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  • Title: The role of β-barrels 1 and 2 in the enzymatic activity of factor XIII A-subunit.
    Author: Hethershaw EL, Adamson PJ, Smith KA, Goldsberry WN, Pease RJ, Radford SE, Grant PJ, Ariëns RAS, Maurer MC, Philippou H.
    Journal: J Thromb Haemost; 2018 Jul; 16(7):1391-1401. PubMed ID: 29675848.
    Abstract:
    UNLABELLED: Essentials The roles of β-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking β-barrel 2, both β-barrels, or full length FXIII, were made. Removing β-barrel 2 caused total loss of activity, removing both β-barrels returned 30% activity. β-barrel 2 is necessary for exposure of the active site cysteine during activation. SUMMARY: Background Factor XIII 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 characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to β-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to β-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of β-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of β-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that 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, whereas 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.
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