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

Abstract

Factor XIII (FXIII) catalyzes formation of γ-glutamyl-ε-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°) 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° 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° toward Q residues within Fbg αC (233–425 WT, Q328P Seoul II, and Q328PQ366N) and actin. FXIII-A* and FXIII-A° displayed similar activities toward α₂-antiplasmin (α₂AP), fibronectin, and Fbg αC (233–388, missing FXIII-binding site αC 389–402). Furthermore, the N-terminal α₂AP peptide (1–15) exhibited similar kinetic properties for FXIII-A* and FXIII-A°. MALDI-TOF mass spectrometry assays with glycine ethyl ester and Fbg αC (233–425 WT, αC E396A, and truncated αC (233–388) further documented that FXIII-A* exerts greater benefit from the αC 389–402 binding site than FXIII-A°. Conformational properties of FXIII-A* versus A° 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.

Keywords

antiplasmin - factor XIII - fibrinogen - fibronectin - actin

Authors Contribution

R.D.S.M, F.D.O.A., and M.C.M. designed the research. R.D.S.M., F.D.O.A., N.M.M., and M.M.H. expressed the proteins and carried out the experiments. R.D.S.M. performed the MDC assays. F.D.O.A., N.M.M., and M.M.H. performed the GEE Mass Spectrometry assays. R.D.S.M. and N.M.M. contributed to the PONDR analysis. M.C.M., R.D.S.M., and F.D.O.A. interpreted the data and wrote the manuscript. All authors reviewed the manuscript and approved its final version.

Publikationsverlauf

Eingereicht: 02. August 2022

Angenommen: 04. Dezember 2022

Accepted Manuscript online:
06. Dezember 2022

Artikel online veröffentlicht:
25. Januar 2023

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