Abstract
In the extracellular environment, the enzyme transglutaminase 2 (TG2) is involved in cell–matrix interactions through association with the extracellular matrix protein, fibronectin (FN). The 45 kDa gelatin-binding domain of FN (45FN) is responsible for the binding to TG2. Previous studies have demonstrated that the FN-binding site of TG2 is located in the N-terminal domain of the enzyme although with conflicting results regarding the specific residues involved. Here we have mapped the FN interaction site of human TG2 by use of hydrogen/deuterium exchange coupled with mass spectrometry, and we confirm that the FN-binding site is located in the N-terminal domain of TG2. Furthermore, by combination of site-directed mutagenesis and surface plasmon resonance analysis we have identified the TG2 residues K30, R116 and H134 as crucial to maintain the high affinity interaction with FN. Mutation of all three residues simultaneously reduced binding to 45FN by more than 2000-fold. We also identified residues in the catalytic core domain of TG2 that contributed to FN binding, hence extending the binding interface between TG2 and FN. This study provides new insights into the high affinity interaction between TG2 and FN.
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Abbreviations
- TG2:
-
Transglutaminase 2
- FN:
-
Fibronectin
- 45FN:
-
45 kDa proteolytic fibronectin fragment (gelatin-binding domain)
- ECM:
-
Extracellular matrix
- HDX-MS:
-
Hydrogen/deuterium exchange coupled with mass spectrometry
- SPR:
-
Surface plasmon resonance
- WT:
-
Wild-type
- RH:
-
TG2 mutant R116A/H134A
- KRH:
-
TG2 mutant K30E/R116A/H134A
- mAb:
-
Monoclonal antibody
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Acknowledgments
This work was supported by the European Commission grants MRTN-CT-2006-036032 and ERC-2010-Ad-268541 and Grants from the Research Council of Norway and the South-Eastern Norway Regional Health Authority. The authors thank Chaitan Khosla (Stanford University, CA; USA) for providing the TG3 plasmid.
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726_2016_2296_MOESM1_ESM.tif
Supplementary material 1 Supplementary Fig. 1 Deuterium uptake plots for all identified TG2 peptides which showed no difference in D-uptake when comparing the 45FN-bound state (red curves) and the unbound state (blue curves). Error bars represent SD based on labeling triplicates (TIFF 2358 kb)
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Cardoso, I., Østerlund, E.C., Stamnaes, J. et al. Dissecting the interaction between transglutaminase 2 and fibronectin. Amino Acids 49, 489–500 (2017). https://doi.org/10.1007/s00726-016-2296-y
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DOI: https://doi.org/10.1007/s00726-016-2296-y