Two-Hybrid Screening Identifies an Interaction between the CUB Domains of Human ADAMTS13 and Factor XII.

The metalloprotease ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers by cleaving a peptide bond in the VWF A2 domain. Unlike the other ADAMTS family members, ADAMTS13 contains two C-terminal CUB domains, and mutations in this region have been found in patients with congenital thrombotic thrombocytopenic purpura (TTP). In vitro studies indicate that CUB domains are dispensable for ADAMTS13 catalytic activity under static conditions with denatured VWF substrate, but they may be crucial for recognition of native VWF under flow. CUB domains in many proteins mediate protein-protein interactions, and to identify potential binding partners we conducted yeast two-hybrid screening using MATCHMAKER GAL4 two-hybrid system 3 (Clontech Laboratories Inc., CA, USA). A cDNA fragment encoding both CUB domains was inserted into the pGBKT7 vector. The resulting GAL4BD-CUB fusion protein was used as bait to screen a human liver cDNA library. Both bait and library vectors were simultaneously introduced into Saccharomyces cerevisiae AH109 and double transformants were selected on minimal synthetic dropout media lacking Trp, Leu and His. A total of approximately 3 × 10⁶ clones were screened. Putative positive colonies were re-streaked and tested for β-galactosidase activity. DNA fragments from positive colonies were sequenced and 26 proteins were identified, 10 of which were plasma proteins including factor XII (FXII), β2-glycoprotein I, and fibrinogen β chain. Interactions were further characterized with a binding assay employing purified ADAMTS13 immobilized on microtiter plates. The FXII-ADAMTS13 interaction was saturable and reversible, with Kd = 17 nM, which is significantly lower than the plasma concentration of FXII (∼ 375 nM). Deletion of CUB domains in ADAMTS13(ΔCUB) reduces its affinity toward FXII by ∼10-fold (Kd = 164 nM). Activated FXII (FXIIa) bound saturably and reversibly to ADAMTS13 with Kd = 19 nM. Binding of FXIIa to ADAMTS13(ΔCUB) was undetectable, suggesting that the FXIIa-ADAMTS13 interaction is more specific than the FXII-ADAMTS13 interaction. This interaction of FXIIa and ADAMTS13 was totally blocked by a heat and acid stable inhibitor in plasma, whereas the interaction of FXII and ADAMTS13 was decreased only 50% by plasma. These data suggest that FXII can bind either to ADAMTS13 CUB domains or with lower affinity to a distinct site that does not involve the CUB domains; FXIIa, a two-chain enzyme formed by a single cleavage of FXII, only retains the structure involved in high affinity binding to ADAMTS13 CUB domains. Fibrinogen and β2-glycoprotein 1 also bound to ADAMTS13 in vitro, but with much lower affinity. However, the plasma concentrations of both proteins (about 9 μM and 4 μM, respectively) are at least 10 times higher than their apparent Kd values. Whether these CUB domain-interacting proteins are important for the regulation of ADAMTS13 activity in vivo remains to be established.