title: Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13.
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).
  
  
  OBJECTIVE
  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 ~3-fold.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Deforche
authors.affiliation: Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: E
authors.initials: E
authors.lastname: Roose
authors.affiliation: Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Vandenbulcke
authors.affiliation: Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: N
authors.initials: N
authors.lastname: Vandeputte
authors.affiliation: Transfusion Research Center, Belgian Red Cross Flanders, Gent, Belgium.
authors.forename: H B
authors.initials: HB
authors.lastname: Feys
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Springer
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: L Z
authors.initials: LZ
authors.lastname: Mi
authors.affiliation: Departments of Medicine, Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA.
authors.forename: J
authors.initials: J
authors.lastname: Muia
authors.affiliation: Departments of Medicine, Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Sadler
authors.affiliation: Research Department 1, The Chemo-Sero-Therapeutic Research Institute, Kikuchi, Kumamoto, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Soejima
authors.affiliation: Baxter Innovations GmbH, Vienna, Austria.
authors.forename: H
authors.initials: H
authors.lastname: Rottensteiner
authors.affiliation: Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: H
authors.initials: H
authors.lastname: Deckmyn
authors.affiliation: Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: S F
authors.initials: SF
authors.lastname: De Meyer
authors.affiliation: Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Vanhoorelbeke
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D000071120:ADAMTS13 Protein; D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D002384:Catalysis; D016384:Consensus Sequence; D004789:Enzyme Activation; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019700:Thrombospondin 1 / Q000737:chemistry; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D019700:Thrombospondin 1; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS-13 protein, human; allosteric regulation; autoantibodies; protein conformation; von Willebrand factor