Role of Glycine 221 in Catalytic Activity of Hyaluronan-Binding Protein 2

Hyaluronan-Binding Protein 2 (HABP2) is a Ca(2+)-dependent serine protease with putative roles in blood coagulation and fibrinolysis. A G221E substitution, known as the Marburg-I polymorphism, reportedly affects HABP2 function and has been associated with increased risk for cardiovascular disease. However, the importance of Gly-221 for HABP2 activity is unclear. Here, we used G221E, G221A, and G221S mutants to assess the role of Gly-221 in HABP2 catalysis. The G221E variant failed to activate single-chain urokinase-type plasminogen activator (scuPA), and the G221A and G221S variants displayed moderately reduced scuPA-activation. Activity toward the peptide substrate S-2288 was markedly decreased in all HABP2 variants, with G221E being the most defective and G221A the least. In the absence of Ca(2+), S-2288 cleavage by wild-type HABP2 was Na(+)-dependent, with Km decreasing from 3.0 to 0.6 mM upon titration from 0 to 0.3 M Na(+) In the presence of 5 mM Ca(2+), Km was further reduced to 0.05 mM, but without appreciable contribution of Na(+) At physiological concentrations of Na(+) and Ca(2+), the three HABP2 variants, and particularly G221E, displayed a major Km increase for S-2288. Chemical footprinting revealed that Ile-16 is significantly less protected from chemical modification in G221E than in wild-type HABP2, suggesting impaired insertion of the N-terminus into the G221E protease domain, with a concomitant impact on catalytic activity. Homology modeling suggested that the Glu-221 side chain could sterically hinder insertion of the N-terminus into the HABP2 protease domain, helping to explain the detrimental effects of Glu-221 substitution on HABP2 activity

Cooperation of Factor VII-Activating Protease and Serum DNase I in the Release of Nucleosomes From Necrotic Cells

Objective. Removal of dead cells is essential in the maintenance of tissue homeostasis, and efficient removal prevents exposure of intracellular content to the immune system, which could lead to autoimmunity. The plasma protease factor VII-activating protease (FSAP) can release nucleosomes from late apoptotic cells. FSAP circulates as an inactive single-chain protein, which is activated upon contact with either apoptotic cells or necrotic cells. The purpose of this study was to investigate the role of FSAP in the release of nucleosomes from necrotic cells. Methods. Necrotic Jurkat cells were incubated with serum, purified 2-chain FSAP, and/or DNase I. Nucleosome release was analyzed by flow cytometry, and agarose gel electrophoresis was performed to detect DNA breakdown. Results. Incubation with serum released nucleosomes from necrotic cells. Incubation with FSAP-deficient serum or serum in which FSAP was inhibited by a blocking antibody was unable to release nucleosomes from necrotic cells, confirming that FSAP is indeed the essential serum factor in this process. Together with serum DNase I, FSAP induced the release of DNA from the cells, the appearance of nucleosomes in the supernatant, and the fragmentation of chromatin into eventually mononucleosomes. Conclusion. FSAP and DNase I are the essential serum factors that cooperate in necrotic cell DNA degradation and nucleosome release. We propose that this mechanism may be important in the removal of potential autoantigen

Role of Glycine 221 in Catalytic Activity of Hyaluronan-Binding Protein 2

Hyaluronan-Binding Protein 2 (HABP2) is a Ca(2+)-dependent serine protease with putative roles in blood coagulation and fibrinolysis. A G221E substitution, known as the Marburg-I polymorphism, reportedly affects HABP2 function and has been associated with increased risk for cardiovascular disease. However, the importance of Gly-221 for HABP2 activity is unclear. Here, we used G221E, G221A, and G221S mutants to assess the role of Gly-221 in HABP2 catalysis. The G221E variant failed to activate single-chain urokinase-type plasminogen activator (scuPA), and the G221A and G221S variants displayed moderately reduced scuPA-activation. Activity toward the peptide substrate S-2288 was markedly decreased in all HABP2 variants, with G221E being the most defective and G221A the least. In the absence of Ca(2+), S-2288 cleavage by wild-type HABP2 was Na(+)-dependent, with Km decreasing from 3.0 to 0.6 mM upon titration from 0 to 0.3 M Na(+) In the presence of 5 mM Ca(2+), Km was further reduced to 0.05 mM, but without appreciable contribution of Na(+) At physiological concentrations of Na(+) and Ca(2+), the three HABP2 variants, and particularly G221E, displayed a major Km increase for S-2288. Chemical footprinting revealed that Ile-16 is significantly less protected from chemical modification in G221E than in wild-type HABP2, suggesting impaired insertion of the N-terminus into the G221E protease domain, with a concomitant impact on catalytic activity. Homology modeling suggested that the Glu-221 side chain could sterically hinder insertion of the N-terminus into the HABP2 protease domain, helping to explain the detrimental effects of Glu-221 substitution on HABP2 activity