Transglutaminase Factor XIII Cross-Links Reactive Glutamines in Disordered Regions of Fibrinogen αC

Abstract

Fibrinogen is the most abundant protein involved in blood coagulation and has been implicated in cardiovascular disease. During the formation of a blood clot, thrombin cleaves fibrinopeptides A and B from fibrinogen (AαBβγ)2 to form fibrin monomers (αβγ)2. Fibrin polymerization proceeds with the formation of protofibrils and a hard clot is only generated when the transglutaminase factor XIII (FXIII) introduces covalent γ-glutamyl-e-lysyl bonds between specific reactive glutamines and lysines in the fibrin clot network. The αC region (Aα221-610) of fibrinogen contains numerous reactive glutamines in the flexible αC-connector (221-392) and a FXIII binding site within αC (371-425). MALDI-TOF mass spectrometry assays and 2D 15N-1H HSQC NMR studies have been carried out on fibrinogen αC (233-425), a segment containing three known reactive glutamines (Q237, Q328, and Q366). FXIIIa crosslinks these three glutamines to the lysine mimic glycine ethyl ester (GEE) in the order Q237 >> Q366 > Q328. To better characterize this system, single site mutants have been generated in which the reactive glutamines (Q) are individually replaced with inactive asparagines (N). Work with these mutants suggests that FXIIIa does not require crosslinking to occur in a sequential manner. Each glutamine can be cross-linked independently of the other. Due to its highly flexible nature, the fibrinogen αC region has not been observed by X-ray crystallography. 2D HSQC NMR studies on 15N-labeled αC (233-425) suggest that this protein is intrinsically disordered. FXIIIa substrate specificity for specific reactive glutamines is predicted to be controlled by a combination of neighboring residues and the presence of regions of disorder. The knowledge gained from these studies can be used for subsequent assay and drug designs.