CTR (4032)

Mass spectrometry QExactive RAW data file; tryptic digest of non-crosslinked TG

Data from: Enhanced B-cell receptor recognition of the autoantigen transglutaminase 2 by efficient catalytic self-multimerization

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

Trimer plus (III+) (4048)

Mass spectrometry QExactive RAW data file; tryptic digest of self-crosslinked TG2 (trimer +, size exclusion fraction III+

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

<div><p>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.</p></div

Trimer (III) (4044)

Mass spectrometry QExactive RAW data file; tryptic digest of self-crosslinked TG2 (trimer, size exclusion fraction III

Dimer (II) (4040)

Mass spectrometry QExactive RAW data file; tryptic digest of self-crosslinked TG2 (dimer, size exclusion fraction II

Monomer (I) (4036)

Mass spectrometry QExactive RAW data file; tryptic digest of self-crosslinked TG2 (monomer, size exclusion fraction I

TG2 multimers are superior to TG2 monomer in activation of TG2-specific A20 B cells

<p>(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).</p

TG2-mediated self-crosslinking sites locate to the catalytic core and C-terminal part of TG2

<p>(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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134922#pone.0134922.ref026" target="_blank">26</a>], 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.</p