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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 self-crosslinking in the absence or presence of competitor substrate.(A) TG2 (2 μM) incubated in the presence of CaCl2 will cross-link itself into complexes that can be resolved by gradient SDS-PAGE (4–20%) but not by standard 12% SDS-PAGE. Self-crosslinking requires TG2 catalytic activity and does not happen in the absence of CaCl2 or presence of an active site inhibitor. (B) TG2 (1 μM) self-crosslinking as seen by gradient SDS-PAGE (at 15min) occurs even in the presence of a high excess of glutamine donor substrate (DQ2.5-glia-α2-QQ-FITC QQ; DQ2.5-glia-α2-QE-FITC, QE) or primary amine (5BP) (Coomassie). Incorporation of the FITC-labeled peptides is observed both in TG2 monomer and TG2 multimers (FITC). (C) Incubation of TG2 (78 kDa) together with human albumin (64 kDa), human IgG (50 kDa + 25 kDa) and ovalbumin (45 kDa) in the presence of CaCl2 results in selective disappearance of the monomeric TG2 band, indicating that self-crosslinking is a specific event. (D) Incubation of TG2 with excess of the known protein substrate 29kDa FN fragment (29FN) results in disappearance of TG2 monomer and incorporation of both TG2 and 29FN into complexes.
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pone.0134922.g001: TG2 self-crosslinking in the absence or presence of competitor substrate.(A) TG2 (2 μM) incubated in the presence of CaCl2 will cross-link itself into complexes that can be resolved by gradient SDS-PAGE (4–20%) but not by standard 12% SDS-PAGE. Self-crosslinking requires TG2 catalytic activity and does not happen in the absence of CaCl2 or presence of an active site inhibitor. (B) TG2 (1 μM) self-crosslinking as seen by gradient SDS-PAGE (at 15min) occurs even in the presence of a high excess of glutamine donor substrate (DQ2.5-glia-α2-QQ-FITC QQ; DQ2.5-glia-α2-QE-FITC, QE) or primary amine (5BP) (Coomassie). Incorporation of the FITC-labeled peptides is observed both in TG2 monomer and TG2 multimers (FITC). (C) Incubation of TG2 (78 kDa) together with human albumin (64 kDa), human IgG (50 kDa + 25 kDa) and ovalbumin (45 kDa) in the presence of CaCl2 results in selective disappearance of the monomeric TG2 band, indicating that self-crosslinking is a specific event. (D) Incubation of TG2 with excess of the known protein substrate 29kDa FN fragment (29FN) results in disappearance of TG2 monomer and incorporation of both TG2 and 29FN into complexes.

Mentions: Human recombinant TG2 incubated with Ca2+ serves as a cross-linking substrate to itself (Fig 1A). Self-crosslinked high molecular weight TG2 complexes could be resolved by gradient SDS-PAGE as distinct bands corresponding to monomer, dimer, trimer and higher-order multimers (Fig 1A). Importantly, we found that TG2 self-crosslinking also occurred in the presence of competing exogenous substrate as observed following incubation of TG2 with a high excess of gluten peptide substrate (DQ2.5-glia-α2-QE-FITC and DQ2.5-glia-α2-QQ-FITC with one or two targeted glutamine residues respectively) and the small primary amine 5-(biotinamido)pentylamine (5BP) (Fig 1B). Of note, gluten peptides were readily cross-linked to TG2 as visualized by FITC fluorescence, in line with previous observations [13]. However, not even a 500 fold excess of gluten peptide substrate and 5000 fold excess of 5BP prevented the parallel reaction of TG2 self-crosslinking. Rather, gluten peptides were concomitantly incorporated into TG2 multimers. The gluten peptide harboring two targeted glutamine residues was cross-linked to a larger extent to TG2 as seen by FITC fluorescence. A comparable degree of self-crosslinking was observed at in vitro saturating and physiological CaCl2 concentrations (5 mM and 1 mM respectively, Fig 1B). To determine the ability of TG2 to self-crosslink in the presence of other proteins, TG2 was incubated with a mix of proteins that could readily be distinguished by SDS-PAGE. In the presence of CaCl2, only the monomeric band of TG2 disappeared (Fig 1C). Thus, TG2 itself is the only protein in this mixture that serves as a cross-linking substrate. Incubation of TG2 with a known protein substrate, the 29kDa proteolytic fragment of fibronectin (FN) [22, 23] also led to a clear decrease in the amount of monomeric TG2 (Fig 1D). Here, several cross-linked products were generated likely consisting of a mix of TG2 multimers and TG2 cross-linked with one or more 29kDa FN fragments. In summary, TG2 self-crosslinking is a specific event that is not readily prevented even by large excess of competing substrates, indicating that TG2 serves as a preferred substrate for itself.


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 self-crosslinking in the absence or presence of competitor substrate.(A) TG2 (2 μM) incubated in the presence of CaCl2 will cross-link itself into complexes that can be resolved by gradient SDS-PAGE (4–20%) but not by standard 12% SDS-PAGE. Self-crosslinking requires TG2 catalytic activity and does not happen in the absence of CaCl2 or presence of an active site inhibitor. (B) TG2 (1 μM) self-crosslinking as seen by gradient SDS-PAGE (at 15min) occurs even in the presence of a high excess of glutamine donor substrate (DQ2.5-glia-α2-QQ-FITC QQ; DQ2.5-glia-α2-QE-FITC, QE) or primary amine (5BP) (Coomassie). Incorporation of the FITC-labeled peptides is observed both in TG2 monomer and TG2 multimers (FITC). (C) Incubation of TG2 (78 kDa) together with human albumin (64 kDa), human IgG (50 kDa + 25 kDa) and ovalbumin (45 kDa) in the presence of CaCl2 results in selective disappearance of the monomeric TG2 band, indicating that self-crosslinking is a specific event. (D) Incubation of TG2 with excess of the known protein substrate 29kDa FN fragment (29FN) results in disappearance of TG2 monomer and incorporation of both TG2 and 29FN into complexes.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134922.g001: TG2 self-crosslinking in the absence or presence of competitor substrate.(A) TG2 (2 μM) incubated in the presence of CaCl2 will cross-link itself into complexes that can be resolved by gradient SDS-PAGE (4–20%) but not by standard 12% SDS-PAGE. Self-crosslinking requires TG2 catalytic activity and does not happen in the absence of CaCl2 or presence of an active site inhibitor. (B) TG2 (1 μM) self-crosslinking as seen by gradient SDS-PAGE (at 15min) occurs even in the presence of a high excess of glutamine donor substrate (DQ2.5-glia-α2-QQ-FITC QQ; DQ2.5-glia-α2-QE-FITC, QE) or primary amine (5BP) (Coomassie). Incorporation of the FITC-labeled peptides is observed both in TG2 monomer and TG2 multimers (FITC). (C) Incubation of TG2 (78 kDa) together with human albumin (64 kDa), human IgG (50 kDa + 25 kDa) and ovalbumin (45 kDa) in the presence of CaCl2 results in selective disappearance of the monomeric TG2 band, indicating that self-crosslinking is a specific event. (D) Incubation of TG2 with excess of the known protein substrate 29kDa FN fragment (29FN) results in disappearance of TG2 monomer and incorporation of both TG2 and 29FN into complexes.
Mentions: Human recombinant TG2 incubated with Ca2+ serves as a cross-linking substrate to itself (Fig 1A). Self-crosslinked high molecular weight TG2 complexes could be resolved by gradient SDS-PAGE as distinct bands corresponding to monomer, dimer, trimer and higher-order multimers (Fig 1A). Importantly, we found that TG2 self-crosslinking also occurred in the presence of competing exogenous substrate as observed following incubation of TG2 with a high excess of gluten peptide substrate (DQ2.5-glia-α2-QE-FITC and DQ2.5-glia-α2-QQ-FITC with one or two targeted glutamine residues respectively) and the small primary amine 5-(biotinamido)pentylamine (5BP) (Fig 1B). Of note, gluten peptides were readily cross-linked to TG2 as visualized by FITC fluorescence, in line with previous observations [13]. However, not even a 500 fold excess of gluten peptide substrate and 5000 fold excess of 5BP prevented the parallel reaction of TG2 self-crosslinking. Rather, gluten peptides were concomitantly incorporated into TG2 multimers. The gluten peptide harboring two targeted glutamine residues was cross-linked to a larger extent to TG2 as seen by FITC fluorescence. A comparable degree of self-crosslinking was observed at in vitro saturating and physiological CaCl2 concentrations (5 mM and 1 mM respectively, Fig 1B). To determine the ability of TG2 to self-crosslink in the presence of other proteins, TG2 was incubated with a mix of proteins that could readily be distinguished by SDS-PAGE. In the presence of CaCl2, only the monomeric band of TG2 disappeared (Fig 1C). Thus, TG2 itself is the only protein in this mixture that serves as a cross-linking substrate. Incubation of TG2 with a known protein substrate, the 29kDa proteolytic fragment of fibronectin (FN) [22, 23] also led to a clear decrease in the amount of monomeric TG2 (Fig 1D). Here, several cross-linked products were generated likely consisting of a mix of TG2 multimers and TG2 cross-linked with one or more 29kDa FN fragments. In summary, TG2 self-crosslinking is a specific event that is not readily prevented even by large excess of competing substrates, indicating that TG2 serves as a preferred substrate for itself.

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