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

We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was...

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Autores principales: Kretz, Colin A., Tomberg, Kärt, Van Esbroeck, Alexander, Yee, Andrew, Ginsburg, David
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809430/
https://www.ncbi.nlm.nih.gov/pubmed/29434246
http://dx.doi.org/10.1038/s41598-018-21021-9
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author Kretz, Colin A.
Tomberg, Kärt
Van Esbroeck, Alexander
Yee, Andrew
Ginsburg, David
author_facet Kretz, Colin A.
Tomberg, Kärt
Van Esbroeck, Alexander
Yee, Andrew
Ginsburg, David
author_sort Kretz, Colin A.
collection PubMed
description We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >10(7) sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3′ interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3′ interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1′. Overall, the P3-P2′ amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.
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spelling pubmed-58094302018-02-15 High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13 Kretz, Colin A. Tomberg, Kärt Van Esbroeck, Alexander Yee, Andrew Ginsburg, David Sci Rep Article We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >10(7) sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3′ interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3′ interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1′. Overall, the P3-P2′ amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases. Nature Publishing Group UK 2018-02-12 /pmc/articles/PMC5809430/ /pubmed/29434246 http://dx.doi.org/10.1038/s41598-018-21021-9 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Kretz, Colin A.
Tomberg, Kärt
Van Esbroeck, Alexander
Yee, Andrew
Ginsburg, David
High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13
title High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13
title_full High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13
title_fullStr High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13
title_full_unstemmed High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13
title_short High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13
title_sort high throughput protease profiling comprehensively defines active site specificity for thrombin and adamts13
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809430/
https://www.ncbi.nlm.nih.gov/pubmed/29434246
http://dx.doi.org/10.1038/s41598-018-21021-9
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