title: Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2.
abstract: HABP2 (hyaluronan-binding protein 2) is a Ca2+-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 the single-chain urokinase-type plasminogen activator, and the G221A and G221S variants displayed moderately reduced single-chain urokinase-type plasminogen activator activation. Activity toward the peptide substrate S-2288 was markedly decreased in all HABP2 variants, with G221E being the most defective and G221A being the least defective. In the absence of Ca2+, S-2288 cleavage by wild-type HABP2 was Na+-dependent, withKmdecreasing from 3.0 to 0.6 mm upon titration from 0 to 0.3 m Na+In the presence of 5 mm Ca2+,Kmwas further reduced to 0.05 mm, but without an appreciable contribution of Na+At physiological concentrations of Na+and Ca2+, the three HABP2 variants, and particularly G221E, displayed a majorKmincrease 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.
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and.
authors.forename: Fabian
authors.initials: F
authors.lastname: Stavenuiter
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and.
authors.forename: Eduard H T M
authors.initials: EHTM
authors.lastname: Ebberink
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and.|the Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG Utrecht, The Netherlands.
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and s.meijer@sanquin.nl.|the Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG Utrecht, The Netherlands.
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Meijer
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D020125:Mutation, Missense; D000072417:Protein Domains; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics
chemical_list: C433550:HABP2 protein, human; D012697:Serine Endopeptidases; D014568:Urokinase-Type Plasminogen Activator; D002118:Calcium; D005998:Glycine
keywords: Marburg I polymorphism; allosteric regulation; coagulation factor; enzyme mechanism; factor seven activating protease (FSAP); fibrinolysis; hyaluronan-binding protein 2 (HABP2); serine protease; sodium binding