Factor XIII activation and substrate crosslinking can be influenced by amino acid substitutions from transglutaminase-2.

Abstract

Factor XIII (FXIII) catalyzes formation of covalent crosslinks between reactive glutamines and lysines in plasma and cellular substrates. Related enzyme Transglutaminase 2 (TG2) also catalyzes crosslinking of cellular substrates, some common with FXIII. FXIII-A2 can be activated proteolytically to FXIII-A∗ by thrombin cleavage of the FXIII activation peptide (AP) and low mM CaCl2. FXIII-A2 can also be activated non-proteolytically by elevated Ca2+ levels (FXIII-A°) with up to three calcium binding sites Ca1, 2, and 3 filled. TG2 is allosterically regulated by Ca2+ and GTP. Kanchan et al (2013) reported that TG2 activity could be increased with a G224V substitution that improves Ca2+ binding. For the current project, two TG2 residues (V224 and N231) located near Ca3 were introduced into comparable positions of FXIII-A2. FXIII WT, G262V, K269N, and (G262V, K269N) were screened for their abilities to catalyze crosslinking of monodansylcadaverine (MDC, lysine mimic) into Fbg αC (233-425, glutamine substrate). For FXIII-A° generated with 100 mM CaCl2, MDC crosslinking ranked as FXIII G262V < FXIII WT ≪ FXIII K269N ≈ FXIII (G262V, K269N). FXIII V262 is proposed to impair a helical segment near Ca3 thus hindering the benefits of effective Ca2+ binding. FXIII K269N with its neutral N269 is hypothesized to enhance the lysine substrate binding surface near Ca3. Comparable crosslinkings occurred for FXIII-A∗ WT and FXIII-A∗ G262V likely due to presence of low mM Ca2+ that would primarily target Ca1 binding. The extent of MDC crosslinking ranked as FXIII-A∗ (WT ≈ G262V) ≪ (G262V, K269N) < K269N. For both FXIII-A∗ and A°, the K269N position in the vicinity of Ca3 and the lysine binding surface generated the greatest improvements in transglutaminase activity. Overall, TG2 substitutions were successfully used to tune properties of FXIII.