Effects of Naturally Occurring Mutations in CUB-1 Domain on ADAMTS-13 Synthesis, Stability and Activity.

Upon stimulation, vascular endothelial cells release von Willebrand factor (VWF) in the unusually large (UL) and prothrombotic forms. The hyperactive ULVWF multimers are rapidly cleaved by the metalloprotease ADAMTS-13 to smaller forms that are hemostatically active, but no longer prothrombotic. Lack of this VWF- cleaving activity results in accumulation of ULVWF, which agglutinates platelets, leading to microvascular thrombosis as demonstrated in patients with thrombotic thrombocytopenia purpura (TTP). ADAMTS-13 deficiency seen in TTP results from either mutations in the ADAMTS13 gene in familial cases or autoantibodies against the metalloprotease in acquired cases. ADAMTS-13 differs from other members of the family metalloproteases by containing two C-terminal CUB domains, which contain the binding site for VWF and whose mutations are associated with TTP. However, recombinant ADAMTS-13 without CUB domains remains active in vitro. To study the functional role of CUB domains, three naturally occurring mutations in the CUB-1 domain (C1213Y, W1245del and K1256frameshift, the latter two removed CUB-2 and the C-terminal part of CUB-1 domain) were generated by site-directed mutagenesis and expressed in the mammalian Hela cells. The release, stability, and activity of the mutants were then examined. We found that the mutations significantly reduced the release of recombinant ADMATS-13 into the culture medium, but not to the extracellular matrix. The intracellular pools of the recombinants detected in cell lysates were comparable to that of wild-type, indicating that the mutations affected the targeted secretion of the metalloprotease. When measured during a course of up to one month at −80°C, C1213Y and K1256framshift significantly accelerated, whereas W1245del delayed, degradation of the recombinants, which was inhibited by 5 mM of EDTA. The observation demonstrates that CUB domains are also critical for the structural stability of the metalloprotease. In comparison, the three mutants remained active in cleaving (UL)VWF under static and flow conditions. Consistent with the activity measurements, all three mutants bound immobilized VWF with similar kinetics in ELISA assay, indicating that the N-terminal sequence (C1192-D1212) of CUB-1, which is not affected by the mutations, contains the binding site for VWF. The conclusion is supported by our previous data that the CUB-1 synthetic peptides derived from the N-terminal region (H1196-P1220) blocked VWF-ADAMTS-13 interaction and cleavage of ULVWF under flow. Together, these results demonstrate that

1. the CUB domains (primarily CUB-2 and the C-terminal part of CUB-1) are crucial for regulating the targeted ADAMTS-13 secretion and the stability of the metalloprotease;

2. the N-terminal sequence of CUB-1 domain contains the VWF binding site that remains intact after deleting the rest of CUB domains; and

3. the primary cause of ADAMTS-13 deficiency found in patients carrying these mutations is likely due to defective secretion of the metalloprotease.