Conformational opening of ADAMTS13 does not necessarily indicate preactivation of its metalloprotease domain

Introduction: ADAMTS13 adopts a closed conformation, maintained by non-covalent interactions between the Spacer (S) and CUB domains. Also, the active site in the metalloprotease (MP) domain is blocked in this autoinhibited conformation. Previously, we showed that certain monoclonal antibodies (mAbs) targeting the S and CUB1 domains not only disrupt this S-CUB interaction, but also preactivate the MP domain. Hereto, we used our mAbs 1C4 (anti-S) and 6A6 (anti-MP) which recognize epitopes that are only accessible upon allosteric ADAMTS13 activation. As a multi-domain enzyme, it remained unknown how targeting other domains than the S and CUB domains affect its conformation and preactivation. To better comprehend its allosteric regulation, we aimed to target each ADAMTS13 domain and asses the effects on its conformation, preactivation and proteolytic function.

Method: A panel of 46 mAbs was used to target each ADAMTS13 domain. In ELISA, their ability to open plasma ADAMTS13 (1C4 exposure) and to preactivate its MP domain (6A6 exposure) was verified. A mAb subset was characterized in a VWF96 proteolysis ELISA to assess mAb-induced changes in catalytic efficiency. Michaelis-Menten kinetics evaluated the mAb effects on VWF turnover and binding.

Results: When targeting each ADAMTS13 domain, mAbs disrupted the S-CUB interaction and conformationally opened ADAMTS13 which exposed the 1C4 epitope. Also, mAbs against most domains preactivated the MP domain, making the 6A6 epitope accessible. Surprisingly, a mAb subset targeting the D, T1, T7 and CUB2 domains, opened ADAMTS13 but did not preactivate the MP domain, as the 6A6 epitope remained concealed. In VWF96 proteolysis assays, some anti-D mAbs, which exposed the 1C4 epitope but not the 6A6 epitope, inhibited the ADAMTS13 catalytic efficiency as shown by a decreased VWF turnover. Alternatively, anti-CUB2 mAbs that exposed the 1C4 but not the 6A6 epitope, enhanced proteolytic activity as shown by increased VWF turnover in Michaelis-Menten analyses.

Conclusion: We investigated how mAbs targeting each ADAMTS13 domain affects its conformation, preactivation and proteolytic activity. Using mAbs targeting each ADAMTS13 domain, the closed, autoinhibited conformation of plasma ADAMTS13 could be opened. Unlike previous anti-S and anti-CUB1 mAbs, some mAbs against the D, T1, T7 or CUB2 domains opened ADAMTS13 without preactivating its MP domain. These mAbs either activated (anti-CUB2) or inhibited (anti-D) the ADAMTS13 proteolytic function, as seen by corresponding changes in VWF turnover. Our data agree with the idea that upon mAb-induced ADAMTS13 opening, diverse mechanisms can not only conformationally preactivate, but also may conformationally restrain the ADAMTS13 MP domain to diminish its enzymatic function. Hence, the molecular mechanism for ADAMTS13 opening is linked to, but differs from the one that allosterically regulates the MP domain.

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Artikel online veröffentlicht:
13. Februar 2025