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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

Homotypic TG2 association is conformation dependent(A) TG2 (2.4 μM) self-crosslinking over time occurs via initial formation of dimer and trimer as observed after 5 and 10 min. (B) Inactive, inhibitor bound TG2 produced in E. coli (TG2-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 size as diameter (d. nm) on the X-axis. Incubation with 9 mM CaCl2 at 37°C results in aggregation formation already after 10 min. (C) Titration of CaCl2 was performed at 37°C and non-covalent complex formation was compared at different time-points: 0 min (black line, grey fill), 10 min (black solid line) and 20 min (black dotted line). For 1 mM CaCl2, 60 min incubation is also shown (grey dotted line). The solid grey line shows TG2-DP3-3 incubated in 1 mM EDTA at time 0 min for comparison. Two datasets from the same experiment are shown for 2.5 mM CaCl2 as variation was observed in the degree of high molecular weight multimer formation for this CaCl2 concentration. (D) Capture of non-covalent TG2-TG2 associations using BS3 cross-linking reagent. TG2 was incubated in the absence of effectors or together with 5 mM CaCl2 or 1 mM GTP for 30 min at 37°C or, where indicated, for 30 min at room temperature (RT) before addition of BS3. To avoid enzymatic self-crosslinking in the presence of Ca2+ the enzyme was pretreated with the active-site inhibitor DP3-3. Incubation of TG2-DP3-3 with CaCl2 at 37°C followed by BS3 treatment results in the formation of large complexes that do not migrate through the gel.
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pone.0134922.g002: Homotypic TG2 association is conformation dependent(A) TG2 (2.4 μM) self-crosslinking over time occurs via initial formation of dimer and trimer as observed after 5 and 10 min. (B) Inactive, inhibitor bound TG2 produced in E. coli (TG2-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 size as diameter (d. nm) on the X-axis. Incubation with 9 mM CaCl2 at 37°C results in aggregation formation already after 10 min. (C) Titration of CaCl2 was performed at 37°C and non-covalent complex formation was compared at different time-points: 0 min (black line, grey fill), 10 min (black solid line) and 20 min (black dotted line). For 1 mM CaCl2, 60 min incubation is also shown (grey dotted line). The solid grey line shows TG2-DP3-3 incubated in 1 mM EDTA at time 0 min for comparison. Two datasets from the same experiment are shown for 2.5 mM CaCl2 as variation was observed in the degree of high molecular weight multimer formation for this CaCl2 concentration. (D) Capture of non-covalent TG2-TG2 associations using BS3 cross-linking reagent. TG2 was incubated in the absence of effectors or together with 5 mM CaCl2 or 1 mM GTP for 30 min at 37°C or, where indicated, for 30 min at room temperature (RT) before addition of BS3. To avoid enzymatic self-crosslinking in the presence of Ca2+ the enzyme was pretreated with the active-site inhibitor DP3-3. Incubation of TG2-DP3-3 with CaCl2 at 37°C followed by BS3 treatment results in the formation of large complexes that do not migrate through the gel.

Mentions: TG2 initially formed cross-linked dimers, extending into multimers over time (Fig 2A) suggesting that the complexes form in an orderly manner through specific TG2-TG2 interactions. To study non-covalent interactions we utilized a catalytically inactive variant of TG2 where the active site is covalently linked to the inhibitor Ac-P-DON-LPF-NH2 (DP3-3) (TG2-DP3-3). By dynamic light scattering (DLS) analysis we found that inactive TG2 incubated with EDTA at 37°C stayed as a monomer (Fig 2B). However, incubation with 9 mM CaCl2 at 37°C resulted in rapid formation of high molecular weight multimers (Fig 2B). Upon titration of CaCl2, we observed rapid multimer-formation also at 5 mM CaCl2 and at a slower rate at 2.5 mM CaCl2. Incubation with 1 mM CaCl2 resulted in a smaller increase in size, likely reflecting formation of low order complexes (Fig 2C). The resolving power of our DLS instrument did not allow us to determine whether this increase in size reflected formation of dimers or trimers. We therefore utilized the bivalent amine reactive cross-linker bis(sulfosuccinimidyl)suberate (BS3) which will covalently cross-link and capture non-covalent protein-protein interactions. BS3 treatment of inactive TG2 (TG2-DP3-3) pre-incubated with 5 mM CaCl2 at 37°C confirmed the formation of non-covalent high molecular weight multimers (Fig 2D) that we had observed by DLS. In addition, TG2 was indeed captured as dimers, trimers and lower order multimers when incubated at 37°C in the absence of CaCl2, or at room temperature with 5 mM CaCl2. Importantly, no self-assembly was observed when TG2 was incubated with GTP to induce the compact, closed conformation. Thus, TG2 can form non-covalent TG2-TG2 complexes depending on the conformational state of the enzyme. Binding of Ca2+ promotes this association, and saturating concentration of CaCl2 and incubation at 37°C shifts the interaction from transient dimer/trimer formation to formation of high molecular weight complexes.


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)

Homotypic TG2 association is conformation dependent(A) TG2 (2.4 μM) self-crosslinking over time occurs via initial formation of dimer and trimer as observed after 5 and 10 min. (B) Inactive, inhibitor bound TG2 produced in E. coli (TG2-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 size as diameter (d. nm) on the X-axis. Incubation with 9 mM CaCl2 at 37°C results in aggregation formation already after 10 min. (C) Titration of CaCl2 was performed at 37°C and non-covalent complex formation was compared at different time-points: 0 min (black line, grey fill), 10 min (black solid line) and 20 min (black dotted line). For 1 mM CaCl2, 60 min incubation is also shown (grey dotted line). The solid grey line shows TG2-DP3-3 incubated in 1 mM EDTA at time 0 min for comparison. Two datasets from the same experiment are shown for 2.5 mM CaCl2 as variation was observed in the degree of high molecular weight multimer formation for this CaCl2 concentration. (D) Capture of non-covalent TG2-TG2 associations using BS3 cross-linking reagent. TG2 was incubated in the absence of effectors or together with 5 mM CaCl2 or 1 mM GTP for 30 min at 37°C or, where indicated, for 30 min at room temperature (RT) before addition of BS3. To avoid enzymatic self-crosslinking in the presence of Ca2+ the enzyme was pretreated with the active-site inhibitor DP3-3. Incubation of TG2-DP3-3 with CaCl2 at 37°C followed by BS3 treatment results in the formation of large complexes that do not migrate through the gel.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134922.g002: Homotypic TG2 association is conformation dependent(A) TG2 (2.4 μM) self-crosslinking over time occurs via initial formation of dimer and trimer as observed after 5 and 10 min. (B) Inactive, inhibitor bound TG2 produced in E. coli (TG2-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 size as diameter (d. nm) on the X-axis. Incubation with 9 mM CaCl2 at 37°C results in aggregation formation already after 10 min. (C) Titration of CaCl2 was performed at 37°C and non-covalent complex formation was compared at different time-points: 0 min (black line, grey fill), 10 min (black solid line) and 20 min (black dotted line). For 1 mM CaCl2, 60 min incubation is also shown (grey dotted line). The solid grey line shows TG2-DP3-3 incubated in 1 mM EDTA at time 0 min for comparison. Two datasets from the same experiment are shown for 2.5 mM CaCl2 as variation was observed in the degree of high molecular weight multimer formation for this CaCl2 concentration. (D) Capture of non-covalent TG2-TG2 associations using BS3 cross-linking reagent. TG2 was incubated in the absence of effectors or together with 5 mM CaCl2 or 1 mM GTP for 30 min at 37°C or, where indicated, for 30 min at room temperature (RT) before addition of BS3. To avoid enzymatic self-crosslinking in the presence of Ca2+ the enzyme was pretreated with the active-site inhibitor DP3-3. Incubation of TG2-DP3-3 with CaCl2 at 37°C followed by BS3 treatment results in the formation of large complexes that do not migrate through the gel.
Mentions: TG2 initially formed cross-linked dimers, extending into multimers over time (Fig 2A) suggesting that the complexes form in an orderly manner through specific TG2-TG2 interactions. To study non-covalent interactions we utilized a catalytically inactive variant of TG2 where the active site is covalently linked to the inhibitor Ac-P-DON-LPF-NH2 (DP3-3) (TG2-DP3-3). By dynamic light scattering (DLS) analysis we found that inactive TG2 incubated with EDTA at 37°C stayed as a monomer (Fig 2B). However, incubation with 9 mM CaCl2 at 37°C resulted in rapid formation of high molecular weight multimers (Fig 2B). Upon titration of CaCl2, we observed rapid multimer-formation also at 5 mM CaCl2 and at a slower rate at 2.5 mM CaCl2. Incubation with 1 mM CaCl2 resulted in a smaller increase in size, likely reflecting formation of low order complexes (Fig 2C). The resolving power of our DLS instrument did not allow us to determine whether this increase in size reflected formation of dimers or trimers. We therefore utilized the bivalent amine reactive cross-linker bis(sulfosuccinimidyl)suberate (BS3) which will covalently cross-link and capture non-covalent protein-protein interactions. BS3 treatment of inactive TG2 (TG2-DP3-3) pre-incubated with 5 mM CaCl2 at 37°C confirmed the formation of non-covalent high molecular weight multimers (Fig 2D) that we had observed by DLS. In addition, TG2 was indeed captured as dimers, trimers and lower order multimers when incubated at 37°C in the absence of CaCl2, or at room temperature with 5 mM CaCl2. Importantly, no self-assembly was observed when TG2 was incubated with GTP to induce the compact, closed conformation. Thus, TG2 can form non-covalent TG2-TG2 complexes depending on the conformational state of the enzyme. Binding of Ca2+ promotes this association, and saturating concentration of CaCl2 and incubation at 37°C shifts the interaction from transient dimer/trimer formation to formation of high molecular weight complexes.

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