Dissociation of Factor XIII Homodimer during Activation Process: Solution Evidence Weighs in on Decades-Long Debate

Abstract

Oligomeric assemblies have been increasingly realized to regulate protein function. As the scope of this research area expands, it has been estimated that 60% of enzymes form oligomers. While many of these enzymes require self-association to fulfill their catalytic function, the spotlight of the current project is on a protein that is inactive in its oligomeric form. Factor XIIIA (FXIIIA) is a transglutaminase that cross-links an array of intra- and extracellular protein substrates in a calcium-dependent manner. FXIIIA is the only member of the transglutaminase family found as a homodimer (A2) in zymogen form. It can be activated by thrombin-mediated cleavage of the activation peptides (AP) or non-proteolytically by Ca2+ ions. The oligomeric composition of activated FXIIIA is controversial. Although the enzyme has long been considered a dimer, recent indirect evidence suggests the possibility of a monomeric state. In the present study, size exclusion chromatography and analytical ultracentrifugation were used to study global rearrangements in FXIIIA2 accompanying its activation. For the first time, a quantitative assessment of FXIIIA intersubunit interactions was performed. Those interactions were tight (Kd 8 nM) in the dimeric zymogen form. Both non-proteolytic and thrombin-mediated FXIIIA activation resulted in monomeric species (with Kd values ranging from 90 to 220 mM). Interestingly, cleavage of a single AP on the FXIII A2-homodimer resulted in dissociation and in expression of full enzymatic activity for both cleaved and noncleaved protomers. By contrast, non-proteolytic activation was much less efficient. Thus, direct experimental evidence was obtained for the monomeric state of activated FXIIIA in solution. Dimerization of the zymogen is proposed to stabilize FXIIIA in a physiological setting and to prevent premature protein cross-linking. Consequently, dissociation of the homodimer is crucial for full expression of FXIIIA function.