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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-mediated self-crosslinking sites locate to the catalytic core and C-terminal part of TG2(A) Glutamine residues of TG2 cross-linked to 5BP. The frequency (%) reflects the abundance of modified residues compared to each other. (B) Lysine residues of TG2 cross-linked to the small peptide Biotin-QLPR. Histograms in (A) and (B) are representative of two and three independent experiments, respectively. (D) Cross-linked peptide adducts identified from in solution tryptic digests of size separated fractions shown in (C). Multiple cross-linked peptide adducts were identified and only peptide adducts identified by two or more independent MSMS scans are listed in (D). Results from the second sample of triplicates are shown. (E) Extracted ion chromatogram for the peptide adducts Q481-K677 (AVKGFR-VGQSMNMGSDFDVFAHITNNTAEEYVC(acetamide)R, 771.15 m/z; EIC window 771.15–771.25 m/z,) and Q234-K677 (AVKGFR-VVSGMVNC(acetamide)NDDQGVLLGR; 648.83m/z, EIC window 648.83–648.93m/z). Signal intensity (y-axis) reflects peptide abundance. (F) Location of glutamine and lysine residues in open conformation TG2 (PDB ID code 2Q3Z). Glutamine residues identified in cross-linked peptide adducts are shown in red, the remaining glutamine residues are shown in pink. Lysine residues identified in cross-linked peptide adducts are shown in blue, the remaining lysine residues are shown in turquoise. Residues E8, R19, K30 and D94, important for recognition of celiac anti-TG2 monoclonal antibodies [26], are shown in yellow. Q307 is not resolved in the crystal structure, and D306 (italics) is highlighted instead. K464 and K468 are also located in unresolved parts.
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pone.0134922.g004: TG2-mediated self-crosslinking sites locate to the catalytic core and C-terminal part of TG2(A) Glutamine residues of TG2 cross-linked to 5BP. The frequency (%) reflects the abundance of modified residues compared to each other. (B) Lysine residues of TG2 cross-linked to the small peptide Biotin-QLPR. Histograms in (A) and (B) are representative of two and three independent experiments, respectively. (D) Cross-linked peptide adducts identified from in solution tryptic digests of size separated fractions shown in (C). Multiple cross-linked peptide adducts were identified and only peptide adducts identified by two or more independent MSMS scans are listed in (D). Results from the second sample of triplicates are shown. (E) Extracted ion chromatogram for the peptide adducts Q481-K677 (AVKGFR-VGQSMNMGSDFDVFAHITNNTAEEYVC(acetamide)R, 771.15 m/z; EIC window 771.15–771.25 m/z,) and Q234-K677 (AVKGFR-VVSGMVNC(acetamide)NDDQGVLLGR; 648.83m/z, EIC window 648.83–648.93m/z). Signal intensity (y-axis) reflects peptide abundance. (F) Location of glutamine and lysine residues in open conformation TG2 (PDB ID code 2Q3Z). Glutamine residues identified in cross-linked peptide adducts are shown in red, the remaining glutamine residues are shown in pink. Lysine residues identified in cross-linked peptide adducts are shown in blue, the remaining lysine residues are shown in turquoise. Residues E8, R19, K30 and D94, important for recognition of celiac anti-TG2 monoclonal antibodies [26], are shown in yellow. Q307 is not resolved in the crystal structure, and D306 (italics) is highlighted instead. K464 and K468 are also located in unresolved parts.

Mentions: To determine self-substrate sites in TG2, we incubated TG2 either with the small gluten peptide analog biotin-QLPR or 5BP followed by in solution trypsin digestion and mass spectrometry (MS) analysis. Several glutamine residues primarily in the catalytic core were cross-linked to 5BP (Fig 4A), of which two have previously been reported (Q234 and Q307;[23]). Using biotin-QLPR we identified lysine residues available for cross-linking (Fig 4B), and six of these have previously been identified [13]. From this it appears that multiple glutamine and lysine residues can act as self-substrate sites in TG2 when probed with small molecule substrates.


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-mediated self-crosslinking sites locate to the catalytic core and C-terminal part of TG2(A) Glutamine residues of TG2 cross-linked to 5BP. The frequency (%) reflects the abundance of modified residues compared to each other. (B) Lysine residues of TG2 cross-linked to the small peptide Biotin-QLPR. Histograms in (A) and (B) are representative of two and three independent experiments, respectively. (D) Cross-linked peptide adducts identified from in solution tryptic digests of size separated fractions shown in (C). Multiple cross-linked peptide adducts were identified and only peptide adducts identified by two or more independent MSMS scans are listed in (D). Results from the second sample of triplicates are shown. (E) Extracted ion chromatogram for the peptide adducts Q481-K677 (AVKGFR-VGQSMNMGSDFDVFAHITNNTAEEYVC(acetamide)R, 771.15 m/z; EIC window 771.15–771.25 m/z,) and Q234-K677 (AVKGFR-VVSGMVNC(acetamide)NDDQGVLLGR; 648.83m/z, EIC window 648.83–648.93m/z). Signal intensity (y-axis) reflects peptide abundance. (F) Location of glutamine and lysine residues in open conformation TG2 (PDB ID code 2Q3Z). Glutamine residues identified in cross-linked peptide adducts are shown in red, the remaining glutamine residues are shown in pink. Lysine residues identified in cross-linked peptide adducts are shown in blue, the remaining lysine residues are shown in turquoise. Residues E8, R19, K30 and D94, important for recognition of celiac anti-TG2 monoclonal antibodies [26], are shown in yellow. Q307 is not resolved in the crystal structure, and D306 (italics) is highlighted instead. K464 and K468 are also located in unresolved parts.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134922.g004: TG2-mediated self-crosslinking sites locate to the catalytic core and C-terminal part of TG2(A) Glutamine residues of TG2 cross-linked to 5BP. The frequency (%) reflects the abundance of modified residues compared to each other. (B) Lysine residues of TG2 cross-linked to the small peptide Biotin-QLPR. Histograms in (A) and (B) are representative of two and three independent experiments, respectively. (D) Cross-linked peptide adducts identified from in solution tryptic digests of size separated fractions shown in (C). Multiple cross-linked peptide adducts were identified and only peptide adducts identified by two or more independent MSMS scans are listed in (D). Results from the second sample of triplicates are shown. (E) Extracted ion chromatogram for the peptide adducts Q481-K677 (AVKGFR-VGQSMNMGSDFDVFAHITNNTAEEYVC(acetamide)R, 771.15 m/z; EIC window 771.15–771.25 m/z,) and Q234-K677 (AVKGFR-VVSGMVNC(acetamide)NDDQGVLLGR; 648.83m/z, EIC window 648.83–648.93m/z). Signal intensity (y-axis) reflects peptide abundance. (F) Location of glutamine and lysine residues in open conformation TG2 (PDB ID code 2Q3Z). Glutamine residues identified in cross-linked peptide adducts are shown in red, the remaining glutamine residues are shown in pink. Lysine residues identified in cross-linked peptide adducts are shown in blue, the remaining lysine residues are shown in turquoise. Residues E8, R19, K30 and D94, important for recognition of celiac anti-TG2 monoclonal antibodies [26], are shown in yellow. Q307 is not resolved in the crystal structure, and D306 (italics) is highlighted instead. K464 and K468 are also located in unresolved parts.
Mentions: To determine self-substrate sites in TG2, we incubated TG2 either with the small gluten peptide analog biotin-QLPR or 5BP followed by in solution trypsin digestion and mass spectrometry (MS) analysis. Several glutamine residues primarily in the catalytic core were cross-linked to 5BP (Fig 4A), of which two have previously been reported (Q234 and Q307;[23]). Using biotin-QLPR we identified lysine residues available for cross-linking (Fig 4B), and six of these have previously been identified [13]. From this it appears that multiple glutamine and lysine residues can act as self-substrate sites in TG2 when probed with small molecule substrates.

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