Activation and Inhibition of Transglutaminase 2 in Mice

Transglutaminase 2 (TG2) is an allosterically regulated enzyme with transamidating, deamidating and cell signaling activities. It is thought to catalyze sequence-specific deamidation of dietary gluten peptides in the small intestines of celiac disease patients. Because this modification has profound consequences for disease pathogenesis, there is considerable interest in the design of small molecule TG2 inhibitors. Although many classes of TG2 inhibitors have been reported, thus far an animal model for screening them to identify promising celiac drug candidates has remained elusive. Using intraperitoneal administration of the toll-like receptor 3 (TLR3) ligand, polyinosinic-polycytidylic acid (poly(I∶C)), we induced rapid TG2 activation in the mouse small intestine. Dose dependence was observed in the activation of TG2 as well as the associated villous atrophy, gross clinical response, and rise in serum concentration of the IL-15/IL-15R complex. TG2 activity was most pronounced in the upper small intestine. No evidence of TG2 activation was observed in the lung mucosa, nor were TLR7/8 ligands able to elicit an analogous response. Introduction of ERW1041E, a small molecule TG2 inhibitor, in this mouse model resulted in TG2 inhibition in the small intestine. TG2 inhibition had no effect on villous atrophy, suggesting that activation of this enzyme is a consequence, rather than a cause, of poly(I∶C) induced enteropathy. Consistent with this finding, administration of poly(I∶C) to TG2 knockout mice also induced villous atrophy. Our findings pave the way for pharmacological evaluation of small molecule TG2 inhibitors as drug candidates for celiac disease

Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia

Transglutaminase 2 (TG2) is well characterized as the main autoantigen of celiac disease. The ability of TG2 to deamidate and crosslink gluten peptides is essential for the gluten-dependent production of TG2 specific autoantibodies. In patients with primarily extraintestinal manifestation of gluten sensitivity the repertoire of autoantibodies may be different. In dermatitis herpetiformis (DH), TG3 appears to be the target autoantigen whereas in gluten ataxia (GA) autoantibodies reactive with TG6 are present. A functional role for TG3 and TG6 in these diseases has yet to be described. It is also not known whether these enzymes can use gluten peptides implicated in the pathology as substrates. We here report that similar to TG2, TG3 and TG6 can specifically deamidate gluten T cell epitopes. However, the fine specificities of the enzymes were found to differ. TG2 can form covalent complexes with gluten by iso-peptide and thioester bonds. We found that both TG3 and TG6 were able to complex with gluten peptides through thioester linkage although less efficiently than TG2, whereas TG6 but not TG3 was able to form iso-peptide linked complexes. Our findings lend credence to the notion that TG3 and TG6 are involved in the gluten-induced autoimmune responses of DH and GA

CTR (4032)

Mass spectrometry QExactive RAW data file; tryptic digest of non-crosslinked TG

Data from: Enhanced B-cell receptor recognition of the autoantigen transglutaminase 2 by efficient catalytic self-multimerization

A hallmark of the gluten-driven enteropathy celiac disease is autoantibody production towards the enzyme transglutaminase 2 (TG2) that catalyzes the formation of covalent protein-protein cross-links. Activation of TG2-specific B cells likely involves gluten-specific CD4 T cells as production of the antibodies is dependent on disease-associated HLA-DQ allotypes and dietary intake of gluten. IgA plasma cells producing TG2 antibodies with few mutations are abundant in the celiac gut lesion. These plasma cells and serum antibodies to TG2 drop rapidly after initiation of a gluten-free diet, suggestive of extrafollicular responses or germinal center reactions of short duration. High antigen avidity is known to promote such responses, and is also important for breakage of self-tolerance. We here inquired whether TG2 avidity could be a feature relevant to celiac disease. Using recombinant enzyme we show by dynamic light scattering and gel electrophoresis that TG2 efficiently utilizes itself as a substrate due to conformation-dependent homotypic association, which involves the C-terminal domains of the enzyme. This leads to the formation of covalently linked TG2 multimers. The presence of exogenous substrate such as gluten peptide does not inhibit TG2 self-cross-linking, but rather results in formation of TG2-TG2-gluten complexes. The celiac disease autoantibody epitopes, clustered in the N-terminal part of TG2, are conserved in the TG2-multimers as determined by mass spectrometry and immunoprecipitation analysis. TG2 multimers are superior to TG2 monomer in activating A20 B cells transduced with TG2-specific B-cell receptor, and uptake of TG2-TG2-gluten multimers leads to efficient activation of gluten-specific T cells. Efficient catalytic self-multimerization of TG2 and generation of multivalent TG2 antigen decorated with gluten peptides suggest a mechanism by which self-reactive B cells are activated to give abundant numbers of plasma cells in celiac disease. Importantly, high avidity of the antigen could explain why TG2-specific plasma cells show signs of an extrafollicular generation pathway

Dimer (II) (4040)

Mass spectrometry QExactive RAW data file; tryptic digest of self-crosslinked TG2 (dimer, size exclusion fraction II

Treatment of C57BL/6J mice with poly(I∶C) results in villous atrophy and activation of TG2 at the villus tips.

<p>Single intraperitoneal injections of 30 (a–d), 20 (e–h), 15 (i–l), 5 (m–p), or 0 (q–t) mg/kg poly(I∶C) were administered. Mice were sacrificed after 9 h, and small intestines collected and frozen in OCT. Sections of 20 µm thickness were cut, fixed, washed, permeabilized, and exposed first to a rabbit anti-TG2 antibody (ab421) followed by a secondary Alexa fluor 488-congugated goat anti-rabbit IgG (c, g, k, o, s). Total TG2 protein (active and non active) is shown. TG2 activity after poly(I∶C) injury, measured using 5BP crosslinking, was visualized by co-staining with Alexa fluor 555-conjugated streptavidin (b, f, j, n, r). Nuclei were stained with DAPI (a, e, i, m, q). Overlays of the three stains are shown in (d, h, l, p, t). At least 3 animals were tested at each poly(I∶C) dose shown; one representative duodenal section is shown.</p

Monomer (I) (4036)

Mass spectrometry QExactive RAW data file; tryptic digest of self-crosslinked TG2 (monomer, size exclusion fraction I

H&E staining of duodenal cross sections from mice treated with poly(I∶C) at 30 mg/kg (A), 15 mg/kg (B), or 0 mg/kg poly(I∶C) (C).

<p>Animals were sacrificed 9 h after poly(I∶C) administration, and their small intestines were harvested.</p

Inhibition of TG2 activity by ERW1041E in the small intestine of poly(I∶C)-treated mice.

<p>Mice were treated with 20 mg/kg (i–p), or 15 mg/kg (q–x) poly(I∶C), followed by two intraperitoneal injections of vehicle (i–l; q–t) or 50 mg/kg ERW1041E (m–p; u-bb) at 6 h and 9 h. Control cohorts were dosed with no pharmacological agent (a–d), only 5BP (e–h) or ERW1041E and 5BP (y-bb). TG2 activity was detected by exposing duodenal cross-sections to Alexa fluor 555-conjugated streptavidin (b, f, j, n, r, v, z). TG2 protein was visualized by co-staining with polyclonal anti-TG2 antibody, followed by a secondary antibody conjugated to Alexa fluor 488 (c, g, k, o, s, w, aa). Nuclei were visualized by staining with DAPI (a, e, i, m, q, u, y). The right column contains overlay images (d, h, l, p, t, x, bb). At least 3 animals were tested in each cohort; one representative duodenal section is shown.</p