Hethershaw, EL orcid.org/0000-0002-4366-278X, Adamson, PJ, Smith, KA orcid.org/0000-0001-5695-7117 et al. (7 more authors) (2018) The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit. Journal of Thrombosis and Haemostasis, 16 (7). pp. 1391-1401. ISSN 1538-7933
Abstract
Background; Factor XIII (FXIII) is composed of an activation peptide segment, a β‐sandwich domain, a catalytic core, and finally β‐barrels 1 and 2. FXIII is activated following cleavage of its A‐subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis.
Objectives; To assess the functional roles of β‐barrels 1 and 2 in FXIII, we expressed and characterised the full‐length FXIII‐A subunit (FXIII‐A) and variants truncated to residue 628 [truncated to β‐barrel 1 (TB1)], 515 [truncated to catalytic core (TCC)] and 184 [truncated to β‐sandwich (TBS)].
Methods; Proteins were analysed by gel electrophoresis, circular dichroism, fluorometric assays, colorimetric activity assays, clot structure by turbidity measurements, confocal microscopy, and clot formation by Chandler Loop.
Results and Conclusions; Circular dichroism spectroscopy and tryptophan fluorometry indicate that full length FXIII‐A and the truncations TCC and TB1 retain their secondary and tertiary structure. Removal of β‐barrel 2 (TB1) resulted in total loss of transglutaminase activity, whilst the additional removal of β‐barrel 1 (TCC) restored enzymatic activity to approximately 30% of full length FXIII‐A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the β‐barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase while the β‐barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual β‐barrel domains in modulating access to the FXIII active site region.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | (c) 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) |
Keywords: | Catalytic domain; enzyme activation; factor XIII; protein conformation; transglutaminases |
Dates: |
|
Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology (Leeds) |
Funding Information: | Funder Grant number British Heart Foundation RG/03/004 British Heart Foundation PG/08/052/25172 |
Depositing User: | Symplectic Publications |
Date Deposited: | 03 May 2018 12:45 |
Last Modified: | 19 Apr 2019 00:38 |
Status: | Published |
Publisher: | Wiley-Blackwell |
Identification Number: | 10.1111/jth.14128 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:130370 |