Abstract
ADAMTS13 is a reprolysin-type metalloproteinase belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motif) family. It specifically cleaves plasma von Willebrand factor (VWF) and regulates platelet adhesion and aggregation. ADAMTS13 is a multi-domain enzyme. In addition to the N-terminal metalloproteinase domain, the ancillary domains, including a disintegrin-like domain, a thrombospondin-1 type 1 repeat, a Cys-rich domain and a spacer domain, are required for VWF recognition and cleavage. In the present study, a fragment of the ADAMTS13 ancillary domains (ADAMTS13-DTCS; residues 287-685) was expressed using CHO Lec cells, purified and crystallized. Diffraction data sets were collected using the SPring-8 beamline. Two ADAMTS13-DTCS crystals with distinct unit-cell parameters generated data sets to 2.6 and 2.8 A resolution, respectively.
Highlights
Introduction von Willebrand factor (VWF) is a plasma glycoprotein that is involved in platelet-dependent haemostasis (Sadler, 1998)
VWF is primarily synthesized in vascular endothelial cells and megakaryocytes and is released into the plasma as ultralarge multimeric forms (UL-VWF) that are highly active in platelet adhesion and aggregation
A deficiency of ADAMTS13 enzymatic activity caused by either genetic mutations in the ADAMTS13 gene or acquired autoantibodies against ADAMTS13 results in the accumulation of UL-VWF in plasma. This leads to the formation of disseminated platelet-rich microthrombi in arterioles, which is one of the characteristic pathogenic features of thrombotic thrombocytopaenic purpura (TTP), a life-threatening systemic disease (Tsai, 2009)
Summary
Initial screening for crystallization conditions for ADAMTS-DTCS was carried out by the sitting-drop vapour-diffusion method using Index Screen, SaltRx Screen, PEG/Ion Screen, Grid Screen MPD and Grid Screen Ammonium Sulfate kits (Hampton Research, Aliso Viejo, California, USA). A volume of 0.1 ml protein solution was manually mixed with an equal amount of reservoir solution and the droplets were allowed to equilibrate against 0.1 ml reservoir solution at 293 K for 24 h
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