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Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP)
Nis V Nielsen1, Elfie Roedel2, Dipankar Manna1
1Oslo University Hospital and University of Oslo, Oslo, Norway.
Abstract:
Factor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.
Insights
The Marburg I (MI) polymorphism in Factor VII activating protease (FSAP) significantly reduces its auto-activation and proteolytic activity. This study used recombinant proteins to reveal how the Gly221Glu substitution impacts FSAP function and regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Factor VII (FVII) activating protease (FSAP) is a serine protease implicated in various diseases.
- The Marburg I (MI) polymorphism (Gly221Glu) is linked to disease pathogenesis.
- Understanding FSAP's molecular function is crucial for disease insights.
Purpose of the Study:
- To investigate the molecular and functional consequences of the Gly221Glu substitution in FSAP.
- To characterize the activity of wild-type (WT) and MI FSAP isoforms using recombinant proteins.
- To explore the role of the MI-single nucleotide polymorphism (SNP) in FSAP regulation.
Main Methods:
- Expression of recombinant wild-type (WT) and Marburg I (MI) FSAP serine protease domains (SPD) in E. coli.
- Assessing auto-catalytic activation and proteolytic activity against various substrates.
- Site-directed mutagenesis and introduction of cleavage sites to analyze protein function.
- Investigating the effect of metal ions (calcium, sodium) on enzymatic activity.
Main Results:
- WT-FSAP SPD showed auto-catalytic activation, while MI-FSAP SPD exhibited significantly reduced auto-activation and proteolytic activity.
- Cleavage at the activation site restored activity to WT-SPD but not MI-SPD.
- Mutations at Gly221, except alanine, led to a loss of FSAP activity, suggesting active site disruption.
- Calcium ions enhanced WT-FSAP SPD activity more than sodium ions.
Conclusions:
- The Gly221Glu substitution in FSAP likely impairs its active site or substrate accessibility, leading to reduced function.
- FSAP activity is modulated by metal ions, with calcium playing a significant role.
- This study provides a novel method for producing recombinant FSAP-SPD to study MI-SNP related regulatory mechanisms.
