High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Proteases like thrombin and ADAMTS13 play critical roles in biological processes.
• Understanding their substrate specificity is essential for deciphering their functions and for therapeutic development.
• Previous methods for defining protease specificity have limitations.

Purpose of the Study:

• To comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13.
• To investigate the role of exosite engagement in substrate recognition by ADAMTS13.
• To establish a robust method for determining protease substrate motifs.

Main Methods:

• Combined random 6 amino acid substrate phage display with high-throughput sequencing.
• Analyzed cleaved peptides to determine substrate motifs for thrombin and ADAMTS13.
• Utilized von Willebrand Factor (VWF73) as a substrate to probe ADAMTS13 exosite interactions.

Main Results:

• Defined a substrate motif for thrombin consistent with prior studies (>6,700 peptides).
• Identified a limited number of ADAMTS13 cleavage events from a large library, initially showing no consensus motif.
• Upon using a VWF73-based substrate, 1670 unique peptides were cleaved by ADAMTS13, revealing a motif with preferences for aliphatic amino acids (P3-P2') and specific tolerance at P2.
• Demonstrated that exosite engagement is critical for ADAMTS13 substrate recognition.

Conclusions:

• Substrate phage display coupled with high-throughput sequencing is a powerful approach for defining protease active site specificity.
• The study defines the substrate recognition motif for ADAMTS13, highlighting the importance of exosite interactions.
• The findings provide insights into the optimal evolution of von Willebrand Factor for ADAMTS13 recognition.