Probing ADAMTS13 Substrate Specificity using Phage Display

Desch, Karl C.; Kretz, Colin A.; Yee, Andrew; Gildersleeve, Robert; Metzger, Kristin; Agrawal, Nidhi; Cheng, Jane; Ginsburg, David

doi:10.1371/journal.pone.0122931

Cited by 10 publications

(11 citation statements)

References 35 publications

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“…S1). This concurs with previous studies of substrate specificity of ADAMTS‐13, in which the substitution of Tyr1605 to lysine did not abolish the proteolysis of a VWF73 expressing phage 29, 30. Our findings suggest proteolysis of fibrinogen by GoF ADAMTS‐13 will be less efficient than its cleavage of unfolded VWF.…”

Section: Discussionsupporting

confidence: 92%

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

South

Freitas

Lane

2016

Journal of Thrombosis and Haemostasis

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Essentials Recently, ADAMTS‐13 has been shown to undergo substrate induced conformation activation.Conformational quiescence of ADAMTS‐13 may serve to prevent off‐target proteolysis in plasma.Conformationally active ADAMTS‐13 variants are capable of proteolysing the Aα chain of fibrinogen.This should be considered as ADAMTS‐13 variants are developed as potential therapeutic agents. Click to hear Dr Zheng's presentation on structure function and cofactor-dependent regulation of ADAMTS‐13 SummaryBackgroundRecent work has revealed that ADAMTS‐13 circulates in a ‘closed’ conformation, only fully interacting with von Willebrand factor (VWF) following a conformational change. We hypothesized that this conformational quiescence also maintains the substrate specificity of ADAMTS‐13 and that the ‘open’ conformation of the protease might facilitate proteolytic promiscuity.ObjectivesTo identify a novel substrate for a constitutively active gain of function (GoF) ADAMTS‐13 variant (R568K/F592Y/R660K/Y661F/Y665F).MethodsFibrinogen proteolysis was characterized using SDS PAGE and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Fibrin formation was monitored by turbidity measurements and fibrin structure visualized by confocal microscopy.Results ADAMTS‐13 exhibits proteolytic activity against the Aα chain of human fibrinogen, but this is only manifest on its conformational activation. Accordingly, the GoF ADAMTS‐13 variant and truncated variants such as MDTCS exhibit this activity. The cleavage site has been determined by LC‐MS/MS to be Aα chain Lys225‐Met226. Proteolysis of fibrinogen by GoF ADAMTS‐13 impairs fibrin formation in plasma‐based assays, alters clot structure and increases clot permeability. Although GoF ADAMTS‐13 does not appear to proteolyse preformed cross‐linked fibrin, its proteolytic activity against fibrinogen increases the susceptibility of fibrin to tissue‐type plasminogen activator (t‐PA)‐induced lysis by plasmin and increases the fibrin clearance rate more than 8‐fold compared with wild‐type (WT) ADAMTS‐13 (EC50 values of 3.0 ± 1.7 nm and 25.2 ± 9.7 nm, respectively) in in vitro thrombosis models.ConclusionThe ‘closed’ conformation of ADAMTS‐13 restricts its specificity and protects against fibrinogenolysis. Induced substrate promiscuity will be important as ADAMTS‐13 variants are developed as potential therapeutic agents against thrombotic thrombocytopenic purpura (TTP) and other cardiovascular diseases.

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Section: Discussionsupporting

confidence: 92%

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

South

Freitas

Lane

2016

Journal of Thrombosis and Haemostasis

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“…The apparent pseudo-first-order rate constant, k app , calculated for WT VWF73 within the library, was 0.2888 ± 0.001, yielding a k cat /K M value of 5.78 × 10 7 M −1 ·min −1 (SI Appendix, Fig. S5), which is comparable to the value determined for the recombinant WT VWF73 peptide (Table 2) (26)(27)(28).…”

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

confidence: 54%

“…Segment A, spanning Pro6-Val12 of VWF73, includes the Tyr10-Met11 scissile bond and showed the greatest enrichment of mutation frequency. Segment B spans Asp19-Asp27 and contains Ile21, a previously identified exosite-binding residue (26). Segment C spans Pro53-Leu69 and contains a number of residues exhibiting mutation enrichment after selection (Fig.…”

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

confidence: 97%

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

Kretz

Dai

Söylemez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Promiscuity may therefore be expected. The entire range of possible residue changes that permit proteolysis of the small VWF substrate VWF73 has been mapped by use of a substrate phage display library . This has highlighted the importance of the scissile bond residues as well as previously identified exosite regions (see below), but has also demonstrated that, within these regions, many possible residue substitutions that permit cleavage are possible.…”

Section: Conformational Activation Of Adamts‐13 By Vwfmentioning

confidence: 99%

ADAMTS‐13 and von Willebrand factor: a dynamic duo

South

Lane

2018

Journal of Thrombosis and Haemostasis

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Summaryvon Willebrand factor (VWF) is a key player in hemostasis, acting as a carrier for factor VIII and capturing platelets at sites of vascular damage. To capture platelets, it must undergo conformational changes, both within its A1 domain and at the macromolecular level through A2 domain unfolding. Its size and this function are regulated by the metalloproteinase ADAMTS‐13. Recently, it has been shown that ADAMTS‐13 undergoes a conformational change upon interaction with VWF, and that this enhances its activity towards its substrate. This review summarizes recent work on these conformational transitions, describing how they are controlled. It points to their importance in hemostasis, bleeding disorders, and the developing field of therapeutic application of ADAMTS‐13 as an antithrombotic agent in obstructive microvascular thrombosis and in cardiovascular disease.

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Probing ADAMTS13 Substrate Specificity using Phage Display

Cited by 10 publications

References 35 publications

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

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

ADAMTS‐13 and von Willebrand factor: a dynamic duo

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