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Enhanced B-Cell Receptor Recognition of the Autoantigen Transglutaminase 2 by Efficient Catalytic Self-Multimerization.

Stamnaes J, Iversen R, du Pré MF, Chen X, Sollid LM - PLoS ONE (2015)

Bottom Line: 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.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.Importantly, high avidity of the antigen could explain why TG2-specific plasma cells show signs of an extrafollicular generation pathway.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

The C-terminal domains are required for homotypic association and self-crosslinking(A) A truncated variant of TG2 lacking the two C-terminal domains (1–465) was expressed in E. coli and treated with DP3-3. The inactive, inhibitor-bound deletion mutant (1-465-DP3-3) was incubated at 37°C in the presence of 1 mM EDTA or 9 mM CaCl2 and aggregation was measured by DLS. The graphs display volume percentage (Y axis) and diameter (d.nm) on the X-axis. No multimer formation of the truncated TG2 was observed following 40 min incubation. (B) Capture of non-covalent complexes of inactive E. coli-produced DP3-3-treated full length (WT) and 1–465 deletion mutant (1–465) TG2 following incubation at 37°C using BS3 as described in Fig 2C. No complex formation was observed for the deletion mutant compared to full-length TG2. (C) Incubation of active E. coli-produced deletion mutant (1–465) with CaCl2 did not result in self-crosslinking as observed for full length E. coli-produced TG2. The gel shows incubation of different enzyme concentrations with 5 mM CaCl2 at 37°C for 60 min: 1) 0.7 μM, 2) 1.4 μM, 4) 4.2 μM and 3) 1.4 μM in the presence of 0.2 mM DQ2.5-glia-α2-QQ-FITC. Incorporated gluten peptides are visualized by FITC fluorescence.
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pone.0134922.g003: The C-terminal domains are required for homotypic association and self-crosslinking(A) A truncated variant of TG2 lacking the two C-terminal domains (1–465) was expressed in E. coli and treated with DP3-3. The inactive, inhibitor-bound deletion mutant (1-465-DP3-3) was incubated at 37°C in the presence of 1 mM EDTA or 9 mM CaCl2 and aggregation was measured by DLS. The graphs display volume percentage (Y axis) and diameter (d.nm) on the X-axis. No multimer formation of the truncated TG2 was observed following 40 min incubation. (B) Capture of non-covalent complexes of inactive E. coli-produced DP3-3-treated full length (WT) and 1–465 deletion mutant (1–465) TG2 following incubation at 37°C using BS3 as described in Fig 2C. No complex formation was observed for the deletion mutant compared to full-length TG2. (C) Incubation of active E. coli-produced deletion mutant (1–465) with CaCl2 did not result in self-crosslinking as observed for full length E. coli-produced TG2. The gel shows incubation of different enzyme concentrations with 5 mM CaCl2 at 37°C for 60 min: 1) 0.7 μM, 2) 1.4 μM, 4) 4.2 μM and 3) 1.4 μM in the presence of 0.2 mM DQ2.5-glia-α2-QQ-FITC. Incorporated gluten peptides are visualized by FITC fluorescence.

Mentions: Comparing the crystal structures of closed, GTP bound TG2 and the open, presumably active conformation of TG2 [24, 25], the most notable structural change involves movement of the two C-terminal beta-barrel domains away from the catalytic core domain. To address if the C-terminal domains were directly involved in TG2 multimerization, we created a deletion mutant (1–465) lacking these two domains. Indeed, in contrast to full length TG2, an inactive inhibitor-bound form of this deletion mutant (1-465-DP3-3) did not form multimers upon incubation with 9 mM CaCl2 at 37°C as observed by DLS (Fig 3A) or by capture with BS3 (Fig 3B). Further, in its active state, this deletion mutant did not cross-link itself into covalent multimers as observed for full length TG2 despite being able to efficiently cross-link FITC-labeled gluten peptides to itself (Fig 3C). The two C-terminal domains of TG2 thus appear pivotal for homotypic TG2 association and also for self-crosslinking. This supports the notion that homotypic association is required for TG2 to efficiently act as a self-substrate.


Enhanced B-Cell Receptor Recognition of the Autoantigen Transglutaminase 2 by Efficient Catalytic Self-Multimerization.

Stamnaes J, Iversen R, du Pré MF, Chen X, Sollid LM - PLoS ONE (2015)

The C-terminal domains are required for homotypic association and self-crosslinking(A) A truncated variant of TG2 lacking the two C-terminal domains (1–465) was expressed in E. coli and treated with DP3-3. The inactive, inhibitor-bound deletion mutant (1-465-DP3-3) was incubated at 37°C in the presence of 1 mM EDTA or 9 mM CaCl2 and aggregation was measured by DLS. The graphs display volume percentage (Y axis) and diameter (d.nm) on the X-axis. No multimer formation of the truncated TG2 was observed following 40 min incubation. (B) Capture of non-covalent complexes of inactive E. coli-produced DP3-3-treated full length (WT) and 1–465 deletion mutant (1–465) TG2 following incubation at 37°C using BS3 as described in Fig 2C. No complex formation was observed for the deletion mutant compared to full-length TG2. (C) Incubation of active E. coli-produced deletion mutant (1–465) with CaCl2 did not result in self-crosslinking as observed for full length E. coli-produced TG2. The gel shows incubation of different enzyme concentrations with 5 mM CaCl2 at 37°C for 60 min: 1) 0.7 μM, 2) 1.4 μM, 4) 4.2 μM and 3) 1.4 μM in the presence of 0.2 mM DQ2.5-glia-α2-QQ-FITC. Incorporated gluten peptides are visualized by FITC fluorescence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526674&req=5

pone.0134922.g003: The C-terminal domains are required for homotypic association and self-crosslinking(A) A truncated variant of TG2 lacking the two C-terminal domains (1–465) was expressed in E. coli and treated with DP3-3. The inactive, inhibitor-bound deletion mutant (1-465-DP3-3) was incubated at 37°C in the presence of 1 mM EDTA or 9 mM CaCl2 and aggregation was measured by DLS. The graphs display volume percentage (Y axis) and diameter (d.nm) on the X-axis. No multimer formation of the truncated TG2 was observed following 40 min incubation. (B) Capture of non-covalent complexes of inactive E. coli-produced DP3-3-treated full length (WT) and 1–465 deletion mutant (1–465) TG2 following incubation at 37°C using BS3 as described in Fig 2C. No complex formation was observed for the deletion mutant compared to full-length TG2. (C) Incubation of active E. coli-produced deletion mutant (1–465) with CaCl2 did not result in self-crosslinking as observed for full length E. coli-produced TG2. The gel shows incubation of different enzyme concentrations with 5 mM CaCl2 at 37°C for 60 min: 1) 0.7 μM, 2) 1.4 μM, 4) 4.2 μM and 3) 1.4 μM in the presence of 0.2 mM DQ2.5-glia-α2-QQ-FITC. Incorporated gluten peptides are visualized by FITC fluorescence.
Mentions: Comparing the crystal structures of closed, GTP bound TG2 and the open, presumably active conformation of TG2 [24, 25], the most notable structural change involves movement of the two C-terminal beta-barrel domains away from the catalytic core domain. To address if the C-terminal domains were directly involved in TG2 multimerization, we created a deletion mutant (1–465) lacking these two domains. Indeed, in contrast to full length TG2, an inactive inhibitor-bound form of this deletion mutant (1-465-DP3-3) did not form multimers upon incubation with 9 mM CaCl2 at 37°C as observed by DLS (Fig 3A) or by capture with BS3 (Fig 3B). Further, in its active state, this deletion mutant did not cross-link itself into covalent multimers as observed for full length TG2 despite being able to efficiently cross-link FITC-labeled gluten peptides to itself (Fig 3C). The two C-terminal domains of TG2 thus appear pivotal for homotypic TG2 association and also for self-crosslinking. This supports the notion that homotypic association is required for TG2 to efficiently act as a self-substrate.

Bottom Line: 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.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.Importantly, high avidity of the antigen could explain why TG2-specific plasma cells show signs of an extrafollicular generation pathway.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus