Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay

Abstract

Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.

Ranking reactive glutamines in the fibrinogen αC region that are targeted by blood coagulant factor XIII

“…47 Final concentrations of 500 nM FXIII, 17 mM GEE, 4 mM CaCl 2 , and MALDI assay buffer (100 mM Tris-acetate, 150 mM NaCl, and 0.1% PEG 8000 , pH 7.4] were incubated at 37°C in a 1.5 mL reaction tube. The FXIII was activated with bovine thrombin (8.4 U/mL final) for 10 minutes and then 200 nM D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone was added to inhibit the thrombin.…”

“…All digested samples were zip-tipped and analyzed using a MALDI-TOF mass spectrometer (Voyager-DE PRO; Applied Biosystems). 47 a-Cyano-4-hydroxycinnamic acid matrix was employed for chymotrypsin digests and ferulic acid matrix for GluC. This assay was done in triplicate using aC (233-425) from 3 independent expression trials.…”

Characterizing reactive glutamines in fibrinogen and elucidating factor XIII substrate specificity.

“…44,48 During the activation of Factor XIII in plasma, the carrier B-subunits dissociate from the catalytic A-subunits following thrombin cleavage of the activation peptide and upon calcium binding. The catalytic core of Factor XIII consists of a Cysteine, Histidine, and Aspartate triad, also conserved in all catalytic transglutaminases.…”

“…48 The Q-containing protein substrate was combined with the lysine mimic glycine ethyl ester (GEE) in the presence of activated Factor XIII. At different time points, aliquots of the reaction were quenched and peptide digests performed to monitor for fragments containing reactive glutamines and its crosslinked product ( Figure 15).…”

“…A mass spectrometry-based assay was designed whereby each reactive glutamine can be monitored individually. 48,108 In this assay, the ability of Factor XIIIa to incorporate a lysine mimic glycine ethyl ester into each reactive glutamine was Furthermore, other Fibrinogen αC and γ segments that could provide additional information on Factor XIII's substrate specificity are discussed.…”