title: Effects of naturally occurring mutations in CUB-1 domain on synthesis, stability, and activity of ADAMTS-13.
abstract: BACKGROUND
  Upon stimulation, endothelial cells release von Willebrand factor (VWF) in the unusually large (UL) and hyperactive forms that are rapidly cleaved by ADAMTS-13. Mutations in the ADAMTS13 gene result in ULVWF-mediated thrombosis found in patients with familial thrombotic thrombocytopenia purpura (TTP). ADAMTS-13 fits in the consensus of the ADAMTS family metalloproteases, but also contains two unique C- terminal CUB domains. Studying mutations in CUB domains could provide insights into the functional role of these domains.
  
  
  METHODS
  Three naturally occurring mutations (C1213Y, W1245del and K1256FS) in the CUB-1 domain found in patients with TTP were expressed in Hela cells. The secretion, stability and VWF-cleaving activity of the mutants under static and flow conditions were examined.
  
  
  RESULTS
  The mutations impaired secretion of ADAMTS-13 to apical surface, but not to extracellular matrix of transfected Hela cells. C1213Y and K1256FS also accelerated, whereas W1245del delayed, extracellular degradation of the mutants. The mutations also resulted in a moderate decrease in cleaving plasma VWF under static conditions. However, the mutated ADAMTS-13 bound to VWF substrate similarly as the wild-type metalloprotease and remained active in cleaving (UL)VWF under flow conditions.
  
  
  CONCLUSIONS
  The CUB-1 domain is critical for ADAMTS-13 secretion and stability upon secretion. ADAMTS-13 deficiency found in TTP patients could be resulted from reduced ADAMTS-13 secretion and, in the case of C1213Y and K1256FS accelerated degradation. W1245del is highly resistant to degradation and active in cleaving VWF.
authors.affiliation: Thrombosis Research Section, Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Zhou
authors.forename: Hua
authors.initials: H
authors.lastname: Jing
authors.forename: Zhenyin
authors.initials: Z
authors.lastname: Tao
authors.forename: Huiwan
authors.initials: H
authors.lastname: Choi
authors.forename: Khatira
authors.initials: K
authors.lastname: Aboulfatova
authors.forename: Joel
authors.initials: J
authors.lastname: Moake
authors.forename: Renhao
authors.initials: R
authors.lastname: Li
authors.forename: Jing-Fei
authors.initials: JF
authors.lastname: Dong
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D004789:Enzyme Activation; D004795:Enzyme Stability; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human