Calcium binding sites of coagulation factor XIII (FXIII) A subunit and their functional relevance to the activation of FXIII A subunit
S. Singh1, C. Krettler2, C. Reinhart 2, J. Dodt 3, V. Ivaskevicius 1, J. Oldenburg 1, A. Biswas1 (1Bonn, Germany, 2Frankfurt, Germany, 3Langen, Germany)
Bleeding disorders - Basic science
Time: 08:00 - 09:15
Objective: To check the functional relevance of three calcium binding sites on FXIIIA subunit by a combination of increasing concentrations of calcium saturated simulations and site directed mutagenesis performed on the recombinant FXIIIA (rFXIIIA).
Methods: rFXIIIA subunit was cloned, expressed and purified in-house, using the yeast based Pichia expression system. Twelve substitutions of two types were generated on the calcium binding site residues: A) substitution with a Lys residue to prevent calcium binding and B) substitution with an Asp residue leading to tighter binding using Geneart site-directed mutagenesis kit (Thermo Fischer Scientific, USA). Positive clones were expressed in yeast based Pichia expression system, and purified using two-step purification strategy (IMAC using His-TRAP, followed by Gel filtration). The purified proteins were tested for identification (by peptide mass fingerprinting) antigenic stability (by western blot), FXIIIA activity (by photometric assay), and FXIIIAa generation (by FXIII generation assay). The complete FXIIIA zymogenic crystal structure was subjected to classical MD simulation with different supra-physiological concentrations of calcium. The same process was repeated also exclusively for only the FXIIIA core domain
Results: In the simulations higher concentrations of calcium led to faster co-ordination of the calcium binding sites. We also observed cross-talk between the three calcium binding sites in terms of correlated motion. So far three clones with implemented mutations in the calcium binding sites were successfully purified. While these three clones were antigenically detectable easily, they had a tendency to degrade faster as compared to the wild type rFXIIIA under same buffer conditions. Amongst the three mutants, only one showed any activity which was very low (almost 1-10% of the wild type).
Conclusion: Calcium binding sites in FXIIIA subunit appear to be highly sensitive areas of the protein, which is why any kind of substitution here appears to compromise the activity or even antigen levels of the protein by causing misfolding of the protein. The three calcium binding sites in agreement to our previous reports also show interdependence and chronology during the activation process.