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.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)