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Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS‐13
Author(s) -
Deforche L.,
Roose E.,
Vandenbulcke A.,
Vandeputte N.,
Feys H. B.,
Springer T. A.,
Mi L. Z.,
Muia J.,
Sadler J. E.,
Soejima K.,
Rottensteiner H.,
Deckmyn H.,
De Meyer S. F.,
Vanhoorelbeke K.
Publication year - 2015
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.13149
Subject(s) - epitope , adamts , chemistry , epitope mapping , linker , conformational epitope , binding site , von willebrand factor , protein structure , antibody , microbiology and biotechnology , biochemistry , metalloproteinase , thrombospondin , biology , enzyme , genetics , immunology , platelet , computer science , operating system
Summary 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‐ CUB 2 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‐ CUB 2 antibodies was studied in the FRETS ‐ VWF 73 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‐ CUB 2 domains, were identified in the FRETS ‐ VWF 73 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‐ CUB 2 antibodies increased the binding to folded VWF up 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‐ CUB 2 domains.