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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

TG2 multimers are superior to TG2 monomer in activation of TG2-specific A20 B cells(A) Celiac TG2-specific monoconal antibodies are able to bind TG2-gluten multimer complexes. TG2 crosslinked in the presence of biotin-33mer peptide was immunoprecipitated using TG2-specific monoclonal antibodies representing all four celiac epitopes, or using a non TG2-specific monoclonal antibody (693-2-F02). TG2 was detected with mouse monoclonal antibody CUB7402 and TG2-bound peptide was detected with HRP-conjugated streptavidin. (B) Intracellular Ca2+ flux was compared following stimulation of transduced A20 B cells expressing HLA-DQ2.5 and TG2-specific (679-14-E06) or non TG2-specific (693-2-F02) IgD BCR with size-separated TG2 complexes or bivalent mouse anti-human IgD antibody. TG2 multimers were superior to monomer and dimers. Calcium flux induced by TG2 was BCR dependent and not observed in control cells. (C) A20 cells expressing TG2-specific (679-14-E06), non TG2-specific (693-2-F02) BCR or only HLA-DQ2.5 were incubated with complexes of TG2 and FITC-labeled 33mer peptide or with anti-human IgD antibody followed by staining for intracellular phoshorylated Erk (pErk). ERK phosphorylation was BCR-dependent and was not observed in A20 cells expressing only HLA-DQ2.5. (D, E) A20 cells expressing HLA-DQ2.5 and TG2-specific (679-14-E06) or non TG2-specific (693-2-F02) IgD BCR were incubated on ice with complexes of TG2 followed by incubation at 37°C. BCR internalization was assessed by flow cytometry staining for surface IgD. Columns in (B) show the means of triplicates from the second experiment (+SD), columns in (D) show the means of two independent experiments (+SD).
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pone.0134922.g005: TG2 multimers are superior to TG2 monomer in activation of TG2-specific A20 B cells(A) Celiac TG2-specific monoconal antibodies are able to bind TG2-gluten multimer complexes. TG2 crosslinked in the presence of biotin-33mer peptide was immunoprecipitated using TG2-specific monoclonal antibodies representing all four celiac epitopes, or using a non TG2-specific monoclonal antibody (693-2-F02). TG2 was detected with mouse monoclonal antibody CUB7402 and TG2-bound peptide was detected with HRP-conjugated streptavidin. (B) Intracellular Ca2+ flux was compared following stimulation of transduced A20 B cells expressing HLA-DQ2.5 and TG2-specific (679-14-E06) or non TG2-specific (693-2-F02) IgD BCR with size-separated TG2 complexes or bivalent mouse anti-human IgD antibody. TG2 multimers were superior to monomer and dimers. Calcium flux induced by TG2 was BCR dependent and not observed in control cells. (C) A20 cells expressing TG2-specific (679-14-E06), non TG2-specific (693-2-F02) BCR or only HLA-DQ2.5 were incubated with complexes of TG2 and FITC-labeled 33mer peptide or with anti-human IgD antibody followed by staining for intracellular phoshorylated Erk (pErk). ERK phosphorylation was BCR-dependent and was not observed in A20 cells expressing only HLA-DQ2.5. (D, E) A20 cells expressing HLA-DQ2.5 and TG2-specific (679-14-E06) or non TG2-specific (693-2-F02) IgD BCR were incubated on ice with complexes of TG2 followed by incubation at 37°C. BCR internalization was assessed by flow cytometry staining for surface IgD. Columns in (B) show the means of triplicates from the second experiment (+SD), columns in (D) show the means of two independent experiments (+SD).

Mentions: Conceivably, formation of TG2-multimers is relevant for activation of TG2-specific B cells in celiac disease, and incorporation of gluten peptides into multimers would allow efficient collaboration between TG2-specific B cells and gluten-specific T cells. A prerequisite for B-cell activation is recognition of the antigen by the BCR. To determine whether celiac anti-TG2 antibodies, and thus BCRs, could still recognize multimerized TG2 crosslinked to gluten peptide, we performed immunoprecipitation of TG2 incubated with CaCl2 in the presence of biotinylated 33mer gluten peptide using monoclonal human IgG antibodies derived from our panel of recombinant celiac anti-TG2 antibodies [14]. Antibodies of all four epitopes could bind and pull down TG2-TG2-33mer complexes (Fig 5A). Thus, the epitopes are conserved in TG2 multimers as predicted from our MS analysis.


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)

TG2 multimers are superior to TG2 monomer in activation of TG2-specific A20 B cells(A) Celiac TG2-specific monoconal antibodies are able to bind TG2-gluten multimer complexes. TG2 crosslinked in the presence of biotin-33mer peptide was immunoprecipitated using TG2-specific monoclonal antibodies representing all four celiac epitopes, or using a non TG2-specific monoclonal antibody (693-2-F02). TG2 was detected with mouse monoclonal antibody CUB7402 and TG2-bound peptide was detected with HRP-conjugated streptavidin. (B) Intracellular Ca2+ flux was compared following stimulation of transduced A20 B cells expressing HLA-DQ2.5 and TG2-specific (679-14-E06) or non TG2-specific (693-2-F02) IgD BCR with size-separated TG2 complexes or bivalent mouse anti-human IgD antibody. TG2 multimers were superior to monomer and dimers. Calcium flux induced by TG2 was BCR dependent and not observed in control cells. (C) A20 cells expressing TG2-specific (679-14-E06), non TG2-specific (693-2-F02) BCR or only HLA-DQ2.5 were incubated with complexes of TG2 and FITC-labeled 33mer peptide or with anti-human IgD antibody followed by staining for intracellular phoshorylated Erk (pErk). ERK phosphorylation was BCR-dependent and was not observed in A20 cells expressing only HLA-DQ2.5. (D, E) A20 cells expressing HLA-DQ2.5 and TG2-specific (679-14-E06) or non TG2-specific (693-2-F02) IgD BCR were incubated on ice with complexes of TG2 followed by incubation at 37°C. BCR internalization was assessed by flow cytometry staining for surface IgD. Columns in (B) show the means of triplicates from the second experiment (+SD), columns in (D) show the means of two independent experiments (+SD).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134922.g005: TG2 multimers are superior to TG2 monomer in activation of TG2-specific A20 B cells(A) Celiac TG2-specific monoconal antibodies are able to bind TG2-gluten multimer complexes. TG2 crosslinked in the presence of biotin-33mer peptide was immunoprecipitated using TG2-specific monoclonal antibodies representing all four celiac epitopes, or using a non TG2-specific monoclonal antibody (693-2-F02). TG2 was detected with mouse monoclonal antibody CUB7402 and TG2-bound peptide was detected with HRP-conjugated streptavidin. (B) Intracellular Ca2+ flux was compared following stimulation of transduced A20 B cells expressing HLA-DQ2.5 and TG2-specific (679-14-E06) or non TG2-specific (693-2-F02) IgD BCR with size-separated TG2 complexes or bivalent mouse anti-human IgD antibody. TG2 multimers were superior to monomer and dimers. Calcium flux induced by TG2 was BCR dependent and not observed in control cells. (C) A20 cells expressing TG2-specific (679-14-E06), non TG2-specific (693-2-F02) BCR or only HLA-DQ2.5 were incubated with complexes of TG2 and FITC-labeled 33mer peptide or with anti-human IgD antibody followed by staining for intracellular phoshorylated Erk (pErk). ERK phosphorylation was BCR-dependent and was not observed in A20 cells expressing only HLA-DQ2.5. (D, E) A20 cells expressing HLA-DQ2.5 and TG2-specific (679-14-E06) or non TG2-specific (693-2-F02) IgD BCR were incubated on ice with complexes of TG2 followed by incubation at 37°C. BCR internalization was assessed by flow cytometry staining for surface IgD. Columns in (B) show the means of triplicates from the second experiment (+SD), columns in (D) show the means of two independent experiments (+SD).
Mentions: Conceivably, formation of TG2-multimers is relevant for activation of TG2-specific B cells in celiac disease, and incorporation of gluten peptides into multimers would allow efficient collaboration between TG2-specific B cells and gluten-specific T cells. A prerequisite for B-cell activation is recognition of the antigen by the BCR. To determine whether celiac anti-TG2 antibodies, and thus BCRs, could still recognize multimerized TG2 crosslinked to gluten peptide, we performed immunoprecipitation of TG2 incubated with CaCl2 in the presence of biotinylated 33mer gluten peptide using monoclonal human IgG antibodies derived from our panel of recombinant celiac anti-TG2 antibodies [14]. Antibodies of all four epitopes could bind and pull down TG2-TG2-33mer complexes (Fig 5A). Thus, the epitopes are conserved in TG2 multimers as predicted from our MS analysis.

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