo-epitope antibodies using sera of selected celiac patients compared to several CD associated single antigens in increasing concentrations, was depicted. Moreover, the rest of the antibodies: tTg, gliadin, DGP and mTg behaved similarly. Only the antibodies against the gliadin docked mTg and tTg complexes present a separated competition graph, most probably competing on shared epitopes.

Discussion

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The present study shows, for the first time, that the mTg, a bacterial enzyme used profoundly as an industrial additive to many processed foods, stimulates the human immune system and induces specific antibodies. The immunogenicity of those antibodies is operative only in celiac patients and not in comparable, nonceliac, symptomatic children and not in the pediatric and adult control groups. This conclusion is further substantiated by the fact that the anti mTg neo-epitope antibodies’ levels positively correlate to the degree of the intestinal injury in CD. More so, this antigenicity is comparable to the endogenous tTg and to the tTg-neo complex. Despite the fact that the human microbiome contains a vast number of various bacteria, and since mTg is a normal constituent of those populations, the normal flora enzyme does not induce antibodies to a self-component, thus establishing a state of tolerance. Only in the CD patients a break of tolerance appears towards the mTg-gliadin complexes.

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Autoantibody production is an important feature of many autoimmune disorders, signifying a breakdown of immune tolerance to self-antigens. However, mounting an antibody to a non-self-environmental constituent, establish the reaction of the endogenous immune system toward an external factor that breaches the bodys’ protective barriers and stimulate the immune cells to react. Taken together, the cross-talks between those mTg neo-epitope antibodies against an environmental nutritional factor in CD surpass the observational frame and mount to cause and effect relationship. Immunogenically, the above described anti-mTg-neo antibodies, are distinguished from the immunoreactivity, observed in celiac patient’s sera against mTg treated gluten peptides. mTg modified gluten proteins were shown to react with celiac sera anti-gliadin IgA antibodies (22), in an age-related manner (23). mTg treatment of wheat prolamines in bread increases the serum IgA reactivity of CD patients. Interestingly enough, IgA from pooled celiac sera were higher against mTg –treated gluten-free breads than against mTg untreated ones. The electrophoretic pattern of gluten-free bread prolamines was altered by the mTg treatment (24). These observations imply that mTg-treated breads or gluten-free breads induce immunogenic peptides that react with human IgA. This is the appropriate place to remind that CD is an IgA mediated disease with specific IgA

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antibodies against wheat gliadins, endomysium, deaminated gliadin peptide, tTg and tTGneo-epitope complexes (25-29). Increased sera immunoreactivity for antigliadin antibodies was detected against mTg deamidated gliadins (30) and most recently, when chymotrypsin was compared to mTg induced transamidation of gluten proteins, the reduction of the immunoreactive gluten was substantially lower with the microbial Tg usage (71 v 42%, respectively)(31). In contrary, mTg treatment during pasta-production, didn’t affect the immunoreactivity of gliadin with CD patient’s sera (32).

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Additionally, mTg deamidated gluten peptides are recognized by gluten-specific T cells, thus enhancing the immunogenicity of gluten (33). Most recently, it was shown that mTg treated gluten peptides applied to cultured intestinal biopsies from CD patients, induced a 15 fold increase in INFγ release, and 2.5 and 2.1 fold increases in medium tTg antibody levels and endomysial antibody positivity, respectively (34). It can be concluded that both mTg-neo and mTg modified gluten proteins are immunogenic to the celiac population.

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On the contrary, most recently, Rothia mucilaginosa, a natural microbial inhabitant of the oral cavity was shown to enzymatically cleave gluten and abolish some gliadin immunogenic properties, however, the immunogenicity was not checked in vivo (35). Accumulating data infer multiple biological effect to celiac disease associated antibodies: inhibition of epithelial cell differentiation and stimulating their proliferation, increasing epithelial permeability especially to gliadin peptides, augmenting blood vessel permeability, defective angiogenesis, interference with gliadin up-take, induction of neuronal and trophoblast apoptosis and induction of ataxia in mice (36). None of those, in vitro and in vivo, effects were studied concerning anti mTg-neo antibodies.

The question arises why there is a difference between CD and the normal populations concerning the immunogenicity of the mTg when complexed with gliadin. Several potential explanations can be hypothesized: 1. The numerous susceptible genes described recently in CD by genome wide association studies or exon sequencing, many of which are involved in

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immune reaction and regulation, might affect antibody production. But this will not explain the recent changes in CD phenotype and its increased incidence. CD microflora is different from the normal population (37). If those bacterial alterations are pathogenic and induce immune reaction, resulting in anti-bacterial proteinomic antibodies, it might represent an explanation. Most recently, seroreactivity to different microbial antigens was observed already in patients with early-stage CD, indicating that microbial targets might have a role in early development of the disease (38). The increased intestinal permeability in CD, due to the gluten-zonulin interplay (39) or to the aberrant bacterial flora, might facilitate the way of the mTg or its immune peptides to affect or cross the tight junction and induce antibody production. Not all anti-tTg activities of patient sera are absorbed by guinea pig or monoclonal tTg (40), pointing to the existence of other submucosal autoantigens (41). In view of their active site, structural and functional homology between endogenous tTg and mTg and their immunogenicity in celiac sera, some shared epitopes presenting cross-antigenicity between the two might exist. A more appealing explanation is present in our recent hypotheses (3,4), whereby, the industrial food mTg increased consumption can explain the recently described epidemiological changes in the phenotype and incidence of CD worldwide. According to the hypotheses, mTg is pathogenic and immunogenic, as presently described. Recently, it was demonstrated that transglutaminase affects dendritic cells in a concentration-dependent manner. High concentrations were associated with a more mature phenotype and increased ability to stimulate T cells, while lower concentrations led to maintenance of an immature phenotype. These data provide support for an additional role of transglutaminase in celiac disease and demonstrate the potential of in vitro modelling of celiac disease pathogenesis. (42). Sharing functional activity with the mTg, one wonder if mTg can similarly affect the intestinal mucosal immune system.

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Most recently, injection of celiac disease patient sera or non IgAimmunoglobulins to mice induced symptoms and early intestinal celiac-like pathology (43). One wonders if the mTg antibodies, harbored in that sera or IgG immunoglobulins did not contribute to the observed intestinal damage. Another aspect highlighting the immunological gap between process and raw food ingredients was describe by Vojdani A (44). Human sera is much more immune reactive to the industrial processed food then to its raw ingredients.

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Based on the present observations, it is suggested that until more knowledge is available and further studies have been performed, there should be a full transparency of mTg use and clear labeling of the mTg used products for the benefit of the consumers and the public health. The regulatory authorities should declare the place of mTg as an additive for industrially processed food for the safety aspect. The discrepancy between the present observations and the official clearance of mTg from toxicity/side effects in relation to celiac disease, as appears in the producer/suppliers electronic and printed media, should be reassessed (45). The resulting product should be labeled accordingly, as was done recently by the Swiss regulatory authorities (46). Gluten-free products are especially important not only for the CD patients but because they are consumed by many non-celiac gluten sensitive populations. After showing the immunogenic effect of the mTg in the present study, the next steps should be directed at its potential pathogenicity.

Conclusions

mTg is immunogenic in children with CD and by complexing to gliadin its immunogenicity is enhanced. Figure No 7 show schematically the differential functions of the tTg and mTg enzymes when they deamidate/crosslink gliadin peptides. Corresponding to the immunogenic products, four specific antibodies are created: anti-tTg/DGP/ tTg-neo and mTg-neo. Anti-neo-epitope mTg antibodies correlate with intestinal damage to the same degree as anti-tTg-neo antibodies. mTg-neo and tTg-neo display comparable immunopotent epitopes. After a long list of CD serological biomarkers, encompassing 5 antibodies: anti gliadin and anti endomysial, tTg, DGP and tTg-neo (7, 9, 10, 18, 25-29, 47-51), a new generation of antibodies, namely anti- mTg-neo are presented. It is only after

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future back to back comparative multi-center study that we will know who the winner is in the long-termed race of CD diagnostic serological marker. The pathogenicity of those antibodies is a subject for future investigations.

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Figure 1 (a) 3D structure alignment of mTg and tTg produced with YASARA [Courtesy of Dr. Christian Meesters, 2012]. Starting from the left side a schematic draw of tTg (PDB-ID 2Q3Z, shown in green) is shown as well as the mTg (PDB-ID 1IU4, shown in magenta). On the right side of the picture, both enzymes are shown in top of each other. The color refers to the apparent degree of sequence similarity as deducted from a Needleman-Wunsch alignment, so that the higher the alignment is stained in the yellow color, the higher is the conservation. (b) Structural alignment of the catalytical triade is shown. The figure shows the mTg (magenta, catalytical triade: Cys 64 – Asp 255 – His 274) and tTg (green active conformation and blue inactive conformation: Cys 277 – His 335 – Asp - 358) alignment on their catalytical triade.

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(c) Docking of gliadin peptide (PDB-ID: 1NNA) with mTg (left side) and tTg (right side). The arisen complexes got stained among their electrostatic profile; the stronger the red staining, the higher the negative charges. [Courtesy of Dr. Christian Meesters, AESKU.KIPP Institute 2012]

Figure 2 (A and B): Immunoreactivity of the anti-mTg/ tTgneo-epitope IgA and IgG and of the IgA and IgG antibodies of the single compounds mTg, tTg and gliadin (not complexed to gliadin complexes).

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Comparing the different IgG antibody activities - lowest to mTg, intermediate to tTg, higher to tTg-neoand highest to mTg-neo - it can be concluded that, most probably, the antibodies against the mTg- neo active site are more clinically relevant, since they are binding to epitopes of the open binding (gliadin docked) sites. In the tTG-neo- case, most probably, they are binding to the epitopes of the binding site but also to some other non-active site determinants. It means that anti-mTg-neo antibodies are more clinically relevant and correlate to intestinal damage. By mTg docking of gliadin, immune reactivity is enhanced leading to two possible explanations: 1. More epitopes are exposed. 2. The epitopes are more immunogenic.

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Figure 3: Sera Immunoreactivity of the enzymes and the resulting neo-epitopes comparing pediatric CD to two different control groups for IgG and IgA isotypes

Figure 4: Antibody titers of tTg neo-epitopes and mTg neo-epitopes in CD sera of different treated or untreated disease stages. (UCD = untreated Celiac patient on gluten containing diet, TCD = treated Celiac patient on gluten free diet).

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Figure 5 (a, b and c): Antibodies’ activity in relation to the degree of intestinal damage in CD. A.

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IgA mTg neo-epitope compared to IgA tTg neo-epitope. B. IgG mTg neo-epitope compared to IgG tTg neo-epitope. C. IgG+IgA (check) mTg neo-epitope compared to tTg neo-epitope.

Figure 6: The dynamic decreases in the IgA antibody % competition in relation to the

corresponding antigens’ concentrations.

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Figure 7: A schematic presentation of the interplay between gliadin, tTg and mTg enzymes and

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their corresponding complexes and specific antibodies.

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Table 1: Correlations between antibodies’ activity in relation to the degree of intestinal damage in CD

Antibody

Correlation coefficient r

P-value

tTg neo check

0.6454

<0.0001

tTg neo IgA

0.6165

<0.0001

mTg neo IgG

0.5633

<0.0001

tTg neo IgG

0.5334

<0.0001

mTg neo check

0.5127

<0.0001

mTg neo IgA

0.3018

0.003

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mTg neo IgA

65.69

98.99

73.68

98.53

0.82

mTg neo IgG

94.95

93.94

94.9

94

0.94

mTg neo check

89.9

87.88

89.69

88.12

0.89

tTg neo IgA

86.87

98.99

88.29

98.85

0.93

tTg neo IgG

77.78

97.98

97.47

81.51

0.88

tTg neo check

97.98

100

98.02

0.99

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Table 2: The sensitivity and specificity values of the single antibodies and the combined antibodies (check). mTg neo-epitope IgG had higher sensitivity than tTg neo-epitope IgG. tTg neo-epitope showed the highest sensitivity as check.

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