Transglutaminase Activities of Blood Coagulant Factor XIII Are Dependent on the Activation Pathways and on the Substrates

Factor XIII (FXIII) catalyzes formation of gamma-glutamyl-epsilon-lysyl crosslinks between reactive glutamines (Q) and lysines (K). In plasma, FXIII is activated proteolytically (FXIII-A*) by the concerted action of thrombin and Ca2+ . Cellular FXIII is activated nonproteolytically (FXIII-A degrees) by elevation of physiological Ca2+ concentrations. FXIII-A targets plasmatic and cellular substrates, but questions remain on correlating FXIII activation, resultant conformational changes, and crosslinking function to different physiological substrates. To address these issues, the characteristics of FXIII-A* versus FXIII-A degrees that contribute to transglutaminase activity and substrate specificities were investigated. Crosslinking of lysine mimics into a series of Q-containing substrates were measured using in-gel fluorescence, mass spectrometry, and UV-Vis spectroscopy. Covalent incorporation of fluorescent monodansylcadaverine revealed that FXIII-A* exhibits greater activity than FXIII-A degrees toward Q residues within Fbg alpha C (233-425 WT, Q328P Seoul II, and Q328PQ366N) and actin. FXIII-A* and FXIII-A degrees displayed similar activities toward alpha(2)-antiplasmin (alpha(2)AP), fibronectin, and Fbg alpha C (233-388, missing FXIII-binding site alpha C 389-402). Furthermore, the N-terminal alpha(2)AP peptide (1-15) exhibited similar kinetic properties for FXIII-A* and FXIII-A degrees. MALDI-TOF mass spectrometry assays with glycine ethyl ester and Fbg alpha C (233-425 WT, alpha C E396A, and truncated alpha C (233-388) further documented that FXIII-A* exerts greater benefit from the alpha C 389-402 binding site than FXIII-A degrees. Conformational properties of FXIII-A* versus A degrees are proposed to help promote transglutaminase function toward different substrates. A combination of protein substrate disorder and secondary FXIII-binding site exposure are utilized to control activity and specificity. From these studies, greater understandings of how FXIII-A targets different substrates are achieved.

Automated summary

Factor XIII (FXIII) catalyzes the formation of crosslinks between reactive glutamines (Q) and lysines (K). FXIII can be activated proteolytically (FXIII-A*) or nonproteolytically (FXIII-A degrees). The activity and specificity of FXIII-A* and FXIII-A degrees towards different substrates were investigated to gain a better understanding of how FXIII-A targets different substrates.