Journal: J Biol Chem / Year: 2015 Title: Structural Basis for Antigen Recognition by Transglutaminase 2-specific Autoantibodies in Celiac Disease. Authors: Xi Chen / Kathrin Hnida / Melissa Ann Graewert / Jan Terje Andersen / Rasmus Iversen / Anne Tuukkanen / Dmitri Svergun / Ludvig M Sollid / Abstract: Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut ...Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease.
Mass: 24026.963 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#2: Antibody
679-14-14E06Fabfragmentlightchain
Mass: 23674.209 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
Mass: 18.015 Da / Num. of mol.: 78 / Source method: isolated from a natural source / Formula: H2O
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION
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Sample preparation
Crystal
Density Matthews: 2.52 Å3/Da / Density % sol: 51.17 % / Description: crystals look like plates
Crystal grow
Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5 Details: 0.01 M nickel (II) chloride hexahydrate, 0.1 M Tris pH=8.5, 20% w/v polyethylene glycol monomethyl ether 2000
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
Wavelength: 0.979093 Å / Relative weight: 1
Reflection
Resolution: 2.4→49.53 Å / Num. obs: 18701 / % possible obs: 99.2 % / Redundancy: 3.4 % / Rsym value: 0.117 / Net I/σ(I): 9.3
Reflection shell
Resolution: 2.4→2.5 Å / Redundancy: 3.5 % / Rmerge(I) obs: 0.605 / Mean I/σ(I) obs: 2.4 / % possible all: 99.5
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Processing
Software
Name
Version
Classification
PHENIX
1.8.4_1496
refinement
MOSFLM
7.1.0
datareduction
Aimless
5.8.0049
datascaling
PHASER
5.8.0049
phasing
Refinement
Method to determine structure: MOLECULAR REPLACEMENT Starting model: Contant and variable domains of heavy chain PDB code 4HPO and a light chain PDB code 1DFB
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