VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13

Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (k cat /K M ) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1′ substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.phage display | protease | high-throughput sequencing | ADAMTS13 | von Willebrand factor P rotease specificity is critical for maintaining diversity and compartmentalization of function, and is tightly controlled. For many proteases, a substrate initially docks to an exosite, which captures and orients the substrate scissile bond toward the active site of the enzyme. At the active site, the Px-Px′ (1) substrate amino acid side chains align with the complementary Sx-Sx′ pockets of the enzyme to optimize recognition by the active site residues that execute the proteolytic reaction (2).Conventional techniques for probing the substrate recognition requirements of a protease are cumbersome and time-consuming and require intimate knowledge of the enzyme/substrate pair. Such methods include engineering deletion mutants (3), use of competitive ligands (4, 5), and site-directed mutagenesis (6, 7). In contrast to these techniques, substrate phage display is a highthroughput, unbiased approach to studying protease substrate specificity (8-10). In this method, a library consisting of 10 6 -10 9 independent phage clones, each expressing a unique potential substrate on its surface, is panned for multiple rounds with a protease, and the cleaved or uncleaved phages after each reaction are removed and amplified for subsequent rounds of selection. In this manner, the library complexity is iteratively reduced and becomes populated by peptide sequences that are most informative. This methodology, although useful, is limited by the number of clones selected for individual Sanger sequencing after the last round of selection, and the selection of phages based on competitive growth advantages unrelated to enzyme specificity. The availability of high-throughput DNA sequencing technology (11) has facilitated detailed analysis of the changing complexity within a phage display library (12-16) without requiring multip...

“…2, Bottom). These data are consistent with previous, much more limited, biochemical studies (3,(27)(28)(29)(30) and provide a high-resolution substrate recognition landscape for ADAMTS13.…”

Section: High-throughput Sequencing Of Uncleaved Phages Defines the Psupporting

confidence: 81%

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

Kretz

Dai

Söylemez

et al. 2015

“…When expressed in E. coli, the refolded A2 domain was resistant to ADAMTS13 in the absence of denaturants, as is full-length, multimeric vWF. A smaller vWF73 construct that included only Asp-1596 to Arg-1668 was cleaved rapidly, but removal of another 9 amino acid residues from the C-terminal end of vWF73 made it completely resistant again (36). Therefore, structures missing from vWF73 normally inhibit access to the Tyr-1605-Met-1606 bond, and the segment Glu-1660-Arg-1668 may contribute to an auxiliary ADAMTS13-binding site.…”

Section: Discussionmentioning

confidence: 99%

“…Also, the von Willebrand disease type 2A mutation R1597Q within the A2 domain increases the rate of vWF cleavage by ADAMTS13, probably by interfering with the normal folding of the domain (15). However, the determinants of vWF recognition need not be limited to exposure of the cleavage site, and some studies of recombinant vWF domain A2 suggest a role for multiple interactions between vWF and ADAMTS13 (36). When expressed in E. coli, the refolded A2 domain was resistant to ADAMTS13 in the absence of denaturants, as is full-length, multimeric vWF.…”

Section: Discussionmentioning

confidence: 99%

Binding of platelet glycoprotein Ibα to von Willebrand factor domain A1 stimulates the cleavage of the adjacent domain A2 by ADAMTS13

Nishio

Anderson

Zheng

et al. 2004

142

135

von Willebrand factor (vWF) is a multimeric plasma glycoprotein with three tandem A domains. Domains A1 and A3 bind to platelet glycoprotein Ib␣ (GPIb␣) and collagen, respectively. Domain A2 contains the Tyr-1605-Met-1606 bond that is cleaved by the metalloprotease ADAMTS13, and this reaction inhibits platelet thrombus growth. Fluid shear stress increases the rate of cleavage, suggesting that productive interaction with ADAMTS13 requires conformational changes within or near domain A2. The influence of the adjacent A1 and A3 domains was assessed by mutagenesis of a recombinant substrate consisting of domains A1A2A3. Deletion of domain A3 did not affect cleavage by ADAMTS13, whereas deletion of domain A1 increased the rate of cleavage Ϸ10-fold. Similar effects were observed with plasma ADAMTS13 and recombinant ADAMTS13 truncated after the spacer domain. Digestion of A1A2A3 by plasma ADAMTS13 was enhanced to a similar extent by a recombinant mutant fragment of platelet GPIb␣ that binds with high affinity to domain A1 or by heparin. Heparin also increased the digestion of purified plasma vWF. Neither GPIb␣ nor heparin increased the cleavage of substrate A2A3 that lacks domain A1. The results suggest that vWF domain A1 inhibits the cleavage of domain A2, and that inhibition can be relieved by interaction of domain A1 with platelet GPIb␣ or certain glycosaminoglycans. Thus, binding of vWF to its major physiological ligands may promote the feedback inhibition of platelet adhesion by stimulating the cleavage of domain A2 by ADAMTS13 independent of fluid shear stress.

“…To investigate further the VWF residues important for cleavage by ADAMTS13, we have considered the activities of previously reported short VWF A2 domain fragments that span the cleavage site. One, VWF64 (1605-1668), could not be cleaved by ADAMTS13 (13), but others, such as VWF73 (1596-1668), VWF76 (1593-1668), and VWF115 (1554-1668 are all proteolysed efficiently (14,18,19). The difference between the substrates that are and those that are not cleaved resides in the sequence N terminal to the scissile bond.…”

mentioning

confidence: 99%

Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13)

Xiang

Groot²,

Crawley³

et al. 2011

The platelet-tethering function of von Willebrand factor (VWF) is proteolytically regulated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which cleaves the Tyr1605-Met1606 (P1-P1′) bond in the VWF A2 domain. To date, most of the functional interactions between ADAMTS13 and VWF that have been characterized involve VWF residues that are C terminal to the scissile bond. We now demonstrate that the substrate P3 position in VWF, Leu1603, is a critical determinant of VWF proteolysis. When VWF Leu1603 was substituted with Ser, Ala, Asn, or Lys in a short VWF substrate, VWF115, proteolysis was either greatly reduced or ablated (up to 400-fold reduction in k cat /K m ). As Leu1603 must interact with residues proximate to the Zn 2+ ion coordinated in the active center of ADAMTS13, we sought the corresponding S3 interacting residues. Substitution of 10 candidate residues in the metalloprotease domain of ADAMTS13 identified two spatially separated clusters centered on Leu198 or Val195 (acting with Leu232 and Leu274, or with Leu151, respectively), as possible subsites interacting with VWF. These experimental findings using the short VWF115 substrate were replicated using full-length VWF. It is hypothesized that VWF Leu1603 interacts with ADAMTS13 Leu198/Leu232/Leu274 and that Val195/Leu151 may form part of a S1 subsite. The recognition of VWF Leu1603 by ADAMTS13, in conjunction with previously reported remote exosites C terminal of the cleavage site, suggests a mechanism whereby the VWF P1-P1′ scissile bond is brought into position over the active site for cleavage. Together with recently characterized remote exosite interactions, these findings provide a general framework for understanding the ADAMTS family substrate interactions. microvascular thrombosis | thrombotic thrombocytopenia purpura V on Willebrand factor (VWF) is a large multimeric glycoprotein that is essential for normal hemostasis (1). Following vessel injury, VWF binds to exposed subendothelial collagen. Thereafter, in response to the shear forces exerted by the flowing blood, it unfolds from its inactive globular conformation into an active string-like form that can specifically recruit platelets (2-4). VWF multimeric size is a primary determinant of its platelettethering function and is proteolytically controlled by the plasma metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) (5-8). The physiological importance of this system is highlighted by the clinical sequelae associated with dysfunction of the VWF/ADAMTS13 axis. Whereas ADAMTS13 deficiency can cause fatal microvascular thrombosis-thrombotic thrombocytopenic purpura (7), excessive VWF proteolysis causes bleeding (i.e., type 2A von Willebrand disease) (1).ADAMTS13 circulates in plasma as a constitutively active enzyme, which is unusual. Despite this behavior, plasma VWF remains essentially resistant to ADAMTS13 proteolysis in free circulation. This resistance is because shear-dependent unfolding...