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 non-proteolytically (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, fibronectin, and Fbg αC (233-388, missing FXIII binding site αC 389-402). Furthermore, the N-terminal α2-AP 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 being achieved.

Fbg αC 389–402 Enhances Factor XIII Cross-Linking in the Fibrinogen αC Region Via Electrostatic and Hydrophobic Interactions

“…Fbg αC 233–425 variant DNA were generated using site-directed mutagenesis with the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA), as described previously. , A pGEX plasmid vector coding for GST-tagged Fbg αC 233–425 was used as the template strand . Primer design sequences are listed in Table S1.…”

“…Mutant plasmids were subsequently transformed into BL21 Gold DE3 (Agilent Technologies, Inc., Cedar Creek, TX) competent cells for expression. Fbg αC 233–425 and its variants were expressed via auto-induction and purified as described previously. , …”

“…FXIII-A* activity was tracked by measuring GEE cross-linking to glutamines within Fbg αC 233–425 via a MALDI-TOF MS kinetic assay. ,,, 50 nM FXIII-A 2 was proteolytically activated by incubation in MALDI kinetic buffer (50 mM Tris-Acetate, 150 mM NaCl, 0.1% PEG-8000, pH 7.4) in the presence of 8.4 NIH units/mL bovine thrombin and 4 mM CaCl 2 at 37 °C for 10 min, followed by reaction quenching via the addition of 190 μM PPACK. The cross-linking reaction was commenced by the addition of 17 mM GEE and 13.6 μM of Fbg αC to the previous solution.…”

“…Assays for each αC species were performed for 30 min and in triplicate. Data were reported as mean ± SD, subjected to statistical analysis by the Student′s t -test, and were fit using a one-phase exponential decay model, [ Q ] remaining, t = ([ Q ] 0 – [ Q ] plateau )­(e – Kt ) + [ Q ] plateau , with GraphPad Prism 9.5.1 as previously described . [ Q ] remaining, t is the concentration of uncrosslinked reactive glutamine in μM for time point t in minutes, [ Q ] 0 is the theoretical initial concentration of uncrosslinked reactive glutamine in μM, [ Q ] plateau is the theoretical concentration of uncrosslinked reactive glutamine in μM when t = ∞ min, and K is the rate constant in min –1 .…”

“…The current study sought to identify additional residues within Fbg αC 389–402 that impact αC cross-linking using a recombinantly expressed Fbg αC 233–425 model system. − Fbg αC D390, W391, F394, and E395 were hypothesized to have an effect on FXIII-A* activity toward αC based on a chemical cross-linking and molecular modeling study previously performed by Smith et al in 2013 . The effect of these residues on cross-linking was now assessed by site-directed mutagenesis, followed by quantification of FXIII-A* activity using a previously designed MALDI-TOF mass spectrometry assay. , Key findings were further supported using a fluorescence-based SDS-PAGE assay. , The impact of αC 327–388 and αC 403–425 on αC cross-linking was similarly assessed by truncations to the model Fbg αC 233–425 system. The results of these studies identified key residues in αC 389–402 that increase αC cross-linking.…”