Deep Protease Profiling to Define the Substrate Specificity of ADAMTS Proteases

Determining the active site specificity of proteases is critically important for defining their role in biological systems. This article outlines a method for coupling substrate phage display with high-throughput sequencing to comprehensively define the active site specificity of proteases. We call this method deep protease profiling. We apply deep protease profiling to the metalloprotease ADAMTS13, a member of the metzincin family of proteases, that helps maintain the integrity of the cardiovascular system. The only known substrate for ADAMTS13 is von Willebrand factor (VWF), a multimeric glycoprotein that recruits platelets and other immune cells to sites of vascular injury. However, our understanding of ADAMTS13 substrate specificity remains limited. Deep protease profiling of ADAMTS13 initially revealed very few significantly cleaved peptides. However, substituting the phage display library into the P3-P3' interval of VWF73, a minimal VWF substrate used to detect ADAMTS13 activity, revealed over 1600 cleaved peptides. These peptides aligned into a clear substrate recognition motif, confirming the importance of exosite engagement and providing a detailed recognition profile that may guide the identification of novel biological substrates for ADAMTS13. This method is broadly applicable to other members of the ADAMTS protease family, whose substrate specificity remains poorly characterized.