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Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13

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Abstract
Background: Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF). Objectives: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13. Methods: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy. Results: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to similar to 3-fold. Conclusion: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.
Keywords
ADAMTS-13 protein, human, allosteric regulation, autoantibodies, protein conformation, von Willebrand factor, VON-WILLEBRAND-FACTOR, THROMBOTIC THROMBOCYTOPENIC PURPURA, CRYSTAL-STRUCTURES, ENDOTHELIAL-CELLS, CATALYTIC DOMAIN, INDUCED CLEAVAGE, SHEAR-STRESS, VWF, PROTEIN, FLOW

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Chicago
Deforche, L, E Roose, A Vandenbulcke, N Vandeputte, Hendrik Feys, TA Springer, LZ Mi, et al. 2015. “Linker Regions and Flexibility Around the Metalloprotease Domain Account for Conformational Activation of ADAMTS-13.” Journal of Thrombosis and Haemostasis 13 (11): 2063–2075.
APA
Deforche, L., Roose, E., Vandenbulcke, A., Vandeputte, N., Feys, H., Springer, T., Mi, L., et al. (2015). Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13. JOURNAL OF THROMBOSIS AND HAEMOSTASIS, 13(11), 2063–2075.
Vancouver
1.
Deforche L, Roose E, Vandenbulcke A, Vandeputte N, Feys H, Springer T, et al. Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13. JOURNAL OF THROMBOSIS AND HAEMOSTASIS. 2015;13(11):2063–75.
MLA
Deforche, L, E Roose, A Vandenbulcke, et al. “Linker Regions and Flexibility Around the Metalloprotease Domain Account for Conformational Activation of ADAMTS-13.” JOURNAL OF THROMBOSIS AND HAEMOSTASIS 13.11 (2015): 2063–2075. Print.
@article{8549070,
  abstract     = {Background: Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF).
Objectives: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13.
Methods: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy.
Results: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to similar to 3-fold.
Conclusion: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.},
  author       = {Deforche, L and Roose, E and Vandenbulcke, A and Vandeputte, N and Feys, Hendrik and Springer, TA and Mi, LZ and Muia, J and Sadler, JE and Soejima, K and Rottensteiner, H and Deckmyn, H and Vanhoorelbeke, K},
  issn         = {1538-7933},
  journal      = {JOURNAL OF THROMBOSIS AND HAEMOSTASIS},
  language     = {eng},
  number       = {11},
  pages        = {2063--2075},
  title        = {Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13},
  url          = {http://dx.doi.org/10.1111/jth.13149},
  volume       = {13},
  year         = {2015},
}

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