PB0541 - Expression of recombinant human Factor VII activating protease (FSAP) and exploration of its 3D structure using AlphaFold.

Background: Factor VII activating protease (FSAP) is a circulating serine protease in blood with homology to coagulation and fibrinolysis factors and is encoded by the HABP2 gene. In animal models, FSAP regulates hemostasis, inflammation, remodeling processes and influences stroke outcome.

Aims: The aim of this study was to express His-tagged human FSAP and its domains in insect cells. AlphaFold-based modelling was used to predict FSAP 3D structure and resolve the activation mechanism of pro-FSAP by extracellular histones.

Methods: Plasmid-based insect cell expression system was utilized to express full length (FL ), heavy chain (HC-) and the serine protease domain (SPD)-FSAP. Secreted proteins were purified from the medium using the His-tag and characterized. AlphaFold and AlphaFold-Multimer was utilized to investigate (i) the structure of FSAP zymogen and (ii) compare it to other members of the family FXII, HGFA and tissue plasminogen activator (tPA) and (iii) study complex formation with histones.

Results: FL- and HC-FSAP was expressed, both, intra-cellularly and secreted into the medium. Inhibition of extracellular serine proteases with aprotinin or intracellular convertase enzymes with a furin inhibitor did not alter this secretion pattern nor the amount. Enzymatically inactive FL- and SPD-FSAP was expressed more robustly than the active counterparts. Codon optimized sequence did not alter this pattern of expression and neither did its fusion with maltose binding protein (MBP). A monoclonal antibody to the extreme N-terminus region blocks pro-FSAP activation by histones. This is a disordered stretch rich in acidic residues in the AlphaFold structure.

Conclusion(s): The drosophila expression system was used to successfully express FSAP and its domains. FSAP is unique in having a disordered stretch in the extreme N-terminus which points to an exclusive mechanism of zymogen activation with histones. The information gained from these studies will help to optimize the expression of recombinant FSAP to promote biochemical-, cellular-, animal-experiments and crystallization studies.