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Carboxyl-terminal truncation of recombinant factor XIII A-chains. Characterization of minimum structural requirement
T S Lai1, K E Achyuthan, M A Santiago
1Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Abstract:
A series of truncation mutants lacking 218, 229, 250, and 269 amino acid residues from the carboxyl terminus of blood coagulation factor XIII A-chains (FXIII A), designated as delta K513, delta A502, delta Y481, and delta K462, respectively, were expressed in Escherichia coli to define the minimum structure required for transglutaminase activity. delta K513 and delta A502 displayed a 3.8-4.7-fold reduction in the Kcat with no change in the Km for the glutamine substrate and a 2-fold increase in the Km of the primary amine substrate. There was no detectable transglutaminase activity for either thrombin-activated delta Y481 or delta K462. The rate of ammonia release of thrombin-activated delta K513 and delta A502 was reduced 6- and 4-fold, respectively, whereas ammonia release was not detected for the delta Y481 and delta 462 mutants. The Kact for calcium ions of the delta K513 mutant was similar to recombinant FXIIIa, whereas, it was increased by approximately 3-fold for the delta A502 mutant. The rate of fibrin gamma-chain dimer formation for the delta K513 and delta A502 mutants was reduced by approximately 19-fold. delta K462 did not bind to fibrin, while all of the other thrombin-cleaved mutants were bound. In conclusion, these results documented that the carboxyl-terminal calcium binding domain (Asp468-Glu495) was important for FXIIIa to adopt the correct conformation to ensure that efficient catalysis occurred.
Insights
The carboxyl-terminal calcium binding domain of blood coagulation factor XIII A-chains (FXIII A) is crucial for its transglutaminase activity. Truncations in this domain significantly reduce or abolish enzyme function and fibrin binding.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Blood coagulation factor XIII A-chains (FXIII A) are essential for stabilizing blood clots.
- Understanding the structure-function relationship of FXIII A is vital for comprehending coagulation mechanisms.
Purpose of the Study:
- To define the minimum structural requirements for the transglutaminase activity of FXIII A.
- To investigate the role of the carboxyl-terminal region in FXIII A function and calcium binding.
Main Methods:
- Expression of various carboxyl-terminal truncation mutants of FXIII A in Escherichia coli.
- Assays to measure transglutaminase activity, including Kcat and Km for substrates.
- Analysis of ammonia release rates and calcium ion activation (Kact).
- Assessment of fibrin binding capabilities of the mutants.
Main Results:
- Truncation mutants delta K513 and delta A502 showed reduced catalytic efficiency (Kcat) and altered substrate affinity (Km).
- Mutants delta Y481 and delta K462 exhibited no detectable transglutaminase activity.
- Calcium ion activation (Kact) was similar to wild-type for delta K513 but increased for delta A502.
- Fibrin gamma-chain dimer formation was significantly reduced, and delta K462 failed to bind fibrin.
Conclusions:
- The carboxyl-terminal calcium binding domain (Asp468-Glu495) is critical for FXIII A to achieve its active conformation.
- Proper conformation is necessary for efficient transglutaminase catalysis and substrate interaction.
- This domain plays a key role in both enzymatic activity and fibrin cross-linking functions.
