Factor XIII Activation Peptide Residues Play Important Roles in Stability, Activation, and Transglutaminase Activity

• Published in: Biochemistry (Easton) 2024-11, Vol.63 (21), p.2830-2841
• Main Authors: Syed Mohammed, Rameesa D., Gutierrez Luque, Lianay, Maurer, Muriel C.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Enzyme Activation; Factor XIII - chemistry; Factor XIII - genetics; Factor XIII - metabolism; Humans; Intercellular Signaling Peptides and Proteins; Peptides - chemistry; Peptides - metabolism; Protein Stability; Proteolysis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Thrombin - chemistry; Thrombin - metabolism; Transglutaminases - chemistry; Transglutaminases - genetics; Transglutaminases - metabolism
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.4c00318

A subunit of factor XIII (FXIII-A) contains a unique activation peptide (AP) that protects the catalytic triad and prevents degradation. In plasma, FXIII is activated proteolytically (FXIII-A*) by thrombin and Ca2+ cleaving AP, while in cytoplasm, it is activated nonproteolytically (FXIII-A°) with increased Ca2+ concentrations. This study aimed to elucidate the role of individual parts of the FXIII-A AP in protein stability, thrombin activation, and transglutaminase activity. Recombinant FXIII-A AP variants were expressed, and SDS-PAGE was used to monitor thrombin hydrolysis at the AP cleavage sites R37–G38. Transglutaminase activities were assessed by cross-linking lysine mimics to Fbg αC (233–425, glutamine–substrate) and monitoring reactions by mass spectrometry and in-gel fluorescence assays. FXIII-A AP variants, S19P, E23K, and D24V, degraded during purification, indicating their vital role in FXIII-A2 stability. Mutation of P36 to L36/F36 abolished the proteolytic cleavage of AP and thus prevented activation. FXIII-A N20S and P27L exhibited slower thrombin activation, likely due to the loss of key interdomain H-bonding interactions. Except N20S and P15L/P16L, all activatable FXIII-A* variants (P15L, P16L, S19A, and P27L) showed similar cross-linking activity to WT. By contrast, FXIII-A° P15L, P16L, and P15L/P16L had significantly lower cross-linking activity than FXIII-A° WT, suggesting that loss of these prolines had a greater structural impact. In conclusion, FXIII-A AP residues that play crucial roles in FXIII-A stability, activation, and activity were identified. The interactions between these AP amino acid residues and other domains control the stability and activity of FXIII.