Effect of Binding Site Mutations in Fibrinogen αC (233-425) On FXIII Substrate Specificity

Abstract

In blood coagulation, the transglutaminase Factor XIII (FXIII) catalyzes the crosslinking of specific glutamine and lysine residues between fibrin and other coagulation proteins to generate fibrin clots resistant to fibrinolysis. The αC region (233-425) of the fibrinogen alpha chain contains three reactive glutamines (Q237, Q328, Q366) and a putative FXIII binding region (αC 389-403), where E396 is reported to be the key binding residue. FXIII can be activated proteolytically by thrombin with low calcium concentration (∼4 mM Ca2+, FXIIIA°) or non-proteolytically with high calcium concentration (>25 mM Ca2+, FXIII A°), both leading to FXIIIA conformational sets that vary in transglutaminase activity. These features were explored further by assaying glutamine reactivities (Fbg αC WT, E396A, and αC 389Stop) with the lysine mimic glycine ethyl ester (GEE) by MALDI-ToF mass spectrometry. Across all αC variants, the reactivity ranking from FXIII A° and FXIII A° was Q237 >> Q366 ∼ Q328. Neither the αC E396A (233-425) nor the αC 389Stop (233-388) mutant prevented transglutaminase activity from FXIII A° or FXIII A°. However, the Q237-GEE crosslinking rate for either mutant was hindered compared to WT for both FXIIIA forms. Removal of the putative FXIII binding region in αC 389Stop had less of an adverse effect on FXIII A°-catalyzed GEE crosslinking to Q237 than αC E396A. Additionally, the Q237-GEE crosslinking in αC 389Stop was slightly faster with FXIII A° than with A°, in contrast to WT and E396A where FXIII A' crosslinking was faster. Overall for the FXIII A° studies, the Q-reactivities were WT > E396A > 389Stop whereas with FXIII A°, they were relatively similar. Differences in transglutaminase function between FXIII A° and FXIII A° involve more than activity and include how they interface with substrates like Fbg αC.